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Objective To investigate the protective effect of vitamin D3on alcoholic liver injury in mice. Methods Forty mice were randomly divided into 4 groups: normal Control(Control) group, vitamin D3(VitD3) group, alcohol model(EtOH) group and alcohol + vitamin D3(EtOH+VitD3) group.The mice were fed with the DeCarlialcohol liquid diet to establish alcoholic liver injury model.Serum levels of alanine aminotransferase(ALT),aspartate aminotransferase(AST) and liver index were detected.HE staining was used to observe the pathological changes of liver.The relative expression levels of tumor necrosis factor α(TNF-α),transforming growth factor β(TGF-β),interleukin-6(IL-6) and interleukin-1β(IL-1β) mRNA were detected by quantitative real-time PCR(qRT-PCR).The expressions of nuclear factor-kappa B(NF-κB) p65 and NF-κB p50 in liver were detected by Western blot. Results The serum ALT,AST vitality, liver index and hepatic TNF-α,TGF-β,IL-6 and IL-1β mRNA in EtOH group were significantly higher than those in Control group.EtOH group disorganized hepatocyte and hepatic lobules boundary was not clear, and the hepatocytes showed apparent inflammatory cells infiltration of liver cells and fat cavitation.NF-κB p65 and NF-κB p50 protein expression increased significantly.Compared with EtOH group, the serum ALT,AST vitality, liver index and hepatic TNF-α,TGF-β,IL-6 and IL-1β mRNA in EtOH+VitD3group decreased significantly.The pathological staining results showed that inflammatory cells infiltration and decrease in the number of fat vacuoles, and the liver cells returned to normal liver cell structure.At the same time the NF-κB p65 and NF-κB p50 protein expression level decreased significantly. Conclusion Vitamin D3has a certain protective effect on alcohol-induced liver injury in mice, and its main mechanisms are related to the inhibition of NF-κB pathway and the reduction of inflammatory response.

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Objective To identify and characterize the g-type lysozyme in G. zanaensis and analyze its role in host immune system. Methods GzLysG cDNA sequence was cloned by Nest PCR technology. Bioinformatics analysis of the GzLysG protein was carried out by ExPASy, SignalP 5.0, CDD, Cluster Omega and other online software. The GzLysG gene expression pattern in G. zanaensis tissues was detected by RT-qPCR. Results The results showed that open reading frame of GzLysG cDNA was 558 bp in length, encoding 185 amino acids. The molecular weight and theoretical isoelectric point of the reduced protein was 20 478.20 and 9.16, respectively. GzLysG was predicted to be a basic and hydrophilic protein, had no signal peptide and contained the typical catalytic active site, GEWL domain and SLT domain of g-type lysozyme. Advanced structural analysis revealed that GzLysG protein was mainly composed of α-helix and random coil. There was a long and narrow crack on GzLysG molecular surface which was closely related to lysozyme activity. Lysozyme was highly conserved in evolution, with GzLysG showing a close topologic relationship with lysozyme from Danio rerio. Quantitative real-time polymerase chain reaction analysis indicated that GzLysG ubiquitously existed in all examined tissues, with higher mRNA expression levels observed in skin, muscle and gill. Conclusion All those results suggest that GzLysG plays a key role in G. zanaensis immune defensive system.

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Objective To detect the expression of serum exosomal miR-30d-5p in occupational dermatitis medicamentosa-like of trichloroethylene(OMDT) patients and its correlation with liver function, then perform bioinformatics analysis and verify the target gene. Methods Serum exosomes were extracted from 6 OMDT patients and 6healthy controls, and miRNA was extracted from exosomes which were identified by transmission electron microscopy, nanoparticle tracking analysis and western blotting.The expression of serum exosomal miR-30d-5p was detected by real-time fluorescence quantitative PCR,then the correlation with liver function was analyzed.The target genes of miR-30d-5p were predicted by the miRWalk and miRBD databases.Gene ontology analysis and KEGG pathways analysis were performed.Finally, RHOB was verified by Dual-luciferase reporter assay. Results The expression of serum exosomal miR-30d-5p significantly decreased in 6 OMDT patients at the peak of the disease(P<0.05),and it was negatively correlated with the level of AST,ALT and GGT(correlation coefficient:r s =-0.943,P=0.005;r s =-0.886,P=0.019;r s =-0.886,P=0.019). Bioinformatics analysis and dual-luciferase reporter assay showed that RHOB was the target gene of miR-30d-5p. Conclusion The expression of serum exosomal miR-30d-5p decreases in OMDT patients, which is negatively correlated with the level of liver function, and the target gene RHOB may be involved in the process of liver injury induced by trichloroethylene.

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Objective To investigate the role and mechanism ofSigirrdeletion in chronic kidney disease complicated with renal interstitial fibrosis(CKD-RIF) in mice. Methods polymerase chain reaction(PCR) was used for identification of gene types of mice.Mice were continuously fed with the foods containing 0.2% adenine for 12 weeks to establish the CKD-RIF models.Then, serum was collected to detect levels of creatinine and nitrogen when mice were killed.H&E staining was used to analyze the pathological changes of kidney tissues.Masson staining was used to observe the degree of renal fibrosis.Immunohistochemistry was used to detect the changes of the interest proteins, such as IL-1β,MyD88,activated NF-κB,TGF-β1,E-cadherin and Vimentin. Results Serum creatinines and urea nitrogens of mice fed with high adenine(CKD-RIF groups) significantly increased, compared with those of the control groups.H&E and Masson staining results showed that there were more infiltrated inflammatory cells and more critical collagen fiber deposition in the renal tissues of theSigirr-/-mice with CKD-RIF.Western blot and Immunohistochemical analysis showed that the expression of IL-1β and its downstream MyD88 increased, and the level of phosphorylated NF-κB(p-P65) significantly increased in the renal tissues of CKD-RIF mice compared with the controls.And upregulation of these proteins in renal tissues ofSigirr-/-mice with CKD-RIF was more obvious than that of the CKD-RIFSigirr+/+mice.TGF-β1,as a key cytokine involved in renal interstitial fibrosis, significantly increased, followed by the increase of vimentin, as well as the decrease of E-cadherin.The results of vimentin and cadherin E detected by Western blot were consistent with those of immunohistochemistry, and α-SMA also increased significantly. Conclusion Adenine diet successfully induces CKD-RIF mice models.Sigirrdeletion is beneficial to activation of the IL-1β mediating NF-κB signal pathway, which promotes TGF-β1 expression in the renal interstitiums to induce renal interstitial fibrosis.

