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Objective To investigate the role of mitochondrial fission and mitophagy in the model of bronchial epithelial cell injury induced by cigarette smoke extract. Methods Bronchial epithelial cells were co-cultured with 5% CSE,pretreated with mitochondrial fission inhibitor Mdivi-1(Md) and mitophagy inducer Urolithin A(UA).Cell proliferation, oxidative stress, mitochondrial structure, the mRNA levels of inflammatory factor(IL-1,IL-6,IL-18,CXCL 1,CXCL 8) and necroptosis components(RIPK1,RIPK3,MLKL),mitochondrial fission protein(DRP1,MFF) and mitophagy protein(p62/SQSTM1,LC3 B)were examined. Results In Beas 2 b cells, 5% CSE induced oxidative stress, increased the mRNA expression of inflammatory factor and necroptosis components, induced the destruction in mitochondrial network, increased the protein expression of mitochondrial fission and mitophagy protein p62/SQSTM1 expression and decreased mitophagy protein LC3 BⅡ/Ⅰ expression.Md/UA pretreatment inhibited oxidative stress, inhibited the expression of inflammatory factor mRNA and necroptosis components mRNA,partially restored mitochondrial network structure and decreased mitochondrial fission protein level.Md pretreatment increased mitophagy protein p62/SQSTM1 protein level, but did not affect LC3 BⅡ/Ⅰ protein expression.Pretreatment with UA inhibited p62/SQSTM1 protein expression, and increased LC3 BⅡ/Ⅰ protein expression. Conclusion Inhibiting mitochondrial fission or promoting mitophagy can reduce CSE-induced oxidative stress and inflammatory response, and restore mitochondrial structure.The mechanism may be related to the inhibition of mitochondrion fission and the mRNA expression of necroptosis components.

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Objective To investigate the role and mechanism of cellular senescence in diffuse large B cell lymphoma(DLBCL) cells resistance to chemotherapy. Methods DLBCL patients in the group were divided into Newly Diagnosed(ND) group, Complete remission(CR) group, relapsed/refractory(r/r) group.HE staining and senescence β-galactosidase staining were used to detect the senescence of lymphatic tissue in the lymph node reactive hyperplasia patients and the r/r DLBCL patients; senescence β-galactosidase staining was used to detect the peripheral blood of DLBCL patients in the ND group and the r/r group; the expression of inflammatory factors in serum of DLBCL patients was detected by ELISA;an senescence model was constructed by repetitively induce senescence of DLBCL cell line LY8 with 10,20,and 40 nmol/L doxorubicin(DOX);CCK-8 was used to detect the cell proliferation of senescent model.Flow cytometry was used to detect the apoptosis of senescent model.ELISA was used to detect cytokine expression in serum of DLBCL patients and supernatant of senescence model. Results Senescent cells in lymph node tissues and peripheral blood of r/r DLBCL patients significantly increased, and the secretion of pro-inflammatory and immunosuppressive cytokines such as interleukin-6(IL-6),interleukin-8(IL-8),interleukin-10(IL-10),interleukin-35(IL-35) and transforming growth factor β1(TGF-β1) in serum increased; 40 nmol DOX was used to induce senescence of LY8 cells, and the proportion of senescent cells was 24.77%.The function detect of control group and DOX induced group showed that the cell proliferation rate of the senescence group was lower than that of the control group but higher than that of the 10 nmol/L and 20 nmol/L DOX groups(P<0.05);The apoptosis rate was higher than the control group but lower than the 10 nmol/L and 20 nmol/L DOX groups(P<0.05).Meanwhile, IL-2,IL-6,IL-8,IL-10,TGF-β1 secretion levels in each group were significantly different(P<0.05).Compared with the control group, the secretion of various pro-inflammatory and immunosuppressive cytokines such as IL-2,IL-6,IL-8,IL-10,IL-35,TGF-β1 in the supernatant of the senescent group increased. Conclusion Cellular senescence promotes DLBCL cell apoptosis resistance by inducing inhibitory cytokine accumulation.

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Objective To study the expression changes of LncRNA-MEG3 in the process of rat cardiac fibrosis and cardiac fibroblasts(CFs) activation and proliferation. Methods SD rats were subcutaneously injected with isoproterenol(ISO) into the abdomen to construct a myocardial fibrosis animal model as the experimental group(ISO group),and the control group was injected with the same amount of normal saline; the CFs of suckling rats were extracted, and transforming growth factor β1(TGF-β1) was used to construct a cell-level fibrosis model as the experimental group(TGF-β1 group),and the control group was CFs cultured normally without TGF-β1;HE and Masson staining were used to observe the changes of myocardial tissue collagen; Western blot method was used to detect the expression of α-smooth muscle actin(α-SMA) and collagen type I(Collagen I) protein; qRT-PCR method was used to detect the expression of Collagen I,α-SMA mRNA and MEG3;The CCK-8 method was used to detect the absorbance(OD value) of CFs after TGF-β1 was used. Results The results of HE and Masson staining indicated that compared with the control group, the volume of cardiomyocytes in the ISO group became larger and disordered, and the area of tissue collagen increased significantly.Compared with the control group, Collagen I,α-SMA protein and mRNA expression in ISO group and TGF-β1 group increased significantly, and the expression of LncRNA-MEG3 decreased significantly; The results of CCK-8 experiments suggested that CFs stimulated by TGF-β1 for 24 h and 48 h significantly increased the OD value of cells compared with the control group, that is, the cell proliferation activity enhanced. Conclusion The expression of LncRNA-MEG3 in rat myocardial fibrosis tissue and the activation and proliferation of CFs cells significantly reduced, suggesting that MEG3 may play a negative regulatory role in rat myocardial fibrosis and activation and proliferation of CFs, providing new ideas for clinical myocardial fibrosis prevention and research.

