Upregulation Of Cannabinoid Receptor-1and Fibrotic Activation Of Mouse Hepatic Stellate Cells During Schistosoma J. Infection:Role Of NADPH Oxidase

Objective To study the effect of shistosomal infection on primary hepatic stellate cells (HSC) to fibrotic phenotype transformation.Method1) The schistosomiasis-associated liver fibrosis (SSLF) model was developed by infecting mice with Schistosoma J. cercariae in the skin. HE and Masson staining check the formation of liver fibrosis.2) HSCs from control and infected mice were then isolated with two-step discontinuous density gradient centrifugation technique.3) Cultured and confirmed HSC by analysis of HSC markers a-SMA with immunofluorescence staining of a-SMA in control and infected HSC.4) Cultured and confirmed HSC by analysis of HSC markers a-SMA and desmin with RT-Real-time PCR to detect the level of mRNA and compare the difference between the control and infected mice isolated HSCs.5) Cultured and confirmed HSC by analysis of HSC markers a-SMA and desmin with western blot to detect the level of protein and compare the difference between the control and infected mice isolated HSCs. At the same time, we detect the difference of fibrotic changes between the control and infected HSC. We also detect the change of fibrotic protein after treated with SEA.Result1) HE staining showed that in livers of the6weeks post-infect mice, we can find eggs deposited in theblood vessel to form round granulomatous, a lot of inflammatory cells diffusated. Masson staining showed blue collage fibers deposited around granulomatous. It has significant difference of collagen deposition between control and infected mice. 2) immunofluorescence staining showed that infected HSC express more α-SMA than control HSC. We used podocytes and smooth muscle cells as positive control of α-SMA and desmin to confirm HSC, the gels shows that the isolated cells is HSC.3) RT Real Time PCR showed that α-SMA and desmin were markedly increased at the mRNA level in infected HSCs compared to those from uninfected mice. Similarly displayed, Western blot analysis demonstrated that the infected HSCs had significantly elevated α-SMA and desmin proteins.Together, these results suggest that primary HSCs, which are enriched with a-SMA and desmin, were successfully isolated from normal and Schistosoma J. infected mice and that Schistosoma J. infection elevates the levels of both markers in HSCs.4) Western blot analysis observed an upregulation of collagen I and TIMP-1expression in infected HSCs indicative of a Schistosoma J. infection-induced fibrogenic phenotype.5) Western blot analysis demonstrated that both fibrotic markers collagen I and TIMP-1increased when HSCs were treated with SEA. RT Real Time PCR also showed mRNA upregulation of CollagenI and TIMP-1.Conclusion From6weeks post-infect, granulomatousand fibrosis are formed in the livers, applying cercaria to the skin of mice can build schistosoma liver fibrosis models. Chronic shistosoma infection and acute SEA stimulation can lead HSC to fibrotic phenotype. Object To study the expression of CB1and CB2between in primary hepatic stellate cells isolated from Ctr1and schistosomal liver fibrosis mice and stimulated by SEA. At the same time to study the effect of CB1and CB2on liver fibrosis.Method1) In mRNA level, we used Real time RT-PCR to detect the mRNA expression of CB1and CB2between in primary hepatic stellate cells isolated from Ctr1and schistosomal liver fibrosis mice and when HSC stimulated by SEA.2)In protein level, we used Western blotting to detect the protein expression of CB1,CB2,CollagenI and TIMP-1.At the same time, to detect the above protein expression after stimulated by SEA in control primary hepatic stellate cells.3) We used siRNACB1transfected to HSC and then to detect the expression of Collagen I, α-SMA�'�TTMP-1in control primary hepatic stellate cells stimulated by SEA.Result1) In mRNA and protein level, contrast to control hepatic stellate cells, liver fibrosis hepatic stellate cells express more CB1(P<0.05), but not the expression of CB2.2) In mRNA and protein level, SEA can promote expression of CB1(P<0.05), but not the expression of CB2.3) When primary HSC transfected siRNACB1to detect the efficient of transfection, the inhibit rate is80%. At the same time to further detect the function of CB1was inhibited by detecting the expression of phospho-AKT after treated with ananamide(AEA), we used western bolt to measure the expression of Phospho-AKT, and finally we found the expression of Phospho-AKT was inhibited by siRNACB1. 