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Objective To study anti-hepatitis C virus NS3/4A protease activity ofLonicera fulvotomentosabased on fluorescence method and molecular docking. Methods The activity of HCV NS3/4A protease inhibition ofL.fulvotomentosaextracts was tested by fluorescence method.The binding of the main active ingredients to HCV NS3/4A virus protease was analyzed based on molecular docking. Results The water extract and alcohol extract ofL.fulvotomentosahad a good inhibitory effect on the activity of HCV NS3/4A protease with IC50of 0.005-0.019 mg/ml.Based on molecular coupling and interaction analysis, it was found that chlorogenic acid, isochlorogenic acid A,cynaroside and luteolin combined well with HCV NS3/4A protease, forming multiple hydrogen bonds. Conclusion The active components ofL.fulvotomentosaagainst HCV NS3/4A protease activity were isochlorogenic acid A and luteolin.

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Objective To study the effect of crisaborole on imiquimod(IMQ)-induced psoriasis in mice. Methods Forty eight Balb/c mice were randomly divided into crisaborole group(7.5, 15, 30 mg/cm2), halometasone group(15 mg/cm2), model group and normal group. IMQ was applied to the back of mice to establish the psoriasis model. Psoriasis area and severity index(PASI) score was calculated, pathological changes, skin epidermal thickness and inflammatory cell infiltration in the dermis were observed by HE staining. The expressions of keratin(K)1, K10, K6, K16 and K17 in skin lesions were detected by Western blot and immunohistochemistry. The levels of cyclic adenosine monophosphate(cAMP), protein kinase A(PKA) and phospho-cAMP response element binding protein(p-CREB) were detected. Results Compared with the model group, the PASI score of the crisaborole group decreased, the expression levels of proliferative keratin(K6, K16 and K17) decreased(F=12.62、19.41、28.39,P<0.01), and the expression levels of differentiation keratin(K1 and K10) increased(F=27.95、9.64,P<0.01). Conclusion Crisaborole plays a therapeutic role in IMQ-induced psoriasis in mice by regulating the abnormal proliferation and differentiation of keratinocytes.

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Objective The research was aimed at exploring the effects of mandelic acid on the proliferation, apoptosis and migration of H1299 lung adenocarcinoma(LUAD) cells and the related molecular mechanisms. Methods CCK-8 was used to detect changes in the proliferation ability of H1299 cells; Hoechst 33258/PI double-staining assay was used to analyze the apoptosis of H1299 cells; Transwell and migration assays were used to analyze changes in the invasive and migration ability of H1299 cells; Western blot was used to detect the expression of proliferation, apoptosis and migration-related pathway proteins. Results Different concentrations of mandelic acid inhibited the proliferation viability and invasion and migration ability of H1299 cells(P<0.05).Mandelic acid induced high expression of bax and cl-caspase-3 and low expression of p-stat3 in proliferation and apoptosis pathways(P<0.05).In addition, it inhibited the expression of MMP-9 and Vimentin proteins, which were related to invasion and migration(P<0.01). Conclusion Mandelic acid inhibited the proliferation of H1299 LUAD cells and elevated the level of apoptosis, and molecular biological mechanism might be closely related to the decreased activation of stat3 and activation of bax/caspase-3 signaling axis.The inhibition of invasive and migrational ability of H1299 cells induced by mandelic acid was associated with the decreased expression of MMP-9 and Vimentin proteins.

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Objective To construct a myosin light chain kinase(MLCK) knockout hypopharyngeal carcinoma FaDu cell line and discuss the knockout effect of MLCK on apoptosis of FaDu cells. Methods MLCK knockout FaDu cell lines were constructed by liposome transfection of sgRNA and Cas9 2NLS Nuclease, and the knockoutcell lines were identified by DNA sequencing and divided into control group, MLCK KO 1 group and MLCK KO 2 group. The knockout efficiency of MLCK was assessed by RT-qPCR and Western blot, and the effect of MLCK knockout on cell cycle and apoptosis was detected by flow cytometry while Western bolt was conducted to detect the effect of MLCK knockout on cell apoptosis. Results DNA sequencing showed that MLCK base sequences were missing or substituted at the sgRNA sequence recognition, and RT-qPCR and Western blot showed lower mRNA and protein levels in MLCK knockout cell lines than those in the control group(P<0.000 1). Furthermore, the flow cytometry assay showed that MLCK knockout did not significantly change the cycle of FaDu cells, but the rate of apoptosis increased(P<0.000 1).Western blot showed that Bax/Bcl-2(P<0.000 1) and Cleaved Caspase-3/Caspase-3(P=0.000 7) ratios raised in the MLCK knockout group. Conclusion The knockout of MLCK induced apoptosis in FaDu cells, but the exact mechanism needs further study.

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Objective To explore the changing characteristics of Wnt2/β-catenin pathway in liver regeneration and repair in C57BL/6 mice. Methods Male C57BL/6 mice were randomly divided into a sham-operated group(Sham group),1 d post-operative group, 2 d post-operative group, 4 d post-operative group, 6 d post-operative group, and 8 d post-operative group.Mice in the operated group underwent partial hepatectomy(PHx) to remove the left and middle lobes of the liver, respectively.Plasma and liver tissues were collected on postoperative days 1,2,4,6,and 8,and plasma ALT and AST activity was measured by ALT(alanine aminotransferase) and AST(aspartate aminotransferase) biochemical analysis kits; Ki67-positive cells were identified by immunohistochemistry in each group; the number of HNF4-α and Ki67 double positive cells, the number of LYVE1 and Ki67 double positive cells, and the number of β-catenin transferred into the nucleus were determined by immunofluorescence method.The expression of Wnt2 protein in each group was detected by Western blot, and the time characteristics of its expression during liver regeneration were analyzed. Results Liver weight and liver/body weight ratio peaked on day 6 after PHx and approached the level of the Sham group.After PHx, there was severe liver damage on day 1,but it had normalized by day 4.On day 2 after PHx, mainly hepatocytes proliferated; and on days 4 and 6,mainly liver sinusoidal endothelial cells proliferated, while the Wnt2/β-catenin pathway was activated. Conclusion The liver has a powerful regenerative repair ability, which is closely related to the rapid proliferation of hepatocytes and liver sinusoidal endothelial cells, and the activation of the Wnt2/β-catenin pathway is activated in the regenerative repair of the mouse liver.