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Objective To explore the mechanism of exosomes derived from glioma cells on cell proliferation and apoptosis. Methods The U251 exosomes were extracted by high-speed centrifugation.The morphology of exosomes was analyzed by electron microscopy and nano particle size, and the characteristic protein expression of exosomes was detected by Western blot.30 μl of exosome suspension with concentration of 100 μg/ml(exosome group) or equal amount of medium without exosomes(control group) was added to U251 cell culture medium, and the double fluorescence staining method was used to verify whether exosomes could enter U251 cells.The changes of cell proliferation and apoptosis were detected by CCK-8 and flow cytometry.The miRNAs differentially expressed in U251 exosomes and human astroglial cells were screened by RT-PCR.Finally, the U251 cells were transfected with miR-NC,miR-125 b mimicsor miR-125 b inhibitor, exosomes were extracted and added into cell culture medium, and the proliferation and apoptosis of U251 cells were detected by CCK-8 and flow cytometry. Results The exosomes isolated from U251 cells were round or oval vesicular bodies, with the diameter around 100 nm and rich in CD63 and CD9 proteins.After adding exosomes suspension in U251 cell culture medium, exosomes could bepackaged by U251 cells.CCK-8 and flow cytometry showed that the proliferation activity of exosomes was higher than that of the control group, and the apoptosis rate was lower.RT-PCR showed that the content of miRNA-125 b in U251 cells exosomes was higher than that in human astrocytes exosomes.After transfection of U251 cells with miR-NC,miR-125 b mimicsor miR-125 b inhibitor, the exosomes of the three groups were extracted and added into U251 cell culture medium.The proliferative activity of miR-125 b inhibitor group was lower than that of miR-NC group, and the apoptosis rate was higher than that of miR-NC group.The cell proliferation activity of miR-125 b mimics group was higher than that of miR-NC group, and the apoptosis rate was lower than that of miR-NC group.The differences were statistically significant(P<0.05). Conclusion By enriching miR-125 b, exosomes derived from glioma cellscan promote the proliferation of glioma cells and inhibit cell apoptosis.

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Objective To explore molecular biological mechanism of Obesity Hypertension based on circNRG-1/miR-193 b-5 p/NRG-1 Axis. Methods 90 SD rats were randomly divided into three groups: control group(n=30),obese hypertension group(n=30) and NRG-1 blocker group(n=30).Blood pressure, body weight, Lee coefficient, fat coefficient, serum triglyceride and total cholesterol levels and the expression of circNRG-1/miR-193 b-5 p/NRG-1 in thoracic aorta were measured, and the expression of circNRG-1 in obese hypertensive rats was analyzed by microarray, the targeting relationship between circNRG-1 and miR-193 b-5 p was analyzed by double luciferase. Results The results of microarray showed that there were 382 circRNAs differentially expressed between the blank control group and the obese hypertension group, of which 235 were up-regulated and 147 were down-regulated(>2 times). Compared with the blank control group, the MMU-CircRNA-42742(whose paternal gene was NRG-1) was up-regulated in the obese hypertension group. Compared with the blank control group, the MMU-CircRNA-42742(paternal gene NRG-1) was up-regulated in the obese hypertension group.Compared with the blank control group, the systolic blood pressure, body weight, Lee coefficient, fat coefficient, serum triglyceride, total cholesterol, and the mRNA expression of NRG-1,circNRG-1 and miR-193 b-5 p in thoracic aorta of obese hypertensive rats all increased, and the vascular wall was proliferated and the intima was thickened. Compared with obese hypertension group, systolic blood pressure, body weight, Lee coefficient, fat coefficient, serum triglyceride, total cholesterol, mRNA expression of NRG-1,circNRG-1 and miR-193 b-5 p in thoracic aorta decreased in NRG-1 blocker group, and vascular remodeling appeared. Conclusion The expression of circNRG-1 and NRG-1 is up-regulated and the expression of miR-193 b-5 p is down-regulated in obese hypertensive rats.After blocking NRG-1,the symptoms of obese hypertensive rats improve, so circNRG-1/miR-193 b-5 p/NRG-1 axis may be a potential target for the treatment of obese hypertension.

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Objective To analyze the changes of gut microbiota during isoniazid(INH)-induced anti-tuberculosis drug-induced liver injury(ADLI) in rats using 16 S rDNA sequencing technology. Methods 24 male SD rats were randomly divided into control group(D0 group),INH gavage 10 d group(H10 group),INH gavage 28 d group(H28 group).After continuous gavage for 0 d, 10 d and 28 d, fresh rat feces were collected for 16 S rDNA sequencing to analyze the structure and composition of the intestinal flora. And liver tissues were taken for HE staining to observe the pathological changes of rat liver tissues. Results Compared with D0 group, the α diversity of H10 group and H28 group did not change(allP>0.05),the β diversity changed(allP<0.05),and the abundance of Verrucomicrobia at the phylum level showed a decreasing trend, while the abundance of Tenericutes showed an increasing trend(allP<0.05),and the abundances of Lactobacillus, Romboutsia, and Akkermansia at the genus level showed a decreasing trend, while the abundance of Ruminococcaceae_UCG-005,Dubosiella, norank_f__norank_o__Mollicutes_RF39,unclassified_f__Ruminococcaceae, Roseburia and Blautia showed an increasing trend(allP<0.05). Conclusion The structure and species composition of intestinal microflora changed in rats with ADLI induced by INH.

