abstract:
Objective To trace and investigate the distribution of hepatic stellate cells by Cre-Loxp technology ofLrat Cre mice. Methods Lrat Cre mice were mated withH11 LoxP - ZsGreen - Stop - LoxP - tdTomato reporter mice and genotype of their progeny was identified by PCR. The liver sections were fixed, the antibody of liver non-parenchymal cells was labeled with immunofluorescence, and the expression and distribution of liver stellate cells and other liver non-parenchymal cells were analyzed by Imaris 3D reduction, as well as positively identify dual positioning of hepatic stellate cells. Results Lrat Cre H11 LoxP - ZsGreen - Stop - LoxP - tdTomato reporter mice specificiallyled to expression of red fluorescent protein tdTomato in hepatic stellate cells, providing a way to analyze the interactions between hepatic stellate cells, Kupffer cells and liver sinusoidal endothelial cells by immunofluorescence localization. Conclusion Lrat Cre strain as a specific reporter mouse for hepatic stellate cells can be uesd to trace hepatic stellate cells.

abstract:
Objective To investigate the effect of paederosidic acid methyl ester(PAME) on postoperative learning and memory impairment in mice and its mechanism. Methods C57BL/6J male mice were randomly divided into Sham group, operation group, operation + PAME group(PAME group), operation + NADPH oxidase 4(Nox4) adeno-associated virus overexpression group(Nox4 overexpression group), operation + Nox4 adeno-associated virus no-laden group(AAV no-load group), and operation + PAME + Nox4 overexpression group(PN group). Exploratory laparotomy was performed. PAME(20 mg/kg) was administered by continuous gavage for 7 days after operation, and adeno-associated virus was injected into the hippocampus 28 days before operation. Morris water maze test and conditioned fear test were used to detect the learning and memory ability of mice. The expression of Nox4 protein was observed by immunofluorescence. The protein expressions of Nox4, long chain acyl CoA synthetase 4(ACSL4) and glutathione peroxidase 4(GPX4) were detected by Western blot. Reactive oxygen species(ROS) and iron content were determined by spectrophotometry. Results Compared with the Sham group, the learning and memory ability of the operation group, the Nox4 overexpression group and the AAV no-load group decreased, the protein expression of Nox4 and ACSL4 increased, the protein expression of GPX4 decreased, and the ROS and iron content increased. After PAME treatment, the postoperative learning and memory ability of mice was improved, and Nox4 and ferroptosis in hippocampal neurons were alleviated. Conclusion PAME treatment can improve the learning and memory ability of postoperative mice, which may be related to the inhibition of hippocampal Nox4-mediated ferroptosis.

abstract:
Objective To explore the effects of inhibition of the inositol 1,4,5-trisphate receptor(IP3R)-Ca2+pathway on acetaminophen(APAP)-induced liver injury in mice and its mitochondrial-associated endoplasmic reticulum membranes(MAMs). Methods 40 SPF-rated male C57BL/6 mice were randomly divided into control group(n=10),model group(n=10),IP3R inhibitor(2-aminoethoxydiphenyl borate, 2-APB)group(n=10) and the Ca2+chelator [1,2-bis(2-aminophenoxy) ethane-N,N,N′N′-tetraacetic acid, BAPTA-AM] group(n=10).Mice in the model group, 2-APB group, and BAPTA-AM group were given a single intraperitoneal injection of APAP(300 mg/kg).Half an hour before APAP injection, mice in the 2-APB group were injected intraperitoneally with 2-APB(20 mg/kg) and mice in the BAPTA-AM group were injected intraperitoneally with BAPTA-AM(2.5 mg/kg).The mice in each group were executed 24 hours after intraperitoneal injection of APAP.Serum alanine aminotransferase(ALT) and aspartate aminotransferase(AST) were measured by chemical method. The pathological change of liver was observed by HE staining.The ultrastructural changes of mitochondria, endoplasmic reticulum and MAMs in liver cells were observed by transmission electron microscopy.The changes of Ca2+in liver tissue homogenate were measured by calcium assay kit.The protein expression levels of mitofusin 1(MFN1),mitofusin 2(MFN2),inositol 1,4,5-trisphate receptor(IP3R1),glucose regulated protein 75(GRP75) were detected by Western blot. Results Compared with the control group, the serum ALT and AST in the model group increased(P<0.01).Disorganized hepatocyte arrangement, hepatocyte degeneration and lobular central necrosis were observed.Breakage and disappearance of mitochondrial cristae, swelling and fracture of the endoplasmic reticulum and increase of the number of MAMs were also observed by transmission electron microscopy.The Ca2+level in liver tissue homogenate increased(P<0.01).MFN1 and MFN2 protein expression decreased(P<0.01),IP3R1 and GRP75 protein expression increased(P<0.01).Compared with the model group, the serum ALT and AST in 2-APB group and BAPTA-AM group decreased(P<0.01),the liver pathological changes were significantly improved, MAMs decreased, the content of Ca2+in liver homogenate decreased(P<0.01),MFN1 and MFN2 protein expression increased(P<0.01),IP3R1 and GRP75 protein expression decreased(P<0.01). Conclusion Inhibition of the IP3R-Ca2+pathway protects against APAP-induced liver injury in mice. Its mechanism is related to reducing hepatic Ca2+levels, reducing the number of MAMs, and regulating the expression of MAMs-related proteins.

abstract:
Objective To construct a conditional knockout target vector for the TDO2 gene in mice, and to lay a foundation for the establishment of conditional knockout mice of the TDO2 gene. Methods According to the principle of CRISPR/Cas9 technology, single guides RNA(sgRNA) and Donor vectors were designed and constructed. Exon 3 of the TDO2-201(ENSMUST00000029645.13) transcript was used as the knockout region, and a Loxp element was placed on each side of the target gene to establish a conditional gene target vector for conditional knockout of TDO2 exon 3. The CRISPR/Cas9 complex and Donor vector were microinjected into the fertilized eggs of C57BL/6 mice to obtain positive F0 mice. Then the positive F0 generation mice were mated with C57BL/6 mice to obtain F1 generation mice, and the F1 generation mice genotypes were identified by PCR and gene sequencing. Results The results showed that the constructed TDO2 gene conditional knockout vector met the design requirements, and B6/JGpt-TDO2em1Cflox/Gpt mice were successfully bred and identified. Conclusion The successful construction of the conditional knockout target vector of the TDO2 gene in mice builds the foundation for the further construction of TDO2 gene conditional knockout mice.