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Objective To explore the effect of FTO(fat mass and obesity-associated protein) and inhibitor on rituximab resistance in DLBCL(diffuse large B cell lymphoma). Methods Two cell lines derived from “double strike” lymphoma were selected, activated B-cell-like(ABC) OCI-LY8(LY8) and germinal center B-cell-like(GCB) OCI-LY18(LY18),and rituximab-resistant DLBCL cell lines(LY8R and LY18R) were constructed by “concentration gradient dosing method”.The CCK-8 method was used to detect the cell survival rate of different concentrations of rituximab after interfering with the parental and drug-resistant cells, and the half maximal inhibitory concentration(IC50) was calculated; AnnexinⅤ/PI double staining method was used to detect the apoptotic rate of parent cells and drug-resistant cells; qRT-RCR,immunofluorescence and Western blot were used to detect the expression of demethylase FTO in rituximab-resistant cells.The expression of FTO was inhibited by adding meclofenamic acid(MA) to drug-resistant cell lines, and the inhibition was verified by RT-qRCR and Western blot.The expression of c-Myc and CEBPA in drug-resistant strains after inhibiting FTO was detected by using Western blot. Results Compared with the parent cells, the expression of demethylase FTO and c-Myc increased, and the expression of CEBPA decreased in the rituximab-resistant cell lines, with statistical significance(P<0.01).After the drug-resistant cell lines treated with MA,the expression of FTO and c-Myc decreased and CEBPA increased, all with statistically significant differences(P<0.01). Conclusion The expression of FTO in drug-resistant DLBCL significantly increases, and MA inhibits the expression of FTO in drug-resistant cells, which may have therapeutic potential for rituximab-resistant DLBCL.

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Objective To investigate the effects of dihydroartemisinin(DHA) on cell growth, migration invasion and vasculogenic mimicry ability of non-small cell lung cancer(NSCLC). Methods Different concentrations of DHA were added to NSCLC cell lines A549 and H3255,and after 24 hours, the cell viability was detected by CCK8 method, and the migration and invasion ability of NSCLC cells was detected by Transwell experiment.qRT-PCR and Western blot detected E-cadherin, N-cadherin, and Vimentin mRNA and protein expression levels, respectively.Three-dimensional cell was cultured to observe the vascular-like morphological generation of cells.qRT-PCR and Western blot detected human vascular endothelial cadherin(VE-cadherin) mRNA and protein expression levels as marker of vasculogenic mimicry. Results DHA inhibited cell growth of A549 and H3255 and showed time-dependent and concentration-dependent.DHA inhibits the invasion and metastasis(allP<0.001) of A549 and H3255 cells; DHA upregulated the expression of E-Cadherin at mRNA(allP<0.001) and protein(P<0.001;P<0.01) levels in A549 and H3255 cells, and downregulated the expression of N-cadherin at mRNA(allP<0.01) and protein(allP<0.001) levels as well as the expression of Vimentin at mRNA(P<0.01;P<0.001) and protein(allP<0.001) levels.The results of three-dimensional cell culture showed that the 12-hour vasculogenic mimicry generation capacity of DHA-treated A549 cells and H3255 cells was reduced, and the expressions of VE-cadherin at mRNA(P<0.001;P<0.01) and protein(allP<0.001) levels were downregulated. Conclusion Dihydroartemisinin inhibits the growth, migration invasion and vasculogenic mimicry ability of non-small cell lung cancer cells.

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Objective To investigate the expression characteristics of mu opioid receptor(MOR) in rat colon smooth muscle cells by cultured rat primary colonic smooth muscle cells. Methods colonic smooth muscle cells were isolated, cultured and identified; immunofluorescence double labeling method was used to observe the distribution characteristics of MOR,Endomorphin-2(EM2),and calmodulin(CaM) in colonic smooth muscle cells; Western Blot method was used to detect the expression of MOR and EM2 in smooth muscle cells of the colon.After the intervention measures Acetylcholine(ACh)(1×10-3mol/L) and EM2(2 μmol/L) were applied, the changes of CaM protein expression were observed; The calciumion imaging method was used to detect the changes of calciumion concentration in smooth muscle cells. Results The colonic smooth muscle cells were cultured and identified.The positive cells labeled with α-smooth muscle actin(α-SMA) accounted for more than 95% of the total number of cells.Immunofluorescence double labeling showed that there were MOR and EM2 distributions in colonic smooth muscle cells, and all MOR and EM2 positive cells coexisted with α-SMA.Western Blot results showed that there were MOR and EM2 expressions in colonic smooth muscle cells.CaM in the ACh group significantly increased at 10 minutes(P<0.05),CaM in the EM2 group significantly decreased(P<0.05); The calciumion imaging results showed that alone applied ACh, the calciumion concentration in smooth muscle cells significantly increased(P<0.05); Alone applied EM2,the calciumion concentration in colonic smooth muscle cells was down-regulated(P<0.05); Applied ACh and EM2 sequentially, EM2 significantly reduced the increase of the calciumion concentration in smooth muscle cells induced by ACh(P<0.05). Conclusion MOR and EM2 are expressed in colonic smooth muscle cells, and EM2 may inhibit the expression of CaM and reduce the concentration of calcium ions through MOR.