4) In this experiment we found that inhibit the expression of CB1can block SEA increasing the expression of Collagen I, α-SMA�'�TIMP-1in control primary hepatic stellate cells.Conclusion CB1play an important role in schistosomal liver fibrosis. Object to investigate whether NADPH Oxidase mediated regulation of the expression of CB1treated by SEA and its related mechanism.Method1) the establishment of schistosomiasis liver fibrosis, liver fibrosis model established by using abdominal applicator with schistosome cercariae, all the mice were divided into three groups:normal control group, schistosomiasis liver fibrosis group and treatment group which treated with DPI (NADPH oxidase inhibition agent) lg/kg/24h,ip. After7weeks, take mouse liver tissue to doHE staining and Masson staining to observe and identify the formation of schistosomiasis liver fibrosis and DPI whether reducing the degree of liver fibrosis.2) To detect the expression in livers of three groups and compare the difference in the three groups using western blotting. We also study the effect of Noxl, Nox2,Nox4which are the subunit of NADPH Oxidase on the expression of CB1, at the same time to study the effect of CB1on the three subunit, and to investigate the effect of Noxl, Nox4on fibrosis change in primary hepatic stellate cells. In this study, other purpose protein were detected by western blot.3) Using ESR to detect the difference of superoxide species in normal HSC, infected HSC and HSC treated with SEA. We also detect the effect of Nox1、Nox4and CB1on the production of superoxide species. 4)using the siRNA transfection to inhibit the expression of Noxl,Nox4and Nrf2, at the same time to using nuclear transfection to inhibit the expression of Nox2and after inhibiting to detect the changes of CB1and fibrosis function in hepatic stellate cells.5)Isolate the nuclear protein to measure the activity of Nrf2.6) Labeled Nrf2immunofluorescence method to detect whether the SEA stimulating Nrf2nuclear transfer, and the impact of Nrf2and metastasis after Nox1and Nox4be suppressed.Result1Establishment of schistosomiasis liver fibrosis model and modeling while giving DPI medication, HE staining and Masson staining showing that the degree of fibrosis in DPI treatment group were significantly lower than in liver fibrosis model group. Use image plot6software statistical analysis of collagen fiber content. The difference is significant (P<0.05).2) We found the production of superoxide species in infected HSC is more than normal HSC, and the HSC treated with SEA also produce more superoxide species than control HSC. In HSC, when Noxl and Nox4were inhibited by siRNA and then treated with SEA, we found the production of superoxide species decreased, but when the CB1were inhibited, the production did not change. We also using DPI to treat HSC, the result is consistent with siRNANox1and siRNANox4.(3) We found when Noxl and Nox4were inhibited by siRNA, the action of SEA increasing CB1expression also was blocked in HSC. At the same time, we found inhibition of Nox2by shRNA cannot effect on CB1expression when HSC stimulated by SEA. When we treat cells with DPI, the result consistent with siNoxl and siNox4.(4) We observed the relationship between Noxl, Nox4and CB1by western blot. We found when Noxl was been inhibited by siRNA, the expression of Nox4decreased, but when Nox4was been inhibited, Noxl expression had no change. At the same time, when CB1was been inhibited, both the expression of Nox1and Nox4did not change. (5)By the immunofluorescence method we detected that after treatment with SEA in HSC, Nrf2ofcan transferred from cytoplasm to the nucleus, and when Noxl and Nox4inhibited nuclear transfer phenomenon will be suppressed.(6) Extract nuclear protein to measure the transcription activity of Nrf2. We found the transcription Nrf2activity significantly increased after stimulating SEA, but when Nox1and Nox4were inhibited by siRNA, the activity of Nrf2stay the same level as control.(7)SEA can promote the expression of Nrf2and when inhibition of Nrf2can suppress the expression of CB1after stimulating by SEA.(8) We found that Noxl and Nox4were suppressed also inhibit various fibrotic changes such as a-SMA, Collagen I and TIMP-1expression after stimulating by SEA using Western blot method. After the use of DPI-treated cells can also suppress a variety of fibrosis such asa-SMA, Collagen I and TIMP-1expression level.Conclusion:Schistosome infections. And SEA stimulation can significantly upregulated the expression of CB1receptors in mouse primary hepatic stellate cells, and upregulation of CB1receptors is dependent on the role of superoxide-mediated Noxl and Nox4generated. Since upregulation of CB1, leading to fibrosis and hepatic stellate cells transformed phenotype. NOX-mediated hepatic stellate cells redox signal can be used for schistosomiasis in a new therapeutic target for the prevention or treatment of cirrhosis.