abstract:
Objective To study the effects of miR-146b-5p and E3 ubiquitin protein ligase(ZNRF3) on the process of hyperglucose-induced epithelial mesenchymal transformation(EMT) and mechanism in mice peritoneal mesoepithelial cells. Methods The mice in normal culture were the control group, and the mice with peritoneal fibrosis were induced by 4.25% glucose dialysate and 0.1 mg/kg lipopolysaccharide. The peritoneal tissues of mice were collected and Masson staining was used to observe the changes of collagen fibers in the peritoneal tissues. The expressions of α-smooth muscle actin(α-SMA) and type I collagen(COL I) were determined by immunohistochemistry. Western blot was used to detect epithelial cadherin(E-cadherin), α-SMA and COL I expression in peritoneal tissues. qRT-PCR was used to detect miR-146b-5p and E3 ubiquitin-protein ligase(ZNRF3) expression in peritoneal tissues. Dual luciferase reporter gene assays were performed to verify the relationship between miR-146b-5p and ZNRF3 targeting. Control mouse peritoneal mesothelial cells were isolated, then high glucose was induced to construct a mouse peritoneal mesothelial cell fibrosis model. The cells were divided into control group, model group, mimic-NC group, miR-146b-5p-mimic group and miR-146b-5p-inhibitor. Transwell chambers were used to detect cell migration and invasion ability. Western blot was used to detect the expression of EMT-related proteins. Results Compared with control mice, model mice showed thickened peritoneal tissue, increased collagen fibers, increased expression of α-SMA and COL I(P<0.05), increased expression of miR-146b-5p(P<0.05), and decreased expression of E-cadherin and ZNRF3(P<0.05). miR-146b-5p targeted to negatively regulate ZNRF3 expression. Cellular level results showed that the number of migrating and invading cells in peritoneal mesothelial cells increased(P<0.05), E-cadherin expression decreased(P<0.05), and Vimentin and N-cadherin expression increased(P<0.05) in model and mimic-NC groups of mice compared with the control group. Compared with model group, the trend of peritoneal mesothelial cell changes was more significant in the miR-146b-5p-mimic group of mice, and the cellular EMT process was inhibited in the miR-146b-5p-inhibitor group. Conclusion MiR-146b-5p can target ZNRF3 gene to promote high glucose-induced EMT process in mouse peritoneal mesothelial cells.

abstract:
Objective To investigate the changes of iron and other metal elements and inflammatory factor γ interferon(IFN-γ) in the liver after taking deferiprone(DFP) in mice with chronic infection ofToxoplasma gondii, and to investigate the efficacy of DFP in the treatment of liver injury caused by chronic infection ofToxoplasma gondii. Methods 6-week-old female C57BL/6 mice were randomly divided into four groups: control group, control + DFP group, TgCtWh6 group, TgCtWh6 + DFP group, with 6 mice in each group, and sterile gavage PBS in control group. Daily gavage was 50 μg/kg DFP in TgCtWh6+DFP group, 20 toxoplasma cysts in TgCtWh6 group, 20 toxoplasmosis cysts in TgCtWh6+DFP group, 20 toxoplasmosis cysts with daily gavage of 50 μg/kg DFP, and mice were sacrificed and liver tissues were taken after 45 days. Inductively coupled plasma mass spectrometry(ICP-MS) was used to detect the content of iron and other metal elements in mouse liver tissues. Real-time PCR(qRT-PCR) and Western blot were used to detect the mRNA levels and protein levels of IFN-γ and ferritin heavy chain(Fth) in mouse liver tissues, respectively. Results Compared with the TgCtWh6 group, the Fth content in the liver(P<0.01) was significantly reduced in the liver of the TgCtWh6+DFP group after taking DFP, the iron metabolism disorder was effectively improved, the IFN-γ content in the liver was significantly lowered(P<0.01), and the inflammatory response of the liver was also reduced. Conclusion DFP can effectively improve iron metabolism disorders in the liver after infection withToxoplasma gondiiand reduce inflammatory damage in the liver.

abstract:
Objective To investigate the expression and clinical significance of tryptophan-2,3-dioxygenase-2 (TDO2) in peripheral blood mononuclear cells(PBMC) and synovial tissue of patients with rheumatoid arthritis(RA).Methods A total of 40 RA patients were selected as the RA group, and 36 healthy subjects were selected as the normal control group during the same period. In RA group and control group PBMC, TDO2 expression was detected by flow cytometry. Immunofluorescence was applied to observe TDO2 expression in synovial tissue of RA groups and control group. The correlation was analyzed between TDO2 expression in PBMC and their inflammatory indicators of RA groups.Results Compared with control group, TDO2 expression in RA group PBMC and synovial tissue significantly increased(P<0.001); TDO2 expression in RA group PBMC was positively correlated with erythrocyte sedimentation rate(ESR)(r=0.405,P=0.045).Conclusion The expression of TDO2 is up-regulated in RA patients PBMC and synovial tissue, and positively correlates with inflammatory indicators ESR, suggesting that TDO2 may participate in RA disease progression.