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Objective To investigate the effect of Porphyromonas gingivallis(Pg) infection of esophageal cancer cells on the polarization of tumor associated macrophages(TAMS) and functional changes.Methods The secretion of tumor-related cytokines in the supernatant of Pg infected and uninfected esophageal squamous cell carcinoma(ESCC) cells was detected by ELISA.A co-culture model of ESCC cells and macrophages in vitro was established,and the changes of TAMs surface markers were detected by qPCR,cellular immunofluorescence and flow cytometry.Cytokines secreted by TAMs after co-culture were detected by ELISA.ESCC cells were cultured using conditioned medium of co-cultured TAMS,and the effects of TAMs on the proliferation,migration and invasion of esophageal cancer cells were evaluated by CCK-8,Wound-healing assay and Transwell assay.Results The expression quantity of IL-6 and IL-10 of Pg-infected ESCC cells increased(P<0.01).The contents of CD 163 and CD206 on the surface of TAMs co-cultured with Pg-infected ESCC cells increased(P<0.001).The cytokines IL-6 and IL-10 secreted by TAMs co-cultured with Pg-infected ESCC cells relatively increased(P<0.01).TAMs co-cultured with Pginfected ESCC cells were able to enhance ESCC cells proliferation,migration and invasion(all P<0.05).Conclusion Pg infection of ESCC cells can induce the secretion of cytokines,remodel TAMs to polarize toward the M2-type immunosuppressive phenotype,thereby promoting the malignant biological behavior of ESCC cells.This study provides data support for the etiology of esophageal cancer and potential target molecules for clinical immunotherapy targeting TAMs.

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Objective To investigate the expression of mannose binding lectin 2(MBL2) in hepatocellular carcinoma(HCC) cell lines and its effects on the proliferation and prognosis. Methods MBL2 expression levels in patients of different sexes and survival analysis of patients were subsequently performed using bioinformatics method.The mRNA and protein expression levels were detected with the qRT-PCR and Western blot.Cell proliferation was examined by MTT assay and cell clone formation assay; Flow cytometry was used to detect the cell apoptosis and cell cycle.Gene set enrichment analysis(GSEA) was used to analyze the signal pathways of MBL2 enrichment. Results Bioinformatics analysis and qRT-PCR results showed that MBL2 was highly expressed in male HCC patients compared with female HCC patients, and was related to the prognosis of HCC patients.Compared with the control group, over-expression of MBL2 inhibited the viability and clone formation rate of Huh7 cells and MHCC-97H cells; In addition, cell cycles were arrested and cell apoptosis increased.GSEA and Western blot results showed that MBL2 was enriched in the cell cycle signal pathway.Overexpression of MBL2 inhibited the expression of CDK4 protein and promoted the expression of P16 and BAX protein. Conclusion The expression of MBL2 is down regulated in HCC,which is related to the prognosis of male patients with HCC and participates in the process of cell proliferation.MBL2 may be a potential therapeutic target for male patients with HCC.

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Objective To construct a mouse derived pcDNA3.1-3 × Flag-c-NUP85 expression plasmid and observe its effect on expression of inflammation factors in LPS-induced RAW264.7 cells,as well as on the proliferation and apoptosis of RAW264.7 cells.Methods The NUP85 gene was amplified by PCR to construct pcDNA3.1-3 × Flagc-NUP85 eukaryotic expression plasmid.The pcDNA3.1-3 × Flag-c vector was divided with enzymes.The purified PCR product was ligated with the vector,and the ligated product was transformed into bacterial competent cells.After identification by enzyme digestion,sequencing and analysis were performed.Then,it was transfected into RAW264.7 cells,and the blank plasmid without NUP85 gene was set as the control group.The effect on cell proliferation and apoptosis were detected by CCK-8 assay and flow cytometry,and the expression of inflammatory cytokines such as tumor necrosis factor-α(TNF-α) and interleukin-6(IL-6) in LPS-induced RAW264.7 cells was detected by Western blot and ELISA.Results Enzyme digestion identification and Western blot results showed that pcDNA3.1-3 × Flag-c-NUP85 eukaryotic expression plasmid was successfully constructed and expressed.The results of CCK-8 assay showed that the cell survival rate of NUP85 overexpression group was significantly lower than that of control group after 24 h [(0.55±0.03) vs(0.67±0.05),F=30.98,P<0.05].The results of flow cytometry showed that the cell apoptosis rate of NUP85 overexpression group was higher than that of control group [(15.78±1.05) % vs(13.40±0.47) %,F=75.38,P<0.05].The results of Western blot and ELISA showed that after transfection of pcDNA3.1-3 × Flag-c-NUP85,the expression of TNF-α and IL-6 in RAW264.7 cells were higher than those in the control group,with statistical significance(P<0.05).Conclusion NUP85 can inhibit the proliferation and promote apoptosis in LPS-stimulated RAW264.7 cells,and NUP85 can promote the expression of inflammatory cytokines IL-6 and TNF-α in LPS-stimulated RAW264.7 cells.

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Objective To investigate the expression of eukaryotic translation initiation factor 2B1(eIF2B1) in HCC and its correlation with clinical prognosis. Methods Through the follow-up of the clinical data of 743 hepatocellular carcinoma patients in the specimen bank, 91 HBV-related liver cancer patients with complete follow-up information were screened out, and their postoperative tumor tissues were selected.Meanwhile, the adjacent tumor tissues with a distance of more than 2 cm from the tumor margin were collected to make tissue chips.Western blot and immunohistochemical experiments were performed.Image J was used to analyze the proportion of positive cells stained by tissue chip and the gray value of Western blot results.Clinical data and follow-up data of included patients were statistically analyzed by R4.0.5 software. Results Immunohistochemical results suggested that eIF2B1 was more strongly expressed in HCC than in para-cancerous tissues.Western blot results showed that eIF2B1 was more strongly expressed in HepG2.2.15 cells than in HepG2 cells.The expression of eIF2B1 in HCC tissues was correlated with tumor number(P<0.05).Cox multivariate regression analysis showed that the expression of eIF2B1 was an independent risk factor for the overall clinical prognosis of HBV-DNA positive patients(HR=4.28,P=0.018). Conclusion The expression of eIF2B1 is significantly enhanced in HBV-associated HCC tissues and is significantly associated with overall survival in HBV DNA positive patients.

