Study On Effects And Mechanisms Of Phenylarsine Oxide Resisting Hepatic Fibrosis

Background: Liver fibrosis is an essential stage to a variety of chronic liver disease progresses to liver cirrhosis,is also the key stage of reverse cirrhosis treatment,mainly is closely related to the HSC activation and activation of HSC outcome,there is no effective prevention and control of drugs.Although the source of activated HSC in liver fibrosis is controversial,but HSC is undoubtedly important targets for anti fibrosis treatment.HSC activation is a gradual process,therefore,inhibition of HSC activation or reverse HSC activation condition is the potential strategy for the treatment of liver fibrosis Our partners-Chinese academy of sciences,Shanghai Advanced Research Institute,professor Huang team's unpublished results show PAO can inhibit ectomesenchymal stem cells in vitro culture of alpha SMA and Calponin1 expression,inhibition of mesenchymal stem cells to the muscle between the fibroblasts direction differentiation,prompt PAO is likely to have the function of liver fibrosis.Objective:(1)to investigate the influence of PAO on HSC-T6 active state;(2)to investigate the influence of PAO on the original generation of HSC activation in vitro and the mechanism.Comprehensive evaluation of PAO resisting hepatic fibrosis and mechanism.Methods:(1)with different concentration of(25,50,100,150 and 200 nmol/L)PAO processing HSC-T6 cells for 24 h,to observe the effects of PAO to HSC T6 vitality;Application determined by MTT method to detect PAO influence on HSC-T6 cell proliferation,to evaluate the cytotoxicity of the PAO.Extraction RNA and total protein of PAO processing cells and the control group;Application of Real-Time PCR and Western-blot test each cell alpha SMA,COLI,COLIII mRNA and alpha SMA protein expression.(2)the SD rat primary HSC isolation and culture;Using Western blot-testing training 1 d,3 d,4 d,7 d primary HSC alpha SMA protein expression and observation of cell morphology,evaluate the original generation dynamic change rules of HSC in vitro activation process;With different concentration of PAO cultivation process in 4 d original generation of HSC,application of Real-Time PCR and Western blot-detection PAO HSC alpha SMA,COLI,after processing COLIII mRNA and alpha SMA and phosphorylated Akt protein expression.Results:(1)MTT results showed that increased with the concentration of PAO,HSC survival rate significantly decreased,when the PAO concentration increased to 150 nmol/L,HSC cell survival rate is 82% in the control group(P < 0.05),when the concentration of PAO increased to 200 nmol/L,the cell survival rate decreased to less than 72% of the control group(P < 0.01),the results showed that the concentration of 25-100 nmol/L PAO has less cell toxicity to HSC and cell survival rate were over 90%,determined as effective work concentration;the western blot results show that compared with the control group,PAO can reduce the expression of HSC-T6 cell alpha SMA,difference is statistically significant;Real-time PCR results showed that compared with the control group,PAO can reduce the expression of alpha SMA and COL1 mRNA,with statistically significant difference,but has no effect on the expression of COL3.(2)Using density gradient centrifugation method to extract the SD rat HSC,with the extension of cultivation time,HSC activation degree increased,most 1d cells were post wall,assumes the circular or elliptic,4 d cells were in initial activation state in vitro culture;the 7 d cells are in completely active state;Prompt that morphology can be used as an effective sign of HSC activation state;the western blot results show that as the extension of cultivation time,HSC alpha SMA expression quantity increased,in vitro culture 4 d expression quantity is(1.51± 0.045)significantly higher than control(0.762 ± 0.062)(P < 0.05),the 7 d alpha SMA expression quantity(1.752±0.053)is further raised in vitro culture;western blot results and the reference(GAPDH)comparative analysis shows that: PAO can decrease alpha SMA protein expression dose dependent,in 50,100 nmol/L concentration,alpha SMA expression quantity respectively(0.63±0.078),(0.41± 0.09)Significantly lower than the control group(1.53 ±0.039),P < 0.05);Real-time PCR results: compared with control,the concentration of PAO has significant inhibitory effect on the expression of HSC alpha SMA and CollegneI mRNA expression,and to increase by concentration dependence(P < 0.01);Western blot results show that compared with the control group,PAO can obviously decrease the expression of 4 d primary HSC PI4 K and p-Akt protein.Conclusion: PAO in 25-100 nmol/L concentration range has no obvious cytotoxicity to HSC-T6,but can obviously reverse HSC activation degree and the inhibition of the spontaneous generation of primary HSC activation,its mechanism may be related to blocking PI3K-Akt signaling pathways,prompt PAO may have a potential to anti liver fibrosis.