Objective To investigate the effect of mutation human proprotein convertase subtilism/kexin type 9(hPCSK9D374Y)in PCSK9 gene on methionine choline deficiency diet(MCD)-induced nonalcoholic steato-hepatitis(NASH)in mice.Methods Sixteen C57BL/6J wild-type mice were selected and randomly divided into the hPCSK9D374Y group and the control GFP group.MCD was fed for 6 weeks,and then the serum level of hepatic triglyceride,alanine aminotransferase(ALT)and aspartate aminotransferase(AST)was examined.Oil Red O and Sirius Red staining microscopy were used to identify hepatic lipid infiltration and fibrosis severity.F4/80-positive cell infiltration was analyzed using immunohistochemistry.Lipid synthesis and inflammatory response-related proteins were detected by Western blot and related mRNA expression was analyzed by RT-qPCR.Results Hepatic hPCSK9 protein and mRNA were significantly up-regulated,LDLR protein expression was down-regulated,and ser-um level of ALT and AST was significantly elevated in the hPCSK9D374Y group of mice(P＜0.05).The degree of he-patic steatosis and fibrosis increased and F4/80-positive cells were significantly increased(P＜0.01).FASN and SCD1 proteins were significantly up-regulated and PPARα was down-regulated in the hPCSK9D374Y group;The ex-pression of TLR4 and p-P65 was elevated,whereas the expression of Iκ Bα was decreased(P＜0.001).RT-qPCR re-sults showed a significant increase of mRNA coding inflammatory factors TNF-α,IL-1β,IL-6,and MCP-1,and a significant up-regulation of fibrosis-associated mRNAs(collagen Ⅰα and collagen Ⅲα)was found(P＜0.001).Conclusions Functionally acquired mutation in the PCSK9 gene(hPCSK9D374Y)exacerbates MCD-induced hepatic steatosis,inflammatory response and fibrosis in mice.

Objective To investigate the effect of mutation human proprotein convertase subtilism/kexin type 9(hPCSK9D374Y)in PCSK9 gene on methionine choline deficiency diet(MCD)-induced nonalcoholic steato-hepatitis(NASH)in mice.Methods Sixteen C57BL/6J wild-type mice were selected and randomly divided into the hPCSK9D374Y group and the control GFP group.MCD was fed for 6 weeks,and then the serum level of hepatic triglyceride,alanine aminotransferase(ALT)and aspartate aminotransferase(AST)was examined.Oil Red O and Sirius Red staining microscopy were used to identify hepatic lipid infiltration and fibrosis severity.F4/80-positive cell infiltration was analyzed using immunohistochemistry.Lipid synthesis and inflammatory response-related proteins were detected by Western blot and related mRNA expression was analyzed by RT-qPCR.Results Hepatic hPCSK9 protein and mRNA were significantly up-regulated,LDLR protein expression was down-regulated,and ser-um level of ALT and AST was significantly elevated in the hPCSK9D374Y group of mice(P＜0.05).The degree of he-patic steatosis and fibrosis increased and F4/80-positive cells were significantly increased(P＜0.01).FASN and SCD1 proteins were significantly up-regulated and PPARα was down-regulated in the hPCSK9D374Y group;The ex-pression of TLR4 and p-P65 was elevated,whereas the expression of Iκ Bα was decreased(P＜0.001).RT-qPCR re-sults showed a significant increase of mRNA coding inflammatory factors TNF-α,IL-1β,IL-6,and MCP-1,and a significant up-regulation of fibrosis-associated mRNAs(collagen Ⅰα and collagen Ⅲα)was found(P＜0.001).Conclusions Functionally acquired mutation in the PCSK9 gene(hPCSK9D374Y)exacerbates MCD-induced hepatic steatosis,inflammatory response and fibrosis in mice.

Objective To investigate the therapeutic effect of CTLA4Ig gene on experimental autoimmune thyroiditis (EAT) by the use of portable synthetic costimulatory molecules of cytotoxic T lymphocyte antigen 4 (CTLA-4) antagonist (CTLA4Ig) eukaryotic expression vector.Methods Thirty C57BL/6J female mice were divided into three groups,named EAT model group (EAT,n =10),CTLA4Ig-treatment group (CTLA4Ig-EAT,n =10) and control group(n =10).At 28 day after first immunization,plasmids mixture with pCI or pCI/CTLA4Ig were injected into thyroid tissues of EAT and CTLA4Ig-EAT by surgery,respectively.Serum,thyroid tissues and spleens were collected as samples.Thyroid autoantibody and expression of interleukin (Th)1,Th2 related cytokinesby were measured by real-time quantitative PCR and ELISA.Results Compared with EAT group,the expression of CTLA-4 in thyroid of CTLA4Ig-EAT group was elevated double folds (P =0.038),and the expression of Th1 cytokine interferon γ and intercellular adhesion molecule-1 decreased significantly (P =0.016,0.042).Meanwhile,Th2 cytokine IL-4 was increased after CTLA4Ig treatment (P =0.044).The same changes were seen in spleen tissues and serum.There was no significant difference in terms of TPOAb between EAT and treated group.Conclusion Local thyroid injection of CTLA4Ig gene shows the therapeutic effect to same degree on EAT through adjusting the underlying Th1/Th2 imbalance.

Objective To investigate the potential role of T follicular helper cells (Tfh) in the pathogenesis of autoimmune thyroid diseases by comparing the expression of C-X-C chemokine teceptor type 5 (CXCR5) and CD57 in Hashimoto's thyroiditis (HT) and Graves' disease (GD) thyroid tissues.Methods The expression of CXCR5 and CD57 proteins was determined by immunohistochemical analysis in 15 HT thyroid samples,18 GD samples and 10 normal thyroid samples.Results Immunohistochemical staining showed that CXCR5 and CD57 were mainly positive in cytomembrane and cytoplasm of the infiltrated lymphocytes both in HT and GD tissues,with much higher levels than that of normal thyroid tissues ( P ＜ 0.05 ).Both CXCR5 and CD57 were not significantly different between the HT and GD tissues.Conclusion CXCR5 and CD57 expressions were increased with a similar expression pattern in both of the two main autoimmune thyroid diseases( AITD),indicating that Tfh may participate inthe development and progression of AITD.