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Objective To investigate the effect of QDPR on renal interstitial fibrosis and Beclin1 expression of unilateral ureteral obstruction(UUO) rat model. Methods The UUO model was constructed by unilateral ureteral ligation.The fibrosis progression, QDPR and Beclin1 expression of UUO model were evaluated by Masson, COL Ⅰ staining and Western blot techniques.UUO model which was over expression QDPR and its control were constructed by retrograde ureteral infusion of corresponding lentivirus.Infection efficiency and QDPR expression level were further evaluated.The QDPR overexpression on fibrosis progression, and Beclin1 was further analyzed. Results Compared with the sham operation group, the collagen fibers level in the UUO model group significantly increased(P<0.05),the QDPR expression level was significantly reduced(P<0.05),and the Beclin1 significantly increased(P<0.05).Retrograde injection of lentivirus through the ureter could successfully infect the kidney and increase the expression level of QDPR in the kidney.The overexpression of QDPR in kidney could significantly decrease collagen fibers(P<0.05) and Beclin1(P<0.05) expression. Conclusion Overexpression of QDPR inhibits the expression level of Beclin1 and progression of fibrosis, suggesting that overexpression of QDPR may relieve renal interstitial fibrosis by inhibiting Beclin1expression.

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Objective To investigate the effect of Ginsenoside Rg1(GsRg1) on proliferation,migration and osteogenic differentiation of human gingival fibroblasts(HGFs) and its molecular mechanism.Methods Human gingival fibroblasts(HGFs) were isolated and cultured by tissue block method,and identified by morphology and immunofluorescence.The effect of six concentrations of GsRg1(0,6.25,12.5,25,50,100 mg/L) on the proliferation of HGFs was detected by CCK-8 method.Trans well assay was used to detect the effects of different concentrations of GsRg1 on the migration ability of HGFs.Alkaline phosphatase(ALP) staining was used to detect osteogenic ability.Alizarin red staining was used to observe and quantify calcium nodules.The expression of COL-I,OCN and OPN osteogenic genes was detected by qRT-PCR.Western blot was used to detect the protein expression of OCN,OPN,COL-Ⅰ and PI3K/AKT signaling pathway.Results Compared with the control group,the proliferation ability of HGFs was significantly improved at the concentrations of 12.5,25,50 and 100 mg/L GsRg1(P<0.05),and the proliferation promotion effect of 100 mg/L GsRg1 was the strongest.There was no significant difference between the6.25 mg/L GsRg1 group and the control group.After GsRg1 treatment,the migration ability of HGFs was enhanced and showed concentration dependence.Compared with the control group,the activity of ALP in 100 mg/L GsRg1group significantly increased(P<0.01).Alizarin red staining showed a significant increase in the number of calcium nodules(P<0.01).The mRNA and egg white expression levels of osteogenic genes OCN,OPN and COL-I increased(P<0.05).The expression levels of p-PI3K and p-Akt were significantly up-regulated with time(P<0.05),while the expression levels of PI3K and AKT had no significant changes.Conclusion GsRg1 can promote the proliferation and migration of HGFs,and 100 mg/L GsRg1 can promote the osteogenic differentiation of HGFs,which may be related to the activation of PI3K/AKT signaling pathway.

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Objective To observe the effects of the tofacitinib on interstitial lung disease(ILD) by regulating the JAK-STAT signaling pathway. Methods Wistar rats were randomly divided into 4 groups: normal group, ILD group, prednisone acetate group, and tofacitinib group.Except for the normal group, the other three groups were given 3 mg/ml bleomycin solution for modeling.After 28 days of intragastric administration, the lung tissues of all rats were collected for hematoxylin-eosin staining(HE) and Western blot(WB) to detect the protein levels of JAK1 and STAT1;Enzyme-linked immunosorbent assay(ELISA) was used to detect tumor necrosis factor in rat serum(TNF)-α,interleukin(IL)-6,IL-10,IL-1β. Results HE staining of lung tissue in ILD,prednisone acetate group and tofacitinib group showed alveolar tissue thickening, alveolar wall capillary congestion, bronchial luminal epithelial cells shedding, and inflammatory cell exudation.The results of WB showed that JAK1 and STAT1 significantly increased in ILD group, and decreased in different degrees compared with ILD group, tofacitinib group and prednisone acetate group(P<0.05).The ELISA results showed that the expressions of serum TNF-α,IL-6 and IL-1β in the ILD group were significantly higher than those in the normal group(P<0.01).The expression of pine group decreased(P<0.05),and the expression of IL-10 was the opposite. Conclusion Tofacitinib reduces lung tissue damage and the inflammatory response in the treatment of ILD by inhibiting the JAK-STAT pathway and down-regulating the expression of inflammatory factors TNF-α,IL-6,IL-1β and up-regulating the anti-inflammatory factor IL-10.

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Objective To investigate the effects of hepatobiliary metabolic dysfunction on gut microbiota in rats with intrahepatic cholestasis of pregnancy(ICP). Methods Forty Sprauge-Dawley(SD) rats at 10 days of gestation were randomly divided into two groups, 25 for ICP induction and 15 as control.Estradiol benzoate, combined with progesterone, was given to the rats by intraperitoneal injection from 10 to 14 days after gestation.Hepatic impairment indicators of total bilirubin(TBIL),alanine aminotransferase(ALT),aspartate aminotransferase(AST),r-glutamyl transferase(GGT),alkaline phosphatase(ALP),and total bile acids(TBA) in sera of the animals were detected in each group to verify the model construction.Simultaneously, the liver tissues were subjected to pathology examination and fresh faeces samples were collected for 16S rDNA sequencing to explore the gut microbiota profiles. Results The serum levels of TBIL,ALT,AST,GGT,ALP and TBA in ICP group were significantly elevated compared with the control(P<0.05);the ICP rats presented obvious characteristics of hepatobiliary disorders, showing prominent steatosis, necrosis of hepatocytes and infiltration of inflammatory cells, with formation of bile thrombus in some animals.The abundance ofEubacterium,Bacteroides,Parabacteroides distasonisandBacteroidesuniformis remarkably decreased whereasPrevotellaandFusobacteriumsignificantly increased in ICP rats when compared with the control(P<0.05). Conclusion The hepatic injuries and bile acid metabolic disorders occurred in ICP rats, which resulted in dysbiosis of gut microbiota.