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Objective To investigate the effect and possible mechanism of microRNA-181 a(miR-181 a) on airway remodeling in the lung tissue of rats with chronic obstructive pulmonary disease(COPD).Methods Sixty-five rats were randomly selected from 80 rats to establish COPD models, 56 rats were successfully modeled.They were randomly divided into model group, NC group, mimics group and inhibitors group with 14 rats in each group, and the remaining 15 rats were sham operation group.Two hours after modeling, the mimics group, the inhibitors group and the NC group were injected with miR-181 a mimics, miR-181 a inhibitors and control mimics through the tail vein, and the sham operation group and the model group were injected with saline.After 24 hours of intervention, RT-qPCR was used to detect the relative expression levels of miR-181 a and maternal signal protein homolog 7(Smad7) mRNA in lung tissue, ELISA method was used to detect the levels of interleukin 1β(IL-1β) and tumor necrosis factor α(TNF-α) in lung tissue homogenate, luciferase reporter gene analysis miR-181 a targeting Smad7,HE staining to observe rat lung tissue disease physiological changes, measure bronchial wall and bronchial wall collagen fiber thickness, Western blot was used to detecte lung tissue matrix metalloproteinase-9(MMP-9),transforming growth factor β1(TGF-β1),Smad7,p-Smad7 protein relative expression level. Results Compared with the sham operation group, the relative expression level of miR-181 a in lung tissues of the model group, NC group and inhibitors group decreased, and the relative expression level of miR-181 a in lung tissue of the mimics group increased, and the mimics group>model group and NC group>inhibitors group(P<0.05).Compared with the sham operation group, the levels of IL-1β and TNF-α in lung tissue homogenate of the model group, NC group, mimics group, and inhibitors group increased, and the inhibitors group>model group and NC group>mimics group(P<0.05).The results of the dual luciferase reporter gene analysis system showed that miR-181 a could significantly inhibit the relative luciferase activity of wild-type Smad7(P<0.05),but had no significant effect on the relative luciferase activity of mutant Smad7(P>0.05).HE staining showed that the alveolar cells of the rats in the sham operation group were normal, and the lung parenchyma of the rats in the model group, NC group and inhibitors group was destroyed, with inflammatory cell infiltration and collagen fiber formation.In the mimics group, alveolar cells tended to be normal, inflammation reduced, collagen fibers decreased, and alveolar structural disorder was significantly improved.Compared with the sham operation group,the bronchial wall and bronchial wall collagen fibers in the model group,NC group,mimics group,and inhibitors group were thickened,and the inhibitors group>model group and NC group>mimics group( P<0. 05). Compared with the sham operation group,the relative expression levels of MMP-9 and TGF-β1 protein in the lung tissues of the model group,NC group,mimics group and inhibitors group increased,while the relative expression level of p-Smad7 protein decreased,and MMP-9,TGF-β1 protein relative expression level inhibitors group>model group and NC group>mimics group,relative expression of p-Smad7 protein mimics group>model group and NC group>inhibitors group( P<0. 05). Comparison of the relative expression levels of miR-181 a,IL-1β,TNF-α levels,bronchial wall and bronchial wall collagen fiber thickness,MMP-9,p-Smad7,and TGF-β1 protein expression levels in the model group and NC group,there was no statistical significance( P>0. 05). Conclusion miR-181 a can improve the airway remodeling in the lung tissue of COPD model rats,and may play a role in the targeted regulation of Smad7 expression.

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Objective To compare the efficacy of laser-activated irrigation(LAI) on the removal of smear layer with different power settings when the fiber tip in the canal entrance. Methods 48 extracted single-rooted teeth were randomly divided into 4 groups(n=12) according to the final irrigation techniques: 0.5 W LAI,1.0 W LAI,ultrasonic and needle irrigation.After the final irrigation, the teeth were split longitudinally into 2 parts and observed under a scanning electron microscope.Images were taken at the coronal, middle and apical thirds of the teeth at a magnification of 1 000×and were scored in the presence of the smear layer using the Hülsmann scoring system. Results In the coronal third, 1.0 W LAI group was significantly more effective in removing SL than the ultrasonic and needle irrigation groups(P<0.05).In the apical and middle thirds, 1.0 W LAI and ultrasonic group were comparable on the removal of the smear layer(P>0.05). 1.0 W LAI group was better than the other groups, and the difference was significant(P<0.05) Conclusion 1.0 W LAI with Er, Cr: YSGG laser in canal entrance is comparable on removal of the smear layer and is better than needle irrigation and 0.5 W LAI,and is better than ultrasonic group in the coronal third.

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Objective To explore the impact of 4-amino-2-trifluoromethyl-phenyl retinate(ATPR),a derivative of all-trans retinoic acid, on the motility of human gastric cancer cell line BGC-823 and the possible mechanisms involved under high-level insulin. Methods Firstly,BGC-823 cells were treated with various concentrations of insulin. Cell counting kit-8( CCK-8) assay was used to detect the effect of insulin on cell proliferation. Then,BGC-823 cells were treated with ATPR combined with different concentrations( with no effect on cell growth but could simulate the high insulin level of humans) of insulin. Wound healing and Transwell assays were used to measure the motility of BGC-823 cells under different treatments. Finally,Western blot assay was performed to detect the expression of proteins related to cell migration. Results CCK-8 results showed that,compared with the control group,0. 5 ng/ml and 5. 0 ng/ml insulin had no effect on proliferation of BGC-823 cells after 48 hours while 10. 0 ng/ml insulin significantly promoted the cell proliferation. Wound healing and Transwell assay showed that 5. 0 ng/ml of insulin promoted the migration of BGC-823 cells,however,ATPR reversed the effect of insulin on BGC-823 cells motility,which could also be attenuated by a specific inhibitor of myosin light chain kinase( MLCK)-ML-7. Western blot revealed that insulin upregulated the expression of MLCK while downregulated Claudin 18. But,after ATPR treated,the protein expression of MLCK was inhibited while the expression of Claudin 18 increased. Conclusion ATPR can reverse the effect of high-level insulin on the motility of BGC-823 cells,which may be attributed to the regulation of ATPR on MLCK and Claudin 18 under high-level insulin.

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Objective To study the effect and mechanism of miR-301 a-3 p in exosomes secreted by osteosarcoma on the invasion of osteosarcoma U2-OS cells and the angiogenesis of vascular endothelial cells. Methods Immunofluorescence staining was used to detect whether exosomes could enter HUVEC to play a role.Western blot method was used to detect the effect of miR-301 a-3 p on the expression of vascular-associated proteins CD31,α-SMA and signaling pathway proteins, and the invasion experiment was used to detect the effect of miR-301 a-3 p on the invasiveness of OS cells. Results The exosomes secreted by U2-OS cells successfully entered HUVEC to play a role.Vascular-associated proteins CD31 and α-SMA were highly expressed, PI3 K/Akt, Mek/Erk pathway proteins were all highly expressed, and blood vessel growth ability relatively increased compared with the control group.After miR-301 a-3 p treatment, the invasion ability of U2-OS cells increased. Conclusion miR-301 a-3 p in osteosarcoma-derived exosomes may promote the invasion ability of U2-OS cells and the angiogenesis of HUVEC through PI3 K/Akt and Mek/Erk signaling pathways.

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Objective To explore the effects of exposure to sevoflurane in neonatal mice on adolescent learning and memory and its related mechanisms. Methods 6-day-old C57 BL/6 mice were randomly divided into control group(CON group),sevoflurane group(SEVO group),control+lithium chloride group(LiCl group),sevoflurane+lithium chloride group(SEVO+LiCl group).The SEVO group was exposed to 3% sevoflurane for 3 consecutive days(day 6 to 8),once a day for 2 hours each time, the CON group was a 60% oxygen control, the LiCl group was intraperitoneally injected with GSK3β inhibitor lithium chloride(100 mg/kg) 30 minutes before 60% oxygen exposure, and the SEVO+LiCl group was given intraperitoneal injection of glycogen synthase kinase 3β(GSK3β) inhibitor lithium chloride(100 mg/kg) 30 minutes before exposure to sevoflurane.New object recognition experiment and Y maze experiment were used to detect the learning and memory ability of mice on day 30 to 32; Western blot was used to detect the content of myelin basic protein(MBP),β-catenin protein and the ratio of pGSK3β/GSK3β in mouse hippocampus. Results The results of behavioral experiments showed that the mouse Discrimination index and the percentage of novel arm in the SEVO group were lower than those in the CON group(P<0.05).After pretreatment with lithium chloride, the Discrimination index and the percentage of novel arm in the SEVO+LiCl group were higher than those in the SEVO group(P<0.05),and the learning and memory function was improved.Western blot results showed that the expressions of MBP protein, β-catenin protein and the ratio of pGSK3β/GSK3β in the hippocampus of the SEVO group were lower than those in the CON group(P<0.05).After pretreatment with lithium chloride, compared with the SEVO group, the expression of MBP protein and β-catenin protein in hippocampus of SEVO+LiCl group mice increased, and the ratio of pGSK3β/GSK3β increased(P<0.05). Conclusion Exposure to sevoflurane in neonatal mice causes impairment of learning and memory in adolescence, which may be related to the myelin damage of hippocampus caused by the inhibition of GSK3β/β-catenin signaling pathway GSK3β(Ser9) phosphorylation.

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Objective To analyze the porcine wound exudatesin proteome and N-terminome at multiple time points after burn, and to determine the candidate biomarkers for evaluating the critical turning point of wound progression. Methods Eight Tibetan miniature pigs were randomly divided into 4 groups, 2 pigs in each group: 0 to 2 days(2 d),2 days to 4 days(4 d),4 days to 6 days(6 d),6 days to 8 days(8 d).4 groups of 5 cm deep second degree skin burn wounds were made by solid alcohol burning for 30 seconds, and the ethylene oxide sterilized foam dressing was cut into appropriate size and placed in the wound to collect the wound exudate.Using a TAILS-iTRAQ,we had established a time-resolved proteome and N-terminome resource from wound exudates in a clinically relevant pig burn wound model that we exploited as a robust template to interpret a heterogeneous dataset from patients, and evaluated the prospective biomarker candidates for assessment of critical turning points in wound progression. Results 1 060 quantifiable proteins and 1667 neo-N-termini from porcine wound exudates were collected.By one-way analysis of variance, we identified 416 proteins and 371 neo–N-termini with significant changes in abundance over time and a subset of 188 neo–N-termini that significantly changed in relation to the abundance of the whole protein(cleavages). IPA showed a prevalence of pathways associated with inflammation(reactive oxygen species/nitric oxide synthase in macrophages, acute phase signaling, complement system) and immediate consequences of injury(coagulation system) in clusters comprising proteins with high abundances in the middle(d4,d6)(clusters 2 and 3) and of pathways associated with cell proliferation and migration(remodeling of epithelial adherens junctions) in clusters including proteins and related neo–N-termini with high abundances at the end(d8) of the monitored healing phase(clusters 4 and 5).The correlation of time-resolved protein abundance profiles derived from the pig wound model and from patient data showed a very high correlation(0.84–0.98) of changes in abundance of proteins associated with dynamics of epithelial adherens junctions.ZYX,IQGA1 and HtrA1 could be used as markers of wound healing process in wound exudate of patients treated with negative pressure suction. Conclusion By using iTRAQ-TAILS to analyze the wound exudate from a pig burn model, many known and novel proteolytic processing eventsin vivoare identified, reflecting the key domain of protease activity in this highly complex tissue reaction.

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Objective To investigate the effect of aryl hydrocarbon receptor(AhR) on the proliferation and chondrogenic differentiation of human umbilical cord derived mesenchymal stem cells(hUC-MSCs). Methods The h UC-MSCs were transfected with AhR-homo-1432 inhibitor and AhR-homo-1432 inhibitor negative control,and set as si-NC group and si-AhR group. The proliferation ability of hUC-MSCs were measured by CCK-8 assay on day1,3,5 and 7 after transfection. Real-time quantitative PCR and Western blot were used to detect the mRNA and protein expression of chondrogenic marker SOX-9 and COL2 A1 after 7 days of the chondrogenic differentiation. Alcian blue staining was used to detect the expression of proteoglycan. Results Compared with si-NC group,the mRNA and protein expression of AhR in si-AhR group decreased which indicated the transfection of hUC-MSCs was successful. The results of CCK-8 showed that compared with si-NC group,the cell viability of si-AhR group improved on 5 and 7 day. After induction of chondrogenic differentiation,the results showed that compared with si-NC group,the expression of SOX-9 and COL2 A1 in si-AhR group increased and Alcian blue staining showed that the siAhR group had more proteoglycan contents,deeper staining and stronger cartilage differentiation ability. Conclusion Knocking down the expression of AhR can promote the proliferation and chondrogenic differentiation of h UCMSCs

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Objective To investigate the effect of inhibition of p38 MAPK signaling pathway on renal cell apoptosis in rabbits with urosepsis and its mechanism. Methods Sixty New Zealand rabbits were randomly divided into 4 groups: sham operation group(sham),model group(model),p38 MAPK inhibitor SB203580 group(SB203580) and SB203580+p53 activator Nutlin-3 group(SB203580 + Nutlin-3),with 15 rabbits in each group.The urosepsis model was made by injecting Escherichia coli into the proximal end of the ureter.Intravenous injection of 30 μg/kg SB203580 or intraperitoneal injection of 128 mg/kg Nutlin-3 was used for intervention at 30 minutes before the operation, once daily, for a total of 3 d.The rectal temperature, respiratory rate and heart rate of the 4 groups of New Zealand rabbits were recorded at 24 h, 48 h and 72 h after operation.The automatic analyzer detected the white blood cell count, serum creatinine and urea nitrogen content.ELISA was used to detect the levels of serum Interleukin-1β(IL-1β),Interleukin-6(IL-6) and Tumor necrosis factor-α(TNF-α). Renal tissue was taken for TUNEL staining to observe the apoptosis; qRT-PCR method was used to detect the expression of MDM2 and p53 mRNA in renal tissue; Western blot method was used to detect the expression of Cleaved caspase-3,Bax, Bcl-2,p38 MAPK,p-p38 MAPK,MDM2,p53 and PUMA protein levels in renal tissue. Results Compared with the Model group, rabbits rectal temperature, respiratory rate, heart rate, white blood cell count, serum IL-1β,IL-6 and TNF-α levels, and serum creatinine and urea nitrogen levels reduced in the SB203580 group(P<0.05),and cell apoptosis rate of renal tissue, the mRNA expression of p53 and the proteins expression of p53,Cleaved caspase-3,Bax, PUMA and p-p38 MAPK decreased(P<0.05),while the expression levels of MDM2 mRNA and MDM2,Bcl-2 proteins increased(P<0.05).Compared with the SB203580 group, the rabbits rectal temperature, respiratory rate, heart rate, white blood cell count, serum IL-1β,IL-6 and TNF-α levels, and serum creatinine and urea nitrogen levels increased in the SB203580+Nutlin-3 group(P<0.05),and renal cell apoptosis rate, the mRNA expression of p53 and the proteins expression of p53,Cleaved caspase-3,Bax and PUMA increased(P<0.05),the protein expression of Bcl-2 significantly decreased(P<0.05),while the expression of MDM2 mRNA and MDM2,p-p38 MAPK proteins had no changes(P>0.05). Conclusion Inhibition of p38 MAPK signaling pathway inhibits the p53-mediated apoptosis pathway by promoting the expression of MDM2,thereby reducing renal cell apoptosis in rabbits with urosepsis.