abstract:
Objective To investigate the expression of programmed death 1(PD1) on CXCR5-CD4+T cells from the patients with systemic lupus erythematosus(SLE) and to analyze the clinical relevance to disease severity. Methods The expression of PD1 on CXCR5-CD4+T cells was examined from 47 SLE patients and 35 healthy controls(HC) by the technique of flow cytometry. The expression of PD1 including percentage of PD1+CXCR5-CD4+T cells and mean fluorescence intensity(MFI) on CXCR5-CD4+T cells was compared between SLE patients and HC. And its correlation with clinical indicators, laboratory inspection and the percentage of plasmablasts was analyzed. Moreover, the predictive value of the expression of PD1 on CXCR5-CD4+T cell was explored. Results (1) The percentage of PD1+CXCR5-CD4+T cells from SLE patients significantly elevated compared with HC(P=0.008 3,U=540.5), and the MFI of PD1 on CXCR5-CD4+T cells from SLE patients significantly elevated compared with HC(P<0.000 1,U=187.0).(2) The percentage of PD1+CXCR5-CD4+T cells was associated with C3(r s =-0.335 2,P=0.022 8), anti-SSA(P=0.016 6,t=2.5), anti-histone(P=0.030 3,t=2.3), treatment(P=0.020 2,t=3.4), plasmablasts(r s =0.387 1,P=0.0072) in SLE patients. The MFI of PD1 on CXCR5-CD4+T cells was associated with SLEDAI(r s =0.403 1,P=0.005 0), ESR(r s =0.356 1,P=0.017 7), CRP(r s =0.337 4,P=0.028 9), RBC(r s =-0.297 0,P=0.042 6), HGB(r s =-0.302 9,P=0.038 5), HCT(r s =-0.381 6,P=0.008 1), L(r s =-0.393 7,P=0.006 2), L%(r s =-0.391 2,P=0.006 5), N%(r s =0.315 0,P=0.031 1), NLR(r s =0.373 0,P=0.009 8), LMR(r s =-0.431 5,P=0.002 5), dNLR(r s =0.315 0,P=0.031 1), anti-Ro52(P=0.029 5,t=63.5), treatment(P=0.035 5, W=21), plasmablasts(r s =0.315 8,P=0.030 6).(3) Logistic regression analysis showed that the MFI of PD1 on CXCR5-CD4+T cells was a risk factor for SLE.(4) ROC analysis showed the AUC of the MFI of PD1 on CXCR5-CD4+T cells was 0.886. A further established model based on combination of the MFI of PD1 on CXCR5-CD4+T cells and HGB showed predictive value in distinguishing SLE from HC with AUC of 0.979. And predictive value was positively associated with SLEDAI(r s =0.313 6,P=0.030 3). Conclusion Increased percentage of PD1+CXCR5-CD4+T cells and increased MFI of PD1 on CXCR5-CD4+T cells in SLE are associated with disease severity and activity, and a model based on combination of the MFI of PD1 on CXCR5-CD4+T cells and HGB shows prominent value for predicting SLE.

abstract:
Objective To investigate the effect and mechanism of exosome(Exo) transported miR-223 on brain tissue injury and microglial activation in rats with traumatic brain injury(TBI). Methods The miR-NC plasmid and miR-223 mimic plasmid were transfected into HEK293 cells by liposome method, and the expression level of miR-223 in the cells was determined by quantitative real-time PCR. Exo was extracted from transfected HEK293 cells and identified by transmission electron microscopy, nanoparticle tracking analysis and Western blot, the expression level of miR-223 in Exo was determined by quantitative real-time PCR. Forty SD rats were randomly divided into sham group, model group, NC-Exo group and miR-223-Exo group, with 10 rats in each group, TBI model was prepared by modified Feeney free fall method in all groups except sham group, rats in NC-Exo group and miR-223-Exo group were injected with cell-derived Exo transfected with miR-NC plasmid and cell-derived Exo transfected with miR-223 mimic plasmidviatail vein, respectively. Two weeks later, hematoxylin-eosin(HE) staining was used to observe the pathological changes of brain tissue in each group, Nissl staining was used to detect the changes and distribution of Nissl bodies in each group, enzyme-linked immunosorbent assay(ELISA) was used to measure the serum levels of tumor necrosis factor-α(TNF-α), interleukin-1β(IL-1β) and interleukin-6(IL-6), immunofluorescence double staining was used to observe the expression of nod-like receptor family pyrin domain containing 3(NLRP3) and ionized calcium binding adaptor molecule 1(Iba-1), Western blot was used to detect the protein expression of NLRP3, apoptosis-associated speck-like protein containing(ASC) and Caspase-1. Results After transfection, compared with control group and miR-NC group, the relative expression of miR-223 in miR-223 group significantly increased(P<0.05). The isolated particles had typical Exo morphology, the peak particle size was about 120 nm, the Exo marker proteins CD9, CD63 and CD81 were significantly overexpressed, and the relative expression of miR-223 significantly increased(P<0.05). Compared with the model group, the damage phenomenon of brain tissue in the miR-223-Exo group was improved, the morphology and number of Nissl bodies were restored, the levels of TNF-α, IL-1β and IL-6 in serum decreased(P<0.05), the intensity of NLRP3 and Iba-1 fluorescence staining in brain tissue decreased(P<0.05), the relative protein expressions of NLRP3, ASC and Caspase-1 in brain tissue were down-regulated(P<0.05). Conclusion Exo operation of miR-223 can significantly improve brain tissue injury and inhibit microglial activation in TBI rats, which may be related to the inhibition of NLRP3.

abstract:
Objective To compare the osteogenic differentiation ability of human apical papilla stem cells(SCAPs), dental pulp mesenchymal stem cells(DPSCs) and alveolar bone mesenchymal stem cells(ABMMSCs) in vitro. Methods The apical papilla stem cells, dental pulp mesenchymal stem cells and alveolar bone mesenchymal stem cells isolated from the third molars and alveolar bone tissues were cultured and passaged. The morphology of primary and P3 generation cells was observed under a microscope. Flow cytometry was used to detect cell immunophenotype. Real-time fluorescent quantitative PCR(qRT-PCR) and cell counting kit-8 were used to analyze cell senescence and proliferation ability. After osteogenic induction, alkaline phosphatase(ALP) and alizarin red staining and qRT-PCR were used to detect osteogenic related genes to compare osteogenic ability. Results There was no significant difference in the morphology of the three cells, which showed the morphology of fibroblasts, long spindle-shaped, smooth and uniform after passage. There were no senescence differences among the 3 types of cells, all of which maintained stable proliferative capacity; the proliferation capacity of SCAPs was significantly higher than that of the other 2 types of cells, and the proliferation capacity of ABMMSCs was weaker; ALP staining and alizarin red staining after 7 and 14 d osteogenesis induction showed that the osteogenic ability of ABMMSCs was significantly stronger than that of SCAPs and DPSCs; qRT-PCR showed that ABMMSCs had the most significant increase in osteogenesis-related genes.Conclusion SCAPs, DPSCs and ABMMSCs have stable biological properties and can undergo osteogenic differentiation, and ABMMSCs have stronger osteogenic ability than DPSCs and SCAPs in vitro.