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Objective To study the effect of long non-coding RNA(LncRNA) HAGLR on the expression of NOD-like receptor pyrin domain-associated protein 3(NLRP3) inflammasome and fracture healing in tibial fracture(TF) mice and to explore the mechanism. Methods First, HAGLR in osteoblast MC3T3-E1 was silenced byin vitro.Cell viability was detected by CCK-8 assay, cell apoptosis was detected by TUNEL assay, and the expressions of bone alkaline phosphatase(BALP) and osteocalcin were detected by qPCR.Western blot assay was used to detect the expressions of RUNX2,phosphorylated RUNX2(p-RUNX2),NLRP3,cysteine aspartic protease 1(Caspase1),apoptosis-associated spot-like protein(ASC) and interleukin-1β.TF mouse models were established by tibial fracture operation in mice.HAGLR was overexpressed in the model mice, and RUNX2 was silenced or an inflammatory body inhibitor MCC950 was added on the basis of overexpression of HAGLR.The expressions of HAGLR and RUNX2 were detected by qPCR,and the expressions of insulin-like growth factor(IGF-1) were detected by Western blot.microCT was used to measure the volume of mouse callus(MBV) and the total tibial wet weight. Results The apoptosis rate of MC3T3-E1 cells increased and the expression levels of RUNX2,p-RUNX2,BALP and osteocalcin decreased(P<0.05).The expressions of NLRP3,Caspase1,ASC and IL-1β increased(P<0.05).Compared with healthy tissue, the expressions of HAGLR and RUNX2 in TF mice decreased.Overexpression of HAGLR promoted the expressions of HAGLR and RUNX2 in TF mice, and increased the expression of MBV and tibia wet weight and IGF-1(P<0.05).Silencing RUNX2 on the basis of overexpression of HAGLR resulted in decreased expression of MBV,tibial wet weight and IGF-1 in TF mice(P<0.05).However, the addition of NLRP3 inflammasome inhibitor MCC950 on top of the overexpression of HAGLR resulted in increased expressions of MBV,full-length tibia wet weight and IGF-1(P<0.05). Conclusion LncRNA HAGLR promotes the healing of tibial fractures by activating RUNX2 and inhibiting NLRP3 inflammasome.

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Objective To investigate the expression of T-cell immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domain(TIGIT) and programmed death 1(PD1) on follicular T helper cells(TFH) from the patients with rheumatoid arthritis(RA) and to analyze the clinical relevance to disease severity. Methods The expression of TIGIT and PD1 on TFH cells were examined from 79 RA patients and 45 healthy controls(HC) by the technique of flow cytometry. The expression of TIGIT and PD1 including percentage of TIGIT+TFH, PD1-TFH, TIGIT+PD1-TFH, TIGIT+PD1+TFH, TIGIT-PD1+TFH cells and the mean fluorescence intensity(MFI) on theses TFH cells were compared between RA patients and HC. Moreover, its correlation with laboratory inspection was analyzed. Results (1) The percentage of PD1+TFH and the MFI of PD1 on PD1+TFH from RA patients significantly increased compared with HC(P<0.01).(2) The percentage of TIGIT+PD1-TFH significantly decreased compared with HC(P<0.01), whereas the percentage of TIGIT+PD1-TFH, the MFI of PD1 on TIGIT+PD1-TFH, the percentage of TIGIT+PD1+TFH and the MFI of PD1 on TIGIT+PD1+TFH significantly increased(P<0.05).(3) The MFI of PD1 on TIGIT+PD1+TFH in RA patients was positively associated with rheumatoid factors(RF)(P<0.05). The percentage of TIGIT-PD1+TFH, the MFI of PD1 on TIGIT-PD1+TFH, the MFI of PD1 on TIGIT+PD1+TFH in RA patients were positively associated with increased RF(P<0.05).(4) The percentage of TIGIT+PD1-TFH in RA patients was negatively associated with erythrocyte sedimentation rate(ESR)(P<0.05). The percentage of TIGIT-PD1+TFH in RA patients was positively associated with ESR(P<0.05). The percentage of TIGIT+PD1+TFH in RA patients was negatively associated with lymphocyte count(Lym)(P<0.05). The MFI of PD1 on TIGIT+PD1+TFH in RA patients was negatively associated with lymphocyte percentage(Lym%), and positively associated with neutrophil count(Net), neutrophil percentage(Net%), neutrophilto-lymphocyte ratio(NLR), systemic immune inflammation index(SII), derived neutrophil to lymphocyte ratio(dNLR), ESR, C-reactive protein(CRP)(P<0.05). The MFI of PD1 on TIGIT-PD1+TFH in RA patients was positively associated with Net, ESR, CRP(P<0.05).(5) The percentage of TIGIT+PD1-TFH in RA patients was negatively associated with disease activity score 28(DAS28)-ESR(P<0.05). The MFI of PD1 on TIGIT+PD1+TFH and the MFI of PD1 on TIGIT-PD1+TFH in RA patients was positively associated with DAS28-ESR, DAS28-CRP, tender joint count(TJC), swollen joint count(SJC), patient visual analogue scale(VAS)(P<0.05).(6) Decreased percentage of TIGIT+PD1-TFH and increased percentage of TIGIT+PD1+TFH were risk of RA. Conclusion The decreased percentage of TIGIT+PD1-TFH, increased percentage of TIGIT+PD1+TFH and TIGIT-PD1+TFH were observed in patients with RA. Aberrations level of TIGIT+PD1-TFH, TIGIT+PD1+TFH, TIGIT-PD1+TFH were associated with the production of antibody, inflammation, and also disease activity.

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Objective Review the independent risk factors of postoperative recurrence in surgical treatment of bladder cancer patients to construct a model of bladder cancer recurrence. Methods A total of 240 surgically treated bladder cancer patients were followed up for at least 1 year and divided into recurrence(n=54) and non-recurrence(n=186).The general data of patients were comparative analyzed, and the different and statistically significant data were further analyzed by ROC curve, and the statistically significant data were included in the multivariate analysis after logistic obtaining univariate analysis results.Risk factors were included in the model construction, and the model correction curve and clinical net benefit analysis were analyzed.The model could be used to predict postoperative recurrence in bladder cancer patients. Results The ROC curves of the statistically significant continuous variables were analyzed in the general data, and the results showed that the AUC of PNI,BLCA-4,BTA,NMP22 and CEA were 0.932,0.979,0.998,0.677 and 0.981,respectively, and the optimal truncation values were ≤40.18%,>140.04 ng/mg, ≤7.22 U/mg,>7.68 μg/mg, and>1.99 ng/mg, respectively.Statistically significant data from univariate analysis were incorporated into the logistic regression model, and the results showed that PNI ≤40.18%,BLCA-4>140.04 ng/mg, BTA≤7.22 U/mg, NMP22>7.68 μg/mg was a risk factor for recurrence in patients with bladder cancer.Subsequently, PNI,BLCA-4,BTA,and NMP22 were incorporated into the construction of the model as predictors of recurrence in patients with bladder cancer.Based on the model correction curve and clinical net benefit analysis, the internal verification results showed that the C-index of the model predicting bladder cancer recurrence was 0.296(95%CI: 0.078-1.329).The calibration curve showed good consistency between the observed and predicted values.The model predicted a risk threshold>0.128 for patients with bladder cancer, and the model provided a clinical net benefit; in addition, the model had a higher clinical net benefit than PNI,BLCA-4,BTA,and NMP22. Conclusion The model correction curve and clinical net benefit analysis, the results of internal verification show that the model can be used to predict recurrence in patients with bladder cancer.

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Objective To investigate the relationship between insulin resistance and the serum level of mesencephalic astrocyte-derived neurotrophic factor(MANF) in patients with polycystic ovary syndrome(PCOS). Methods There were 120 patients with PCOS and 40 healthy women recruited as the experimental group and the control group respectively.The serum levels of sex hormones, fasting blood glucose(FBG),fasting insulin(FINS),and MANF in the experimental group and the control group were compared.The serum MANF level of patients with or without insulin resistance in the experimental group was also compared and the relationship between homeostasis model assessment for insulin resistance(HOMA-IR) and the serum MANF level was analyzed. Results Compared to the control group, the serum levels of luteinizing hormone, testosterone, androstenedione, dehydroepiandrosterone sulfate, FBG,FINS,and HOMA-IR in the experimental group were significantly higher(P<0.05),whereas the serum levels of follicle stimulating hormone, sex hormone binding globulin, and MANF were significantly lower(P<0.05).Compared to patients without insulin resistance(59 cases),the serum MANF level of patients with insulin resistance in the experimental group(61 cases) was significantly lower(P<0.05),and there was a negative correlation between the serum MANF level and HOMA-IR of PCOS patients in the experimental group(P<0.05). Conclusion MANF possibly plays an important role in the occurrence and progression of insulin resistance in PCOS patients, which will be able to provide new approaches to the diagnosis and treatment of PCOS.

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Objective To explore the clinical application value of deep convoluti onal neural network for automatic detection and classification of benign and malignant thyroid nodules ultrasound images. Methods A total of 1 012 ultrasound images of thyroid nodules were retrospectively selected and labeled.The YOLOv5 network model was constructed to accurately locate the location of thyroid nodules and automatically trim the area of the nodules.At the same time, a GoogLeNet network model was constructed to classify benign and malignant nodules after reduction. Results In the collected data set, the mean accuracy of the target detection network for thyroid nodule location detection was 96.2%.Meanwhile, the sensitivity, specificity, accuracy and AUC of the classification network for benign and malignant nodules were 0.885,0.822,0.866 and 0.92 respectively, which were significantly higher than those of the AlexNet model(0.81),VGG model(0.86) and MobileNet model(0.76). Conclusion The deep convolutional neural network model has high localization and recognition ability for benign and malignant thyroid nodules in ultrasound images, which is helpful to improve the accuracy of automatic image diagnosis.

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Objective To explore the direct-on-target micro-droplet growth assay(DOT-MGA) for rapid detection of the susceptibility of tigecycline and polymyxin. Methods A total of 67 strains ofKlebsiella pneumoniaewere collected for DOT-MGA.6 μl droplets with or without tigecycline or polymyxin(The final drug concentration is 2 μg/ml) were added in triplicate into the wells of the MALDI plate with four incubation time points(3 h, 4 h, 6 h, and 8 h).The results were classified as susceptible(score<1.7) and non-susceptible(score≥1.7) according to the Bruker Biotype software. Results After incubation for 4 h, the growth efficiency, specificity and positive predictive value of tigecycline and polymyxin were both 100.00%.The classification consistency rate was 98.15% and 96.15%,the sensitivity was 96.30% and 92.31%,and the negative predictive value was 96.45% and 92.86%,respectively. Conclusion DOT-MGA can provide rapid and reliable drug susceptibility diagnosis of tigecycline and polymyxin, which is of great significance for the clinical anti-infective treatment.

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Objective To investigate the correlation of uteroglobulin-related protein 1(UGRP1) with latent autoimmune diabetes in adults(LADA).Methods 124 participants were selected,including 45 LADA patients as LADA group,41 type 2 diabetes mellitus(T2DM) patients as T2DM group and 38 healthy people without diabetes as control group.General information,diabetes related indicators and serum UGRP1 levels were compared among the three groups.Spearman correlation was used to analyze the relationship between serum UGRP1 and diabetes related indicators.Binary logistic regression was used to analyze whether UGRP1 could be an independent risk factor for LADA.The diagnostic value of UGRP1 in LADA was discussed by ROC curve analysis.Results There were significant differences in BMI,systolicpressure,diastolicpressure,fasting blood glucose,2h postprandial blood glucose,fasting C-peptide,2h postprandial C-peptide,HbA1c,diabetes antibodies(GADAb,IA-2,ZnT8) among the three groups(P<0.05).The level of serum UGRP1 in LADA group was significantly higher than that in control group and T2DM group(P<0.05).Spearman correlation analysis showed that the serum UGRP1 level of diabetes patients was related to fasting C-peptide,2h postprandial C-peptide,GADAb,the number of positive diabetes antibodies.Binary Logistic regression analysis showed that serum UGRP1 level was an independent risk factor for LADA.ROC curve analysis results suggested that UGRP1 could be used as a better assistant indicator for the diagnosis of LADA.Conclusion UGRP1 may be involved in the pathogenesis of LADA.

