Dahuang zhechong pills regulate ASICla/VEGF pathway and alleviate hepatic fibrosis / 中国药理学通报

Aim To examine the therapeutic effects of DHZCP on carbon tetrachloride(CCl4)-induced chemical hepatic fibrosis model in rats and the mechanism of acid-sensitive ion channels 1a(ASIC1a)and vascular endothelial growth factor(VEGF)-related mechanisms.Methods The rats were injected intraperitoneally with CCl4 vegetable oil mixture to establish hepatic fibrosis model,and randomly divided into six groups:control group,hepatic fibrosis model group,DHZCP low dose group,DHZCP medium dose group,DHZCP high dose group and colchicine(Col)positive control group.HE staining was used to observe the pathological changes of hepatic structures in each group,Masson staining to view the production of collagen fibers in each group,and immunohistochemistry,Western blot,q-PCR to investigate the expression level of ASIC1a,CaMKKβ,VEGF,α-SMA,Collagen-I proteins.Results In model group,serum ALT and AST levels were obviously up-regulated,liver tissue structure was severely damaged,and ASIC1a,CaMKKβ,VEGF,α-SMA,Collagen-I gene and protein expression levels were significantly elevated.Compared with model group,each treatment group of DHZCP could markedly alleviate the pathological changes of liver fibrosis caused by CCl4,significantly reduce the serum ALT and AST levels,and dose-dependently down-regulate the gene and protein expression levels of ASIC1a,CaMKKβ,VEGF,α-SMA,Collagen-I,etc.Conclusions DHZCP ameliorates hepatic fibrosis in rats,and its mechanism of action may be associated with the regulation of ASIC1a/VEGF.

Expression,Purification of PUMA-BH3 Death Domain Peptide in E.coli and Identification of Its Pro-apoptotic Activity / 中国生物工程杂志

The Bcl-2 family of proteins play a central role in the control of apoptosis, a fundamental process for both human health and disease, by mitochondrial pathway. PUMA(p53 up-regulated modulator of apoptosis protein) is one of BH3-only members of Bcl-2 family , its function is to promote cell apoptosis. To obtain BH3 death domain peptide of PUMA and detect its biological activity, the synthesized double-stranded oligomeric nucleotide encoding PUMA-BH3 peptide was cloned into expression vector pTYB2,thus generating a construct of pTYB2-PUMA-BH3 which expressed PUMA-BH3-intein-chitin binding domain fusion protein. Then the recombinant plasmid was transformed into E.coli BL-21 (DE3) and fusion protein was expressed under induction by IPTG. The soluble PUMA-BH3 peptide was purified from chitin affinity chromatography by DTT reduction. Through measuring mitochondria viability(MTT),mitochondria permeability transition(MPT) and the translocation of cytochrome c(Cyt c ) assayed by western blotting, the biological pro-apoptotic activity of PUMA-BH3 peptide was studied. The PUMA-BH3 peptide has the effects on decreasing the mitochondria viability remarkably , inducing mitochondrial swelling and promoting Cyt c releasing from isolated mitochodria . Mitochondrial swelling and the release of Cyt c induced by PUMA-BH3 peptide concerned with the opening of MPT,which can be improved by cyclosporine A(CsA).These results indicated that recombinant PUMA-BH3 peptide might possess pro-apoptosis activity and paved a reasonable way for the study of new apoptosis regulators.