abstract:
Objective To detect the expression of protein arginine methyltransferase 5(PRMT5) in gastric cancer tissues and gastric cancer cells, and to investigate the regulation of PRMT5 on the proliferation, migration, invasion and epithelial mesenchymal transition(EMT) of gastric cancer cells. Methods (1) The expression of PRMT5 in pathological tissues of chronic non-atrophic gastritis, and pregastric cancer, including chronic atrophic gastritis and intestinal metaplasia, and early gastric cancer was analyzed based on Bioinformatics Analysis by GEO database. Ualcan and HPA databases were employed to analyze the expression of PRMT5 in gastric cancer tissues. The expression of PRMT5 in gastric cancer cells was detected by Western blot.(2) The expression of PRMT5 in gastric cancer cells was regulated by plasmid, and its efficiency was verified by Western blot and RT-PCR. The protein expression of PRMT5 in gastric cancer cells was analyzedviaWestern blot. The abilities of migration and invasion were examined by scratch assay, Transwell and BD Matrigel invasion assays. Clone formation assay and CCK-8 assay were used to examine the proliferation of gastric cancer cells.(3) The expression of interstitial-related proteins and epithelial-related proteins was evaluatedviaWestern blot. Results GEO database found that PRMT5 expression increased gradually in chronic non-atrophic gastritis, precancerous lesions and early gastric cancer. Ualcan and HPA databases found that PRMT5 in gastric cancer tissues were higher than that in normal gastric tissues both at the mRNA and protein levels. PRMT5 upregulation elevated the migration, invasion and proliferation of gastric cancer cells, while PRMT5 downregulation inhibited those. In addition, PRMT5 upregulation raised the expression of interstitial-related proteins and decreased the expression of epithelial-related proteins while PRMT5 downregulation was the opposite. Conclusion PRMT5 is relatively highly expressed in gastric cancer tissues. Furthermore, PRMT5 can enhance the migration, invasion and proliferation ability of gastric cancer cells, and promote EMT in gastric cancer.

abstract:
Objective To investigate whether serine/threonine kinase 3(AKT3)is able to reverse the synergistic inhibition of miR-22-3p/29a-3p on the activation of human hepatic stellate cell LX-2. Methods TargetScan, Starbase, miRDB and DIANA were used to predict the common target genes of miR-22-3p and miR-29a-3p, and the selected target genes were analyzed by Kyoto encyclopedia genes and genomes(KEEG),gene ontology(GO), protein-protein interaction(PPI). The binding free energy of candidate target genes with two miRNAs was analyzed by RNAhybrid, and their targeting relationship was determined by double luciferase reporting experiment. CCK-8, Transwell and qRT-PCR were used to detect the proliferation, migration and the expression of fibrosis markers of LX-2 cells. Results There were 24 common target genes of miR-22-3p and miR-29a-3p, and they were enriched in the hepatic fibrosis-related signaling pathways and biological processes. The binding free energy analysis of candidate target gene AKT3 with miR-22-3p and miR-29a-3p combined with the double luciferase experiment results showed that AKT3 was the common target gene of miR-22-3p and miR-29a-3p. The proliferation, migration and the mRNA expression of fibrosis markers of α-smooth muscle actin(α-SMA) and type I collagen α1 chain(COL1A1) of LX-2 cells were inhibited by miR-22-3p and miR-29a-3p mimics independently or cooperatively(P<0.05), and AKT3 overexpression could reverse the synergistic inhibition of miR-22-3p and miR-29a-3p mimics on LX-2 cells mentioned-above(P<0.05).Conclusion The overexpression of AKT3 can reverse the synergistic inhibition of miR-22-3p and miR-29a-3p on the activation of LX-2 cells.

abstract:
Objective To explore the effects of piperlongumine(PL) on the proliferation and apoptosis of triple-negative breast cancer MDA-MB-231 cellsin vitroandin vivoas well as its possible molecular mechanism. Methods MDA-MB-231 cells were treated with different concentrations of PL. MTT assay was performed to detect the level of cell proliferation. The apoptotic level was detected by flow cytometry. Western blot was performed to detect protein expression levels of proliferating cell nuclear antigen(PCNA), B-cell lymphoma-2(Bcl-2), cyclin-dependent kinase inhibitor 1A(CDKN1A/p21), phosphorylated signal transducer and activator of transcription 3(p-STAT3), signal transducer and activator of transcription 3(STAT3), hypoxia inducible factor-1α(HIF-1α) and Survivin. MDA-MB-231 cells were used to model tumor bearing nude mice and then PL was injected by intraperitoneal(i.p.). The tumor protein expression levels of PCNA, p-STAT3, STAT3, HIF-1α and Survivin were detected by Western blot. Results PL inhibited cell proliferation and induced apoptosis in a dose-dependent manner. PL down-regulated the protein expression levels of PCNA, Bcl-2, p-STAT3, HIF-1α and Survivin, and up-regulated the protein expression level of p21. Furthermore, PL inhibited tumor growth and down-regulated the protein expression levels of PCNA, p-STAT3, HIF-1α and Survivin in nude mice. Conclusion PL inhibits the proliferation of MDA-MB-231 cellsin vitroandin vivo, and the underlying mechanism can be related to the negative regulation of STAT3/HIF-1α pathway.