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Objective To develop a nano particle delivery system which combined deacetylated poly(N-acetylglucosamine)(DEAC-PNAG) polymer with miR-150 by self-assembly and the corresponding vascular protection mechanism were discussed. Methods Nano polymer was obtained by self-assembly, characterized by transmission electron microscopy and DLS.The gel displacement method was used to evaluate the encapsulation efficiency.Cell uptake was detected by fluorescence microscopy. RT-PCR was used to detect the expression of miR-150 in transfected cells.Western blot was used to detect the effect of nano-polymer on the expression of Ang-Ⅱ and DLK-1 in HUVECs. Results DEAC-PNAG polymer with nano size and positive surface potential could protect miRNA from RNase A degradation.In addition, DEAC-PNAG@miRNA could promote the cell uptake of miRNA and play a role in vascular protection by inhibiting the expression of Ang-Ⅱ and DLK-1 in HUVECs. Conclusion DEAC-PNAG is an effective miRNAs delivery platform, which may have positive significance for sepsis treatment.

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Objective To study the effect and mechanism of liposomal curcumol(LC) combined with cisplatin on human ovarian cancer cells. Methods The experiment took ovarian cancer cell lines SKOV3and HO8910 as the research objects, combined with different concentrations of cisplatin(0,2,4,8,16,32,64) μg/ml based on 10 μg/ml LC for grouping. MTT,Transwell and flow cytometry assay were used to detect cell proliferation, invasion, migration and apoptosis ability respectively in each group.Western blot assay was used to detect AKT,p-AKT and cleaved Caspase 8,9,3 protein expression in each group. Results MTT results showed that compared with the cisplatin alone or LC group, the tumor cell proliferation rate of the combined drug group reduced(P<0.05),the IC50of cisplatin to SKOV3in the combination was(12.67±2.03) μg/ml(vsthe IC50of cisplatin alone:(38.28±5.98) μg/ml, the IC50of cisplatin to HO8910 in the combination was(10.55±1.55) μg/ml [vsIC50of cisplatin alone:(26.41±2.30) μg/ml]. In the combination group(8 μg/ml cisplatin+10 μg/ml LC),the invasion and migration rates of ovarian cancer cells reduced(P<0.05),and the apoptosis rate was the highest(P<0.05). Western Blot results showed that the expression level of p-AKT protein in ovarian cancer cells in the combined drug group decreased(P<0.05),and the protein levels of cleaved-Caspase8,9,3 increased(P<0.05). Conclusion Low-dose LC can enhance the ability of cisplatin to inhibit the proliferation, migration and invasion of human ovarian cancer cells, and promote tumor cell apoptosis.The combination of drugs may promote apoptosis of ovarian cancer cells through the PI3 K/AKT pathway.

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Objective To analyze the relationship between E26 transformation-specific(ETS) variant 4(ETV4) expression and clinicopathological parameters of colon cancer, and to explore its effect and molecular mechanism on colon cancer invasiveness. Methods Over expression genes in colon cancer were identified by GEPIA and R2 online bioinformatics analysis.The ETV4 protein level of colon cancer tissues and adjacent normal tissues was detected by immunohistochemistry, and the relationship between ETV4 level and pathological parameters of colon cancer was analyzed.The mRNA expression of ETV4 in colon cancer cell lines and immortal normal colonic epithelial cells.After silencing or overexpression of ETV4 in LOVO cells, the invasiveness was evaluated by Transwell assay.The downstream genes of ETV4 were predicted by bioinformatic analysis, and confirmed by Western blot. Results GEPIA and R2 online bioinformatics analysis showed that ETV4 and MMP7 were highly expressed in colon cancer tissues.Immunohistochemical results showed that ETV4 expression was higher in cancer tissues than in paired paracancer tissues, and its expression level was positively correlated with T,N,and M grades of tumors.RT-qPCR results showed that ETV4 mRNA level in colon cancer cell lines HCT116,LS174 T,SW480,LOVO,SW620,and RKO were significantly higher than that in immortal normal colonic epithelial cells FHC.Transwell assay showed that ETV4 promoted the invasiveness of LOVO cells.R2 online bioinformatic analysis showed that ETV4 was positively correlated with MMP7.Western blot results showed that ETV4 upregulated MMP7 in LOVO.Meanwhile, rescue experiments showed that silencing MMP7 reversed the increased invasiveness of LOVO by ETV4. Conclusion The expression of ETV4 increases in colon cancer tissues and cell lines and promotes the invasiveness of colon cancer cells by upregulation of MMP7.

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Objective To investigate the correlation between the level of S-nitroglutathione reducing enzyme(GSNOR) in hippocampus and the changes of spatial learning and memory ability induced by exposure to inflammation in late pregnancy in elderly female rats. Methods Female CD-1 rats with 15 days of pregnancy were randomly divided into bacterial lipopolysaccharide exposure(LPS) and control group(CON),which were intraperitoneally injec-ted bacterial lipopolysaccharide(LPS,50 g/kg; LPS group) or equal volume normal saline(CON group). When female rats were 6 and 18 months,the spatial learning and memory ability was detected by Morris water maze( MWM),the level of hippocampal GSNOR was detected by Western blot. Results Compared with the 6-CON group,the 18-CON group had a longer average swim length in the water maze( P<0. 01),lower percentage of target quadrant swimming distance and hippocampal GSNOR level( P<0. 01),and the comparison between 18-LPS group and 18-CON group also showed the same results. Conclusion Exposure to inflammation in late pregnancy aggravates age-related changes of GSNOR levels in the hippocampus of mice,which may be related to spatial learning and memory impairment in old age.

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Objective To investigate the protective effect and possible mechanism of inhibiting miR-128-3 p expression on oleic acid-induced lung injury in rats. Methods One hundred SD rats were randomly divided into five groups: sham operation group(sham), Model group(Model), antagomir NC group(NC), miR-128-3 p antagomir group(antagomir) and miR-128-3 p antagomir+ML385 group(antagomir+ML385), with 20 rats in each group. ARDS rat model was established by injecting oleic acid(0.15 ml/kg) into tail vein. miR-128-3 p antagomir or ML385 were given for intervention 1 h before surgery. After 24 h of successful modeling, arterial blood oxygen partial pressure(PaO2), oxygenation index(OI) and lung tissue wet/dry weight ratio(W/D) were measured. Lung histopathological changes were observed and scored by hematoxylin-eosin(HE) staining. The contents of tumor necrosis factor-α(TNF-α), interleukin-1β(IL-1β) and interleukin-6(IL-6) in bronchoalveolar lavage fluid(BALF) were determined by enzyme linked immunosorbent assay(ELISA). Superoxide dismutase(SOD), malondialdehyde(MDA) and reactive oxide species(ROS) levels in lung tissue were determined by colorimetric method. The expression levels of miR-128-3 p and mRNA levels of Nrf2 and HO-1 in lung tissues were detected by RT-PCR, and the protein expression levels of E2 related factor 2(Nrf2) and heme oxygenase-1(HO-1) in lung tissues were detected by Western blot. Luciferase reporting experiments verified the targeting relationship between miR-128-3 p and Nrf2. Results Compared with the sham group, the lung tissues of the Model group were seriously damaged, and the lung pathological scores, W/D values, TNF-α, IL-1β and IL-6 contents in BALF, ROS and MDA levels and miR-128-3 p expression levels in the lung tissues increased(P<0.01), However, the values of PaO2, OI, SOD activity, mRNA and protein expression levels of Nrf2 and HO-1 reduced(P<0.01). Compared with the Model group, the degree of lung tissue injury in antagomir group improved, the lung pathological scores, W/D values, TNF-α, IL-1β and IL-6 contents in BALF, ROS and MDA levels and miR-128-3 p expression levels in the lung tissues decreased(P<0.01), However, the values of PaO2, OI, SOD activity, mRNA and protein expression levels of Nrf2 and HO-1 were induced(P<0.01), there was no significant difference in antagomir NC group(P>0.05). Compared with the antagomir group, the variation trend of antagomir+ML385 group was reversed(P<0.01). Luciferase reporting experiments confirmed that miR-128-3 p could target and regulate Nrf2 expression. Conclusion Inhibition of miR-128-3 p expression can reduce lung injury in rats, and its mechanism may be related to the inhibition of inflammation in lung tissues through targeted regulation of Nrf2 expression.

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Objective To investigate the relationship between the expression of thymidine synthase(TS) in gastric cancer and clinicopathological factors, survival and prognosis, as well as the relationship between the blood TS concentration and the timing of intraperitoneal administration of raltitrexed in patients with gastric cancer. Methods Immunohistochemistry was used to detect TS expression in 80 cases of gastric cancer and adjacent tissues. Enzyme-linked immunosorbent assay(ELISA) was used to determine the concentration of TS in 20 cases of fresh gastric cancer and paracancer tissues, and the concentration of TS in blood before and after intraperitoneal administration of raltitrexed in 40 cases. q-PCR was used to detect TS mRNA expression in 20 cases of fresh frozen gastric cancer and its adjacent tissues. Intraperitoneal chemotherapy: Abdominal puncture was given to the group 2 d before surgery, and before abdominal closure was given to the group after surgery. Results The positive rate of TS in 80 gastric cancer tissues(48.75%, 39/80) was higher than that in the adjacent tissues(23.75%, 19/80), and the difference was statistically significant(P<0.001).The average TS concentration in the fresh gastric cancer tissues(1.105×10-3±0.177×10-3)μmol/L was significantly higher than that in the adjacent gastric tissues(0.811×10-3±0.192×10-3)μmol/L, and the difference was statistically significant(P<0.001). TS mRNA expression in 20 fresh gastric cancer tissues(3.08±1.70) was higher than that in adjacent gastric tissues(1.02±0.23), and the difference was statistically significant(P<0.001). The expression of TS in gastric cancer tissue was correlated with the degree of tumor differentiation(P<0.05), and the expression of TS in blood was correlated with N stage and the degree of tumor differentiation(P<0.05). The survival rate of patients with TS negative gastric cancer was higher than that of patients with TS positive gastric cancer(P<0.001), and TS was also an independent risk factor for the prognosis of gastric cancer. After intraperitoneal administration of raltitrexed, the blood TS concentration dropped to the lowest value on the second day, and the disease-free survival rate of the group two days before surgery was higher than that of the group after surgery(P<0.05). Conclusion The positive expression rate of TS increases in gastric cancer, and its expression is related to the degree of tumor differentiation and survival prognosis of patients. Changes in blood TS concentration after intraperitoneal administration of Retictrexate in patients with gastric cancer can be used to predict the optimal time for intraperitoneal administration, and the optimal time for intraperitoneal administration of retictrexate is two days before surgery.

