| Objective To observe prevention and treatment effect of Cistanche Phenylethanoid glycosides(CPh Gs)to bovine serum albumin(BSA)-induced liver fibrosis in rats. Analysis of serological indexes, liver histopathology and immunohistochemical index, evaluation of CPh Gs anti hepatic fibrosis effects. To observe the cell viability of CPh Gs on proliferation and apoptosis of HSC, to explain the anti hepatic fibrosis effects of CPh Gs on cell level; at the same time, to study the effects of CPh Gs on TGF β1/smad and PDGF/Erk1/2 signal transduction pathway and investigate their target points for resisting hepatic fibrosis, to elucidate the molecular mechanism in molecular level, and provide basic data for the further development of new drugs.Methods l) Seventy-five male SD rats in a SPF grade were randomLy divided into six groups, normal(distilled water-treated), model(BSA-treated), positive drug(BSA-treated + Bie Jia Ruan Gan Pian 600 mg/kg/day), and BSA-treated + CPh Gs(125, 250, 500 mg/kg/day) groups. BSA-treated + CPh Gs(125, 250, 500 mg/kg/day) groups had 13 rats in each group, and other group had 12 rats in each group. The BSA-induced rat immune hepatic fