Recombinant adeno-associated virus 8-mediated inhibition of microRNA let-7a ameliorates sclerosing cholangitis in a clinically relevant mouse model

BACKGROUND Primary sclerosing cholangitis(PSC)is characterized by chronic inflammation and it predisposes to cholangiocarcinoma due to lack of effective treatment options.Recombinant adeno-associated virus(rAAV)provides a promising platform for gene therapy on such kinds of diseases.A microRNA(miRNA)let-7a has been reported to be associated with the progress of PSC but the potential therapeutic implication of inhibition of let-7a on PSC has not been evaluated.AIM To investigate the therapeutic effects of inhibition of a miRNA let-7a transferred by recombinant adeno-associated virus 8(rAAV8)on a xenobiotic-induced mouse model of sclerosing cholangitis.METHODS A xenobiotic-induced mouse model of sclerosing cholangitis was induced by 0.1% 3,5-Diethoxycarbonyl-1,4-Dihydrocollidine(DDC)feeding for 2 wk or 6 wk.A single dose of rAAV8-mediated anti-let-7a-5p sponges or scramble control was injected in vivo into mice onset of DDC feeding.Upon sacrifice,the liver and the serum were collected from each mouse.The hepatobiliary injuries,hepatic inflammation and fibrosis were evaluated.The targets of let-7a-5p and downstream molecule NF-κB were detected using Western blot.RESULTS rAAV8-mediated anti-let-7a-5p sponges can depress the expression of let-7a-5p in mice after DDC feeding for 2 wk or 6 wk.The reduced expression of let-7a-5p can alleviate hepato-biliary injuries indicated by serum markers,and prevent the proliferation of cholangiocytes and biliary fibrosis.Furthermore,inhibition of let-7a mediated by rAAV8 can increase the expression of potential target molecules such as suppressor of cytokine signaling 1 and Dectin1,which consequently inhibit of NF-κB-mediated hepatic inflammation.CONCLUSION Our study demonstrates that a rAAV8 vector designed for liver-specific inhibition of let-7a-5p can potently ameliorate symptoms in a xenobiotic-induced mouse model of sclerosing cholangitis,which provides a possible clinical translation of PSC of human.

HBx activates FasL and mediates HepG2 cell apoptosis through MLK3-MKK7-JNKs signal module

AIM:To investigate the possible mechanism by which hepatitis B virus X protein(HBx)mediates apoptosis of HepG2 cells.METHODS:HBx expression vector pcDNA3.1-X was transfected into HepG2 cells to establish an HBx highexpression cellular model as pcDNA3.1-X transfected group.The pcDNA3.1-X and pSilencer3.1-shHBX(HBx antagonist)were cotransfected into HepG2 cells to establish an HBx low-expression model as RNAi group.Untransfected HepG2 cells and HepG2 cells transfected with negative control plasmid were used as controls.Apoptosis rate,the expression of Fas/FasL signaling pathway-related proteins and the phosphorylation levels of MLK3,MKK7 and JNKs,which are upstream molecules of death receptor pathways and belong to the family of mitogen-activated protein kinases(MAPKs),were measured in each group.RESULTS:Compared with HepG2 cell group and RNAi group,apoptosis rate,the expression of Fas and FasL proteins,and the activation of MLK3,MKK7 and JNKs were increased in the pcDNA3.1-X transfected group.The activation of JNKs and expression of FasL protein were inhibited in the pcDNA3.1-X transfected group when treated with a known JNK inhibitor,SP600125.When authors treated pcDNA3.1-X transfected group with K252a,a known MLK3 inhibitor,the activation of MLK3,MKK7 and JNKs as well as expression of FasL protein was inhibited.Furthermore,cell apoptosis rate was also significantly declined in the presence of K252a in the pcDNA3.1-X transfected group.CONCLUSION:HBx can induce HepG2 cell apoptosis via a novel active MLK3-MKK7-JNKs signaling module to upregulate FasL protein expression.

Genetic diversity and phylogenetic analysis of EG95 sequences of Echinococcus granulosus:Implications for EG95 vaccine application

Objective:To analyse the genetic variability of EG95 sequences and provide guidance for EG95 vaccine application against Echinococcus granulosus(E. granulosus). Methods:We analysed EG95 polymorphism by collecting total 97 different E. granulosus isolates from 12 different host species that originated from 10 different countries. Multiple sequence alignments and the homology were performed by Lasergene 1(DNASTAR Inc.,Madison,WI),and the phylogenetic analysis was performed by using MEGA5.1(CEMI,Tempe,AZ,USA). In addition,linear and conformational epitopes were analysed,including secondary structure,NXT/S glycosylation,fibronectin type ecoⅢ(Fnndary Ⅲ) domain and glycosylphosphatidylinositol anchor signal(GPIanchor). The s structure was predicted by PSIPRED method. Results:Our results indicated that most isolates overall shared 72.6-100% identity in EG95 gene sequence with the published standard EG95 sequence,X90928. However,EG95 gene indeed has polymorphism in different isolates. Phylogenetic analysis showed that different isolates could be divided into three subgroups. Subgroup 1 contained 87 isolates while Subgroup 2 and Subgroup 3 consisted of 3 and 7 isolates,respectively. Four sequences cloned from oncosphere shared a high identity with the parental sequence of the current vaccine,X90928,and they belonged to Subgroup 1. However,in comparison to X90928,several amino acid mutations occurred in most isolates besides oncosphere,which potentially altered the immunodominant linear epitopes,glycosylation sites and secondary structures in EG95 genes. All these variations might change their previous antigenicity and thereby affecting the efficacy of current EG95 vaccine. Conclusions:This study reveals the genetic variability of EG95 sequences in different E. granulosus isolates,and proposed that more vaccination trials would be needed to test the effectiveness of current EG95 vaccine against distinct isolates in different countries.

Gut microbiota-derived metabolites as key mucosal barrier modulators in obesity

A significant breakthrough in the field of obesity research was the demonstration that an obese phenotype could be manipulated by modulating the gut microbiota.An important next step is to elucidate a human-relevant"map"of microbiota-host interactions that regulate the metabolic health of the host.An improved understanding of this crosstalk is a prerequisite for optimizing therapeutic strategies to combat obesity.Intestinal mucosal barrier dysfunction is an important contributor to metabolic diseases and has also been found to be involved in a variety of other chronic inflammatory conditions,including cancer,neurodegeneration,and aging.The mechanistic basis for intestinal barrier dysfunction accompanying metabolic disorders remains poorly understood.Understanding the molecular and cellular modulators of intestinal barrier function will help devise improved strategies to counteract the detrimental systemic consequences of gut barrier breakage.Changes in the composition and function of the gut microbiota,i.e.,dysbiosis,are thought to drive obesity-related pathogenesis and may be one of the most important drivers of mucosal barrier dysfunction.Many effects of the microbiota on the host are mediated by microbiota-derived metabolites.In this review,we focus on several relatively well-studied microbial metabolites that can influence intestinal mucosal homeostasis and discuss how they might affect metabolic diseases.The design and use of microbes and their metabolites that are locally active in the gut without systemic side effects are promising novel and safe therapeutic modalities for metabolic diseases.

真核起始因子4E对小鼠动脉粥样硬化发生、发展的炎症调节作用

目的探讨真核起始因子4E(eIF4E)在小鼠动脉粥样硬化发生、发展过程中的炎症调节作用。方法分别用200 ng/μl脂多糖(LPS)和20 ng/μl白细胞介素-4(IL-4)诱导RAW264.7细胞12 h后提取mRNA,24 h后提取蛋白,采用实时荧光定量聚合酶链反应(qRT-PCR)和Western blotting检测eIF4E mRNA和蛋白在M1型和M2型巨噬细胞中的表达变化。复制ApoE^-/-动脉粥样硬化小鼠模型,检测小鼠腹主动脉eIF4E mRNA和蛋白表达变化;组织免疫荧光检测eIF4E在主动脉根部中分别与M1型和M2型巨噬细胞中的共表达。结果M1型巨噬细胞eIF4E mRNA和蛋白表达高于M2型巨噬细胞(P<0.05)。在ApoE^-/-小鼠动脉粥样硬化模型中,ApoE^-/-小鼠腹主动脉eIF4E mRNA和蛋白表达高于C57BL/6J小鼠(P<0.05);eIF4E与CD86的共定位表达高于eIF4E与CD206的共定位表达。结论eIF4E在动脉粥样硬化的发展过程中主要在M1型巨噬细胞中起作用,进而对动脉粥样硬化产生影响。