abstract:
Objective To explore the effects of interleukin(IL)-17/interleukin-17 receptor(IL-17R) on the angiogenesis of the endothelial cells co-cultured with cancer cells through DLL4-Notch signaling pathway and its molecular mechanism.Methods Cancer-endothelial cell co-culture model was established using the Transwell system. The effects of human recombinant IL-17A at 50 ng/ml on the migration of human umbilical vein endothelial cells(HUVECs) in the co-culture system were determined, the proliferation and angiogenesis of HUVECs was observed in co-culture system stimulated by human recombinant IL-17A, the angiogenesis ability of HUVECs was tested by tube formation assay, the proliferation of HUVECs was detected using CCK-8 assay. The expression of DLL4-Notch signaling pathway and angiogenesis protein in each group were detected by Western blot.Results 50 ng/ml human recombinant IL-17A could promote the migration of HUVECs in co-culture system. Treatment with 50 ng/ml human recombinant IL-17A significantly enhanced the proliferation and angiogenesis ability of the HUVECs in coculture system(P<0.01). The expression of DLL4-Notch signaling pathway and angiogenesis-related proteins in co-culture system significantly increased compared with those in control group.Conclusion IL-17/IL-17R promotes DLL4-Notch signaling pathway in the angiogenesis of papillary thyroid carcinoma.

abstract:
Objective To investigate the role of mast cell(MC) in the progression of inflammatory bowel disease(IBD) and the possible regulatory mechanisms. Methods Wild type mice, MC-deficient mice and MC reconstituted mice were used to construct dextran sodium sulfate(DSS)-induced colitis models. Assays such as toluidine blue staining, flow cytometry and Real-time quantitative PCR were used to analyze results. Results MCs migrated and infiltrated to the intestine under the induction of stem cell factor(Scf) and matrix metalloprotein 9(Mmp-9) factors. In the mouse IBD models, the integrity of intestinal mucosal villi in MC-deficient mice was worse than that in WT mice. In MC-deficient mice, the intestinal inflammation was more severe and the ability of mucosal repair was worse. After MC reconstruction, intestinal inflammation was reduced and mucosal repair ability was restored. The ratio of macrophage M1/M2 in WT mouse was lower than that in MC-deficient mouse. The genes interleukin-4(Il4) and interleukin-10(Il10) associated with M2 macrophages were higher in the WT mouse than in the MC-deficient mouse, and other anti-inflammatory factor genes interleukin-13(Il13) and GATA binding protein 3(Gata3) were also higher than those in the WT group. The pro-inflammatory factor gene inducible nitric oxide synthase(Inos) was opposite. Conclusion MC polarizes macrophages to exert anti-inflammatory and promote mucosal repair in the process of IBD.

abstract:
Objective To investigate the molecular mechanism of Toll-like receptor 4(TLR4)/Ras homologue A(RhoA) signaling pathway involved in regulating the effect of septic serum on vascular endothelial cell permeability before and after continuous hemofiltration. Methods The serum of 5 patients with sepsis before and after continuous hemofiltration treatment was collected, and the levels of inflammatory cytokines in serum before and after hemofiltration were detected.Human umbilical vein endothelial cells(HUVEC) were treated with serum before and after continuous hemofiltration for 24 hours. The expression of VE-cadherin, F-actin, TLR4 and RhoA in vascular endothelial cells were detected by Western blot. A TLR4 low expression cell line was constructed to detect the effect of TLR4 low expression on the expression of VE-cadherin, F-actin and RhoA and the permeability of endothelial cells.Results After continuous blood treatment, the serum levels of TLR4, RhoA, interleukin-1(IL-1), interleukin-6(IL-6) and tumor necrosis factor-α(TNF-α) significantly decreased. The expression levels of VE-cadherin, F-actin, TLR4 and RhoA in the serum intervention group after continuous hemofiltration treatment significantly decreased, and the cell permeability significantly decreased. Low expression of TLR4 significantly promoted the expression of VE-cadherin and F-actin, and inhibited the expression of RhoA protein.Conclusion TLR4/RhoA signaling pathway is involved in the regulation of changes in vascular endothelial cell permeability induced by septic serum after continuous hemofiltration treatment.

abstract:
Objective To investigate the expression, clinical significance, progression, and prognosis of miR-381-3p in acute myeloid leukemia(AML), as well as its impact on AML cell proliferation and apoptosis, in order to provide theoretical basis for the treatment of AML. Methods Bioinformatics analysis was used to identify differentially expressed miRNAs, clinical data and blood samples of AML patients were collected, and the expression levels of miRNAs in the bone marrow fluid of the included patients were measured to further elucidate the relationship between miRNAs and AML. The included patients were followed up to calculate overall survival(OS) and disease-free survival(DFS); AML cells were culturedin vitro, miR-381-3p plasmids were constructed, miR-381-3p was overexpressed and miR-381-3p was knocked down in AML, and they were divided into five groups: control, miR-381 mimics, mimics NC, miR-381 inhibitor, inhibitor NC. The proliferation and apoptosis of AML cells were detected using CCK-8 and flow cytometry. Results Differentially expressed miRNAs were identified using bioinformatics analysis, and miR-381-3p was ultimately determined as the study molecule. A total of 90 AML patients were included. The expression level of miR-381 in AML patients was lower than that in the control group, and all FAB subtypes were lower than those in the normal group. The expression level of miR-381 was not related to the age, gender, peripheral blood leukocytes, lymphocytes, and FAB typing of AML patients, and the OS and DFS of miR-381 patients with high expression were significantly prolonged, with statistically significant differences.In vitroexperiments had shown that knocking down miR-381 could inhibit apoptosis and promote proliferation of AML cells. Overexpression of miR-381 could promote apoptosis and inhibit proliferation of AML cells. Conclusion MiR-381-3p is low expressed in AML patients, and its overexpression can significantly prolong OS and DFS. miR-381-3p can promote apoptosis of AML cells, inhibit proliferation, and may become a targeted molecule for the treatment of AML.