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Objective To investigate the clinical features and prognostic factors of gastric cancer patients under the age of 40. Methods The clinical data of 126 young gastric cancer patients under the age of 40 were retrospectively analyzed.Log-Rank test and Cox regression model were used to analyze prognostic risk factors, respectively. Results The average age of gastric cancer patients in our group was 33.6 years, and there were more female patients than male patients(female ∶male=1.25 ∶1),Upper abdominal pain and fullness discomfort were the most common clinical manifestations, and the predominant sites were gastric antrum angle(43.7%) and gastric body(42.9%).Lauren type was mainly diffuse type(89.7%),Nakamura type was mainly undifferentiated type(91.3%),and WHO type was the most common type of low-adherence carcinoma(46.8%).71.4% of patients were diagnosed with advanced tumor.The most common site of metastasis was the peritoneum.The 1-year and 3-year survival rates were 67.3% and 46.7%, respectively. The results of univariate analysis showed that the levels of serum CA199,tumor length, depth of tumor invasion, lymphatic metastasis, vascular invasion, and surgical approach were risk factors affecting the prognosis of patients.Multivariate analysis revealed that the levels of serum CA199,the depth of tumor invasion and surgical approach were independent prognostic factors. Conclusion Young gastric cancer has a high degree of malignancy and strong biological invasiveness.Most patients are diagnosed at the advanced stage of the tumor, and the overall prognosis is poor.The levels of serum CA199,the depth of tumor invasion and surgical approach were independent prognostic factors.

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Objective To investigate the expression of secreted protein acidic and rich in cysteine-like1(SPARCL1) in atherosclerosis(AS) and the association between SPARCL1 gene rs7695558 and rs1049539 polymorphism with the susceptibility to AS. Methods In this case-control study, 209 AS patients were selected as the case group, and 208 healthy matched in age and sex were selected as the control group.The expression level of serum SPARCL1 was measured by enzyme-linked immunosorbent assay(ELISA).Linear and Logistic regression analysis were used to evaluate the correlation between SPARCL1 level and vascular risk factors, lifestyle and demographic variables.Expression of SPARCL1 in tissue specimens was assessed by immunohistochemistry.Single nucleotide polymorphisms(SNPs) were genotyped by high resolution melting method.Chi-square test was used to analyze the relationship between rs7695558 and rs1049539 polymorphism and susceptibility to AS. Results The serum expression level of SPARCL1 in AS patients was lower than that in healthy controls(Z=-2.916,P=0.004).The level of SPARCL1 was related to age(P=0.027) and diastolic blood pressure(P=0.008),but not to sex and other cardiovascular risk factors(P>0.05).The expression level of SPARCL1 in atherosclerotic lesions of coronary artery tissue increased.There was no significant difference in gene distribution of rs7695558 and rs1049539 between the case group and the control group by chi-square test(P>0.05).In the recessive genetic model of rs7695558,there was a difference in the distribution of genes with and without A.Patients without A allele(GG) had a lower risk of AS than patients with A allele(AA+AG).TheORvalue was 0.417,95%CI:0.184～0.945,which was significant at 10% confidence level(P=0.034). Conclusion Rs7695558,a new susceptible site related to AS risk, located in the intron of human SPARCL1 gene is identified for the first time in Anhui population of China, suggesting that SPARCL1 may play an anti-AS role as a vascular protective factor.

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Objective To study the positive effect and mechanism of histone deacetylase Sirtuin 1(Sirt1) on the osteogenic capacity of inflammatory PDLSCs. Methods PDLSCs were isolated and cultured from the healthy individuals and periodontitis patients, the expression of Sirt1 in two types of PDLSCs was observed; osteogenic induction of inflammatory PDLSCs was performed, concurrently Sirt1 agonist resveratrol was used to up-regulate the expression of Sirt1,then the osteogenic differentiation was observed, the expressions of Runx2,acetylated NF-κB,acetylated FoxO1 and FoxO1 were assessed by Western blot. Results The protein expression of Sirt1 was suppressed in inflamed PDLSCs(P<0.01);resveratrol could up-regulate the expression of Sirt1 in inflamed PDLSCs; compared with the osteogenic induction group, the osteogenesis+resveratrol group increased the expression of BSP(t=14.045,P<0.01),Runx2(t=3.349,P<0.01),OCN(t=7.218,P<0.01) and Osx(t=4.544,P<0.01) mRNA in inflamed PDLSCs, and enhanced ALP staining(P<0.01);expression of FoxO1(t=8.737,P<0.01) and Runx2(t=6.152,P<0.01) increased after osteogenesis of inflamed PDLSCs; in the osteogenesis+resveratrol group, the expression of Sirt1 was up-regulated, and the expression of FoxO1(t=5.912,P<0.01) and Runx2(t=6.277,P<0.01) further increased compared with the osteogenic induction group, while the expression of acetylated NF-κB(F=184.033,P<0.01) and acetylated FoxO1(F=301.454,P<0.01) was significantly lower than that of control group and osteogenic induction group. Conclusion The deacetylase Sirt1 could promote the osteogenic differentiation of inflammatory PDLSCs through deacetylating NF-κB and FoxO1.

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To investigate the differential diagnostic value of Chinese thyroid imaging reporting and data system(Chinese-TIRADS, C-TIRADS) and the combination with ultrasonic elastic strain rate(SR)andBRAFV600Egene detection in the diagnosis of benign and malignant thyroid nodules. A total of 137 patients with 142 thyroid nodules were analyzed retrospectively, which included observing two-dimensional ultrasound and elastic image characteristics and recording the elastic strain rate. The malignant risk of thyroid nodules was stratified by C-TIRADS. To use pathology results as the gold standard, the efficacy of the above methods in diagnosing the nodules was evaluated. The diagnostic efficacy of C-TIRADS combined with elastic strain rate in the diagnosis of thyroid nodules was higher than that of C-TIRADS and elastic strain rate diagnosis methods independently, and the difference was statistically significant(P<0.05). There was no statistical difference between C-TIRADS andBRAFV600Egene detection(P>0.05).