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Objective To analyze the risk factors of coronary atherosclerosis(CAS),and combine hematological indicators and carotid artery plaque to form a new nomogram model to further predict the risk of CAS. Methods This study is a retrospective case-control study.147 patients with suspected coronary artery disease(CAD) were selected from the First Affiliated Hospital of Anhui Medical University.Based on the results of coronary CT angiography(coronary CTA),they were divided into non-CAS group(50 cases) and CAS group(97 cases).Color Doppler ultrasound was used to measure the carotid artery intima-media thickness(cIMT) and plaque,and the results of blood routine test,basic blood chemistry test and coagulation test were collected. Nomogram was used to enstablish the prediction model,and the prognostic value was validated by ROC curve. Results There were differences of age,gender,smoking history and hypertension between the non-CAS group and the CAS group. The red blood cell distribution width-SD value(RDW-SD) of the CAS group,cystatin C,creatinine,fasting plasma glucose,the neutrophil/lymphocyte ratio(NLR),carotid plaque,and cIMT in CAS group were significantly higher than those in the nonCAS group,and the lymphocyte in CAS group was lower than that in non-CAS group(P<0. 05). Multivariate binary logistic regression analysis found that the factors of men,carotid plaque,elevated RDW-SD,elevated fasting plasma glucose and decreased lymphocyte count were independent risk factors for the occurrence of CAS(P<0. 05).Based on the above five indicators,a new nomogram was generated to predict the risk of CAS. The total points of the new nomogram could effectively predict the risk of CAS(AUC: 0. 870,95% CI: 0. 805-0. 936,Hosmer-Lemeshow P = 0. 553). Conclusion This study established a new type of nomogram to predict the risk of CAS. By analyzing the patients' hematological indicators and carotid plaque,the risk of CAS can be accurately predicted to provide guidance for clinical diagnosis and treatment.

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Objective To explore the feasibility of a dilated convolution U-Net model for accurate segmentation of pancreas. Methods A kind of multi-scale dilated convolution U-Net model was improved based on standard U-Net. Radiotherapy positioning CT of 100 patients with pelvic tumors, containing the whole pancreas, were included in this study, which were used to train and test two kinds of models, and finally the results were compared. The quantitative indicators for the result evaluation were Dice Similarity Coefficient(DSC), Jaccard Similarity Coefficient(JSC), Hausdorff Distance(HD), and Average Surface Distance(ASD). Results The average DSC was 0.87, JSC was 0.78, ASD was 1.62 mm, HD was 9.85 mm. Comparing the standard U-Net, the dilated convolution U-Net had a better performance. T test was used in result analysis and the P value was less than 0.05 which meant that there were significant differences between the two results. Conclusion Building the dilated convolution model based on U-Net can accurately segment healthy pancreas, which is of great significance for improving the computer aided diagnosis system and the efficiency of radiotherapy.

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Objective To analyze the levels of interleukin 6(IL-6) in different groups of patients with severe fever with thrombocytopenia syndrome(SFTS) and to investigate the clinical predictive value of IL-6 levels oon the severity and prognosis of the disease. Methods According to the severity and prognosis, 105 patients with confirmed SFTS were divided into mild group VS severe group and improved severe group VS the poor prognosis group.The difference between IL-6 and each clinical indicator in different groups was compared and the correlation between IL-6 and various indexes was analyzed.Multivariate Logistic regression was used to analyze the independent risk factors of the severity and prognosis, and receiver operating characteristic curve(ROC) was used to assess the predictive value of IL-6 for severity and prognosis in SFTS patients. Results The lymphocyte number(L),platelet number(PLT) and albumin(ALB) were lower in the severe group than those in the mild group, and the age, IL-6,aspartate aminotransferase(AST),creatine kinase isoenzyme(CKMB),lactate dehydrogenase(LDH),mitochondrial aspartic transaminase(mAST),blood amylase(AMY),lipase(LPS),α-hydroxybutyrate dehydrogenase(HBDH) and calcitonin(PCT) were higher in the severe group(allP<0.05); age, IL-6,HBDH,LPS and PCT were higher in the poor prognosis group than those in the mild group(allP<0.05),while the differences in other indicators were not statistically significant(allP>0.05). Levels of IL-6 were associated with AST,creatine kinase(CK),CKMB,LDH,HBDH,mAST AMY,LPS and PCT were positively correlated(allP<0.05),and negatively correlated with L,monocyte count(M),PLT and ALB(allP<0.05),while there was no significant correlation with other indexes.IL-6 was found to be an independent predictor of severity and prognosis in the multifactorial logistic regression analysis.When predicting severe disease, the area under the curve(AUC) was 0.883,with a sensitivity and specificity of 61.54% and 98.04%,respectively; when predicting poor prognosis, the AUC was 0.937,with a sensitivity and specificity of 88.00% and 88.89%,respectively. Conclusion IL-6 level has an important clinical value in evaluating the severity and prognosis of SFTS patients.

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Objective By collecting various clinical data during chemotherapy of breast cancer patients, we can discover the influencing factors of liver function abnormalities in breast cancer patients during chemotherapy, and establish a risk prediction model.To find out the influencing factors of liver function abnormalities in breast cancer patients during chemotherapy by collecting various clinical data during chemotherapy, and to establish a risk prediction model. Methods Epidemiological questionnaire surveys, clinical biochemical tests and blood sample collection were conducted for admitted breast cancer patients, and the age, tumor type, tumor TNM staging, chemotherapy regimen and other data of breast cancer patients were analyzed during chemotherapy, and decision trees were used to construct breast glands cancer chemotherapy drug-induced liver injury risk prediction model.During this period, Tangshan People′s Hospital admitted a total of 675 breast cancer patients, and all were women.According to the inclusion and exclusion criteria of the study subjects, it was found that demographic information, clinical biochemical indicators, and blood samples were collected from complete a total of 640 breast cancer patients.EpiData 3.0 was used to establish a database, and R software was used for statistical analysis. Results A total of 640 breast cancer patients were investigated.After chemotherapy, 106 breast cancer patients developed liver damage, the prevalence rate was 16.56%.The proportion of patients with drug-induced liver injury(22.82%) in the 60-80-year-old age group was higher than that in other age groups, and the incidence of liver injury showed an upward trend with age(P<0.05).The rates of liver injury in breast cancer patients who had been menopausal and breast cancer patients with a long-term medication history were 21.17% and 17.32%,respectively, which were higher than those of non-menopausal and liver-impaired patients without long-term medication history.The difference was statistically significant(P<0.05).Tumor pathology classification, tumor TNM staging, and chemotherapy regimens were all related to the incidence of drug-induced liver injury in breast cancer patients.Among them, the incidence of liver injury in the combined drug group in the chemotherapy regimen was 20.67%,which is significantly higher than that of other drug groups.There was statistical significance(P<0.05).The decision tree model for breast cancer chemotherapy drug-induced liver injury risk screened out three observational indicators for predicting liver injury risk: age, tumor TNM stage and combination medication group.These three indicators were correct in predicting the risk of breast cancer chemotherapy drug-induced liver injury The rate was 92.19%,and the sensitivity and specificity were 86.36% and 93.40%. Conclusion Age, menopause, long-term medication history, tumor pathology classification, tumor TNM staging, and chemotherapy regimens are the influencing factors for liver injury in breast cancer patients during chemotherapy.Combining the above influencing factors, a risk prediction model for breast cancer chemotherapy drug-induced liver injury is established to realize individualized treatment of patients and ensure the smooth progress of chemotherapy for breast cancer patients.