abstract:
Objective To investigate the expression and clinical significance of classic vascular endothelial growth factor A(VEGFA) and vasotropic mimicry factor matrix metalloproteinase 14(MMP-14) in lung adenocarcinoma(LUAD). Methods The expression levels of MMP-14 and VEGFA genes and proteins in LUAD and their correlation with survival and prognosis were analyzed using TCGA and UALCAN databases. Serum of 69 patients with LUAD and 20 healthy subjects(control group) was collected, and the contents of MMP-14 and VEGFA were detected by ELISA and chemiluminescence, respectively, to analyze the correlation between the two and the clinicopathological features of tumors and their value in prediction and diagnosis of LUAD. Results The levels of MMP-14 and VEGFA in LUAD tissues and serum were higher than those in control group. There were significant differences in serum VEGFA and MMP-14 expression levels among early stage group, advanced stage group and control group(P<0.001). MMP-14 was higher in T3/T4stage than that in T1/T2stage(P=0.045), higher in N2/N3stage than that in N0/N1stage(P=0.035), and higher in the pleural metastasis group than that in the non-pleural metastasis group(P=0.034). VEGFA level was higher in M1than M0(P=0.025). Elevated VEGFA level was a risk factor for LUAD(P=0.002). The area under the curve(AUC) of MMP-14, VEGFA and CEA alone was 0.793,0.849 and 0.851, respectively, and the AUC of the combined test was 0.952. The overall survival(OS) and disease specific survival(DSS) of MMP-14 and VEGFA low expression group were longer than those of MMP-14 and VEGFA high expression group(P<0.05). Conclusion High expression of MMP-14 and VEGFA in LUAD is associated with the growth, invasion and metastasis of LUAD, respectively, and has implications for survival prognosis determination.

abstract:
Objective To construct the sodium alginate-chitosan polyelectrolyte multilayer membrane structure on the surface of alkaline heat treated titanium by LBL technology, and to combine the peptide that specifically bind to osteoblasts with this structure to explore their slow-release effects. Methods After the smooth titanium surface was heat treated with NaOH, sodium alginate and chitosan were alternately adsorbed and deposited on the surface by layer by layer self-assembly method to form a polyelectrolyte multilayer film structure, and the multilayer film was used to load the peptide that specifically bind to osteoblasts, construct slow-release system, and evaluate the slow-release effect. Results The sample surface was tested by contact angle measuring instrument, field emission scanning electron microscope, fluorescence microscope, X-ray photoelectron spectrometer, Fourier transform infrared spectroscopy and ultraviolet spectrophotometer to confirm that the sodium alginate-chitosan multilayer film structure was successfully constructed on the surface of titanium sheets, and the peptide that specifically bind to osteoblasts were successfully loaded. Conclusion The surface of titanium implants is self-assembled layer by layer to form a multilayer membrane structure and successfully loaded with the peptide that specifically bind to osteoblasts, and the sustained release system of the peptide is constructed, which has a remarkable sustained release effect and is expected to promote osseointegration on the surface of titanium implants.

abstract:
Objective To investigate the effects of gestational diabetes mellitus(GDM) on birth outcome and umbilical artery(UA) blood flow parameters in the third trimester, and to analyze the role of UA blood flow parameters in GDM and birth outcome. Methods Based on the birth cohort from Wuhu, Anhui, China, 189 pregnant women with GDM were collected as the case group. The non-GDM pregnant women were matched 1 ∶1 according to age and pre-pregnancy body mass index, and 189 normal pregnant women were selected as the control group. Pregnant women with GDM were divided into poorly controlled group and well controlled group according to fasting blood glucose in the third trimester. The UA blood flow parameters and fetal birth outcomes in the third trimester were tracked. Results Compared with the control group, UA parameters in poorly controlled and well controlled groups significantly increased(F=6.63,P<0.05;F=4.43,P<0.05;F=5.57,P<0.05). Poor glycemic control of GDM was associated with increased birth weight and risk of larger than gestational age. The multi-factor linear regression model showed that the Z score of the peak systolic velocity/end diastolic velocity(S/D) in the poorly controlled group was negatively correlated with birth weight(β=-209.78, 95%CI:-301.48-118.07). S/D index Z score mediated the relationship between poor blood glucose control and birth weight. The intermediate effect value was-58.41(95%CI:-106.40～-19.65), accounting for 25.98% of the total effect. Conclusion Poor glycemic control in GDM is a risk factor for fetal weight gain, and UA function plays a partial mediating role in influencing neonatal birth weight. GDM pregnant women should strictly control blood glucose level to better protect maternal and infant health.

abstract:
Objective A convolutional neural network(CNN) model was established to automatically analyze flow cytometry(FCM) data to achieve the preliminary diagnosis of acute myeloid leukemia(AML), and explore the feasibility of applying CNN model to FCM data analysis. Methods The exploratory study of CNN application was carried out using the bone marrow FCM data obtained by the FlowRepository database and the Clinical Testing Center of Xinjiang Uygur Autonomous Region People′s Hospital, and the data had been clinically confirmed whether AML was present. Among them, the public data was divided into training sets, validation sets and test sets according to 6∶2∶2, and local data was used for external test; In order to adapt the FCM data to the CNN model, an FCM data structure based on the image matrix principle was proposed, and after preprocessing the original data, the variables related to the preliminary diagnosis of AML were extracted, including sidescattered light and the expression levels of CD45, CD13, CD33, HLA-DR, CD117, CD34, and each variable was written into the matrix. Cell sampling and data augmentation methods were used to increase the sample size of the training set, the keras software package was used to build the LeNet-5 CNN model in Python, and the training set and the validation set were used for model training and parameter tuning respectively to evaluate the performance of the model on the test set. Results The accuracy of CNN to identify AML on the two test sets was 0.931, 0.851, the sensitivity was 0.667, 0.636, the specificity was 0.968, 0.940, and the area under the receiver operating characteristic curve was 0.940 and 0.917. Conclusion Based on the proposed FCM data structure, the CNN model can realize the preliminary diagnosis of AML, indicating that CNN has certain application value in FCM data analysis.

abstract:
Objective To explore the role of T cell 17/regulatory T cell(Th17/Treg) related cytokine imbalance in the pathogenesis of preeclampsia. Methods Serum samples from 50 preeclampsia pregnant women and 30 normal pregnant women were collected, and the concentration of interleukin-23(IL-23)and interleukin-2 recepter(IL-2R) in serum was tested using ELISA. Additionally, the concentration of interleukin-6(IL-6)and interleukin-10(IL-10)in serum was measured using cell microsphere array(CBA) technology.Then placental and decidual tissues of 10 pregnant women with preeclampsia and 6 normal pregnant women were selected for further verification of the expression of related cytokines by using QPCR and immunohistochemistry. Results The concentrations of IL-10, IL-2R, IL-23 and IL-6 in the serum of preeclampsia pregnant women were higher than those of normal pregnant women(P<0.05). The QPCR results showed that, compared to normal pregnant women, there was no statistically significant difference in the expression of IL-23 mRNA in the placenta and decidua tissues of preeclampsia patients(P>0.05), while the expression level of IL-10 mRNA in the placenta and decidua tissues of preeclampsia patients was lower than that of normal pregnant women, with a statistically significant difference(P<0.05). The expression level of IL-2R mRNA in the placenta and decidua tissues of preeclampsia patients was higher than that of normal pregnant women, with a statistically significant difference(P<0.05). The immunohistochemical results showed that the expression of IL-10 in the placenta and decidua of preeclampsia pregnant women was lower than that of normal pregnant women, with a statistically significant difference(P<0.05). The expression of IL-2R and IL-23 in the placenta of preeclampsia was higher than that of normal pregnant women, with a statistically significant difference(P<0.05). However, there was no statistically significant difference in the expression of IL-23 and IL-2R in the decidua of the two groups(P>0.05). Conclusion Compared with normal pregnant women, there are differences in the expression levels of IL-10, IL-23, and IL-2R in preeclampsia pregnant women. This difference may be related to Th17/Treg cellular immune imbalance, suggesting that Th17/Treg immune imbalance mediated changes in related cytokines may be involved in the pathogenesis of preeclampsia.

abstract:
Objective To investigate the efficacy of standardized allergen subcutaneous immunotherapy(SCIT) in children with asthma sensitized to single dust mite allergens versus multiple allergens and to assess the safety of SCIT. Methods 62 children with confirmed allergic asthma who received standardized allergen SCIT were retrospectively analyzed and divided into the monosensitized group(dust mite results≥+++) and the polysensitized group(dust mite results ≥+++ combined with other positive allergens) according to the results of skin prick test, we observed the changes of pulmonary function, medication score and visual analog scale(VAS) scores, children asthma control test(C-ACT)scores, asthma control questionnaire(ACQ) scores before and after treatment in both groupsand compared the efficacy of the two groups. The incidence of local and systemic adverse effects was recorded during treatment in all children to assess the safety of SCIT. Results Standardized allergen SCIT treatment improved lung function parameters, medication scores and VAS scores, C-ACT scores, ACQ scores in both the monosensitized and polysensitized groups, with statistically significant differences before and after treatment(P<0.05). In comparison between the two groups, lung function parameters [forced expiratory flow at 50% vital capacity(FEF50%), maximum midexpiratory flow(MMEF)], medication scores, C-ACT scores and ACQ scores improved significantly in the monosensitized group compared with the polysensitized group after treatment(P<0.001). 62 patients received a total of 2 606 injections during the treatment of SCIT, 6 children had a total of 10 local adverse reactions and 3 children had 3 mild to moderate systemic adverse reactions, with an incidence of 0.38% for local adverse reactions and 0.12% for systemic adverse reactions. Conclusion The children with asthma in both the monosensitized group and polysensitized group achieved significant and safe clinical outcomes under standardized allergen SCIT. The children in the monosensitized group had more obvious clinical effects than the polysensitized group under standardized allergen SCIT.

abstract:
Objective To investigate the factors influencing the purity of peripheral blood natural killer(NK) cellsculturedin vitroand to establish a prediction model for the purity of peripheral blood NK cells. Methods The peripheral blood of 93 healthy donors was collected, the purity of NK cells was detected by flow cytometryafterin vitroculture, and the clinical physical examination indicators of the donors were collected. 77 cases were randomly selected as the training set, and the remaining 16 cases were used as the test set. Pearson′s correlation test was used to analyze the indexes related to the purity of NK cells, and multivariate regression analysis was used to establish a model for predicting the purity of NK cells. We analyzed the correlation between model predicted purity and actual purity and the area under the receiver operating characteristic curve(ROC) of the model. Results The indicators correlated with the purity of NK cells were age, triiodothyronine(T3), red blood cell distribution width(RDW), red blood cell volume distribution width standard deviation(RDW-SD), creatinine(Cr), lymphocyte percentage(LY%), glucose(Glu), percentage of eosinophils(EOS%), number of eosinophils(EOS) and platelet volume distribution width(PDW). For regression model, NK index was-164.557+2.544 RDW-SD +3.730 PDW +4.389 Glu +10.237 T3(R2=0.494,P<0.05). In the test set, the coefficient of determination(R-Square) between the predicted value and the true value of the NK index model was 0.725,Pwas 0.001, and the area under the ROC curve of the NK index model in the training set and test set was 0.815 and 0.938. Conclusion The NK index model can better predict the purity of peripheral blood-derived NK cells culturedin vitro, and provide theoretical guidance for the subsequent formulation of clinical NK cells reinfusion treatment plans.