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Objective To compare the outcomes of “Trifecta” and “Pentafecta” in laparoscopic partial nephrectomy between patients receiving modified and traditional technique, and to further evaluate the value of safety and renal function preservation of the modified technique and determine its indication. Methods 80 patients including 40 receiving modified technique and 40 receiving traditional technique were recruited before clinical data of baseline, perioperative and follow-up were collected and analyzed. Results No significant difference in the incidence of complications, positive margin, recurrence, as well as the trifecta achievement had been found between groups.The achievement of pentafecta in group of modified technique was better than the group of traditional technique.Older age, larger diameter of tumor and lower preoperative eGFR had been found to be the independent risk factors of pentafecta failure in the group of traditional technique, with the cut-off value being 64 years, 35 mm and 73 ml/(min·1.73 m2) respectively. Conclusion The modified technique does not increase the risk of complications and has advantages of renal function preservation and pentafecta achievement in patients with older age, larger tumor and worse renal function.

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Objective To evaluate the value of color doppler ultrasonography and shear wave elastography in the acute kidney injury after the kidney transplantation. Methods The data of general demographic, ultrasound and blood index had been collected.t test, χ2test and logistic regression model had been used to analyze the data. Results 202 patients were included in this survey, and 67 patients were AKI.Univariate analysis found that AKI after the kidney transplantation had an older age(t=2.87,P<0.01),shorter length of transplanted kidney(t=2.56,P=0.01),lower peak systolic velocity(P<0.01),higher resistance index and pulsatility index(P<0.01),higher scores of max, mean and min values of renal cortex(t=4.21,P<0.01;t=4.92,P<0.01;t=4.34,P<0.01).Logistic regression model analysis found that length of transplanted kidney(OR=0.96,[95%CI,0.90-0.99]) and PSV of renal aorta(OR=0.98,[95%CI,0.97-0.99]) were protective factors. Conclusion The technique of doppler ultrasonography has a certain application prospect in evaluating the occurrence of AKI after kidney transplantation, while the technique of shear wave elastography needs further study.

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Objective To explore the prognostic factors of primary gastrointestinal diffuse large B-cell lymphoma(PGI-DLBCL). Methods A total of 126 patients with PGI-DLBCL were selected.The demographic data, clinical characteristics and laboratory characteristics were collected for retrospective analysis.Kaplan-Meier survival analysis was used for univariate analysis and Cox proportional hazard models were used for multivariate analysis to determine prognostic related factors. Results Among 126 patients with PGI-DLBCL,22 patients died, and the 1,3,5 year cumulative survival rates were 90.5%,87.0%,82.6% respectively.Age, Ann Arbor staging, NCCN IPI score, mid-term efficacy evaluation, lactate dehydrogenase( LDH),hemoglobin count(Hb),albumin(ALB),prealbumin, stool occult blood test, β2microglobulin and lymphocyte percentage were all factors associated with the poor prognosis of PGI-DLBCL.Hb, prealbumin and mid-term efficacy evaluation were independent prognostic factors in patients with PGI-DLBCL. Conclusion Anemia, low prealbumin and poor mid-term assessment are independent prognostic factors of PGI-DLBCL.

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Objective To explore the associations between rs11225163 and rs1820453 of Yes associated protein(YAP) gene and the risk of Helicobacter pylori(H.pylori) infection and non-cardia gastric cancer, and to provide new ideas for the prevention and treatment of gastric cancer and diseases related to H.pylori infection. Methods Taqman method was used to genotype 427 cases with non-cardia gastric cancer and 381 controls.Enzyme linked immunosorbent assay(ELISA) was used to detect the titer of the anti-H.pylori antibody in the peripheral blood of normal people.The Unconditional Logistic regression method was used to calculate the odds ratio(OR) and 95% confidence interval(CI) to evaluate the associations betweenYAPSNPs and the infection of H.pylori and non-cardia gastric cancer under Codominant, Dominant, Overdominant, Recessive, and Log-additive genetic models.Haploview 4.2 software was used to construct haplotypes. Results No associations were found between SNP rs11225163 and rs1820453 and infection of H.pylori under Codominant, Dominant, Overdominant, Recessive, and Log-additive genetic models.No haplotype was constructed between the two sites.No associations were found between SNP rs11225163 and rs1820453 and the risk of non-cardia gastric cancer under Codominant, Dominant, Overdominant, Recessive, and Log-additive genetic models.There was no haplotype between the two sites. Conclusion YAPrs11225163 and rs1820453 may not play a major role in H.pylori infection and the risk of non-cardia gastric cancer.

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Objective To evaluate the indication and efficacy of ESD in the treatment of early esophageal squamous cell carcinoma by analyzing the clinicopathological features and short-term outcome of endoscopic submucosal dissection(ESD) in the treatment of early esophageal squamous cell carcinoma. Methods This study retrospectively analyzed the general clinical data, ESD treatment, histological and pathological results of resected specimens and follow-up results of patients with early esophageal squamous cell carcinoma treated with ESD. Results It was found that the lesion size, the circumference of the lumen, the depth of invasion and the resection margin were related to lymphovascular invasion(LVI).Multivariate analysis showed that the circumference of the lumen and the depth of invasion were independent risk factors for LVI.The en bloc resection rate was 100.00%(328/328),the complete resection rate was 94.51%(310/328),and the curative resection rate was 89.33%(293/328).The postoperative complications were as follows: delayed bleeding 0.75%,esophageal stenosis 4.49%,and no perforation occurred.The overall recurrence rate was 0.75%(2/267).A total of 23 patients underwent additional surgery or adjuvant treatment after ESD.There was not any additional treatment in group A(pCurA),radiotherapy was the main additional treatment in group B(pCurB),and surgery was the main additional treatment in group C(pCurC). Conclusion For patients with early esophageal squamous cell cancer whose depth of invasion do not exceed SM1 and there is no lymph node metastasis, ESD is safe and effective, and can be used as the first choice for treatment.Patients with lesion circumference ≥3/4 or deep invasion are independent risk factors for LVI,which should be paid attention to in preoperative evaluation.In postoperative evaluation, patients with pCur A or pCur B should be closely followed up, while patients with pCur C should be actively supplemented with surgery or adjuvant treatment.