abstract:
Objective To investigate the natural evolution of the sciatic bone thickness in pediatric untreated unilateral developmental dysplasia of the hip(DDH) aged 1-15 years. Methods 329 cases of DDH children aged 1-15 years with unilateral dislocation were retrospectively recorded. The connection lines were defined on the coronal plane or axial plane of CT. The connection lines of the Y-shaped cartilage center on both sides were line H, the connection lines of the lowest edge of the ischia on both sides were line b, and the middle part of the two lines were divided into Zone 1 and Zone 2. Zone 1 represented the marginal area, and Zone 2 represented the central area. The thickness of ischium, epiphyseal plate, iliac bone thickness and epiphyseal quotient of femoral head on both sides were measured and compared. Results In coronal CL1-CL4, the ischial thickness gradually increased with age from 1 to 10 years old, and decreased from 11 to 15 years old. The range of ischial thickness of CL1-CL4 was 2.1-16.7 mm, 3.3-18.9 mm, 2.4-13.6 mm and 3.0-14.9 mm, respectively. The width of the epiphyseal plate in coronal position, the width of the epiphyseal plate in axial position, and the thickness of the iliac bone in the affected side were greater than those in the opposite side and had statistical differences. In the correlation test of ischial thickness with age and degree of dislocation, the thickness of ischial bone in coronal and axial positions was moderately correlated with age(r=0.413-0.570,P<0.05), and had no correlation with the degree of dislocation(r=0.024-0.073,P>0.05). In the correlation tests of ischial thickness and epiphyseal thickness CD, epiphyseal quotient, coronal iliac thickness IL on the affected side, the thickness of ischial bone in different parts and sections were positive(r=0.427-0.681,P<0.05), and the thickness of ischial bone was negatively correlated with the epiphyseal quotient(r=0.130-0.241,P<0.05). Conclusion The ischial thickness in coronal zone 1 and zone 2 of 1-10 years old children with unilateral DDH increased at a stable rate with age, and the growth rate decreased gradually in 11-15 years old. The thickness of ischia on the affected side in different sections and areas were greater than that on the opposite side. The difference in the central area of the hip joint was greater than that in the marginal area. The thickness of ischia was positively correlated with acetabular cartilage index, epiphyseal plate thickness, and coronal iliac bone thickness.

abstract:
Objective To study the correlation between short stature and ocular biological parameters in children. Methods This study adopted a cross-sectional study. Eighty-two children(82 eyes) with height lower than the tenth percentage and normal vision were selected as the short group. One hundred and thirty healthy children(130 eyes) with age and sex matching were served as the control group. Ocular biological parameters were measured for all subjects, including visual acuity, axial length, corneal curvature, astigmatism, intraocular pressure, central corneal thickness, anterior chamber depth, lens thickness and the distance of whiteness to whiteness. Then axial length/corneal radius ratio was calculated. The differences in ocular biological parameters between the two groups and the correlation between height and parameters were analyzed. Results The measurement of ocular biological parameters showed that the axial length of the short group was lower than that of the control group, and the difference was statistically significant(t=-3.161,P=0.002). Also there were significant differences in corneal curvature, anterior chamber depth between the two groups(t=2.996,-2.449,P<0.05). The corneal curvature of the short group was greater than that of the control group, and the anterior chamber depth of the short group was smaller than that of the control group. There were no significant differences in other ocular biological parameters(P>0.05).In the short group, axial length(t=-2.435,P=0.015), axial length/corneal radius ratio(t=2.577,P=0.012) and anterior chamber depth(t=2.563,P=0.012) of male were higher than those of female, and the difference was statistically significant. Axial length, axial length/corneal radius ratio, intraocular pressure, and anterior chamber depth were positively correlated with age and height, and lens thickness was negatively correlated with age and height. After controlling for age and sex, axial length and axial length/corneal radius ratio were positively associated with height and no correlation with age. Conclusion There are differences between ocular biological parameters in short children and healthy children, which suggests that on the one hand, parents should pay attention to the development of the eyes of their children as soon as possible; On the other hand, there should be different screening standards for short children in eye screening. Moreover, more comprehensive studies need to be included to supplement this data in the future.

abstract:
Objective To analyze the overall survival(OS) of sequential osimertinib treatment in patients with epidermal growth factor receptor(EGFR)exon 20 T790M mutant advanced non-small cell lung cancer(NSCLC) and risk factors of the efficacy of sequential osimertinib treatment.Methods The data of 138 advanced NSCLC patients with acquired EGFR exon 20 T790M mutation who took sequential osimertinib as second-line treatment. KaplanMeier variable was used for survival analysis. The Log-rank method was used for univariate analysis. The COX risk regression model was used for multivariate analysis. The survival status and influencing factors of patients treated with sequential osimertinib were analyzed.Results At the last follow-up, 99 of the 138 patients died. Median progression free survival(PFS1)of first-line of first-or second-generation epidermal growth factor receptor tyrosine kinase inhibitors(EGFR-TKIs) was 11 months(95%CI:10.1-11.9); median PFS2 of osimertinib was 10 months(95%CI: 8.5-11.5); The median PFS with sequential osimertinib treatment was 24 months(95%CI:21.7-26.3), the median OS was 32 months(95%CI: 28.9-35.1). In univariate and multivariate analysis, PFS1 was an independent prognostic factor for PFS and OS(P<0.001).Conclusion Sequential osimertinib treatment for advanced NSCLC patients with acquired EGFR exon 20 T790M mutation achieved good PFS(24 months) and OS(32 months).

abstract:
Objective To study the resistance pattern of streptomycin and ethambutol inMycobacterium tuberculosisin Luoyang area, guide clinical medication and supplement epidemiological data on local drug-resistant tuberculosis. Methods The positive results of high-resolution melting curve(HRM) in 2 941 cases in Luoyang area were analyzed to assess the risk factors associated with streptomycin and ethambutol resistance. Results Of the 2 941 HRM-positive patients, 18.4% were resistant to streptomycin and 8.0% were ethambutol. Both streptomycin and ethambutol and resistance rates were higher in men than those in women(19.0%vs16.9%,P=0.129; 8.0%vs7.9%,P=0.987). The resistance rates to streptomycin and ethambutol were higher in urban than those in rural areas(21.3%vs16.6%,P=0.002; 9.8%vs6.9%,P=0.004). The resistance rate was much higher in previously treated patients than those newly diagnosed for MTB infection(25.8%vs17.3%,P<0.001; 12.1%vs7.4%,P=0.002). The resistance rates to streptomycin were higher in the<51 years than those in the>50 years group(21.1%vs16.1%,P<0.001). According to age, the highest resistance rates to streptomycin and ethambutol occurred in the age range of 31-35 years and 56-60 years in men, respectively, while in the age range of 21-25 years and 56-60 years in women, respectively. In multivariate models, prior treatment history, age less than 51 years, and urban area were positively associated with streptomycin and ethambutol resistance after adjusting for smear results and year testing. Conclusion Men, prior treatment history, age less than 51 years, and urban residents are key monitoring targets for streptomycin and ethambutol resistant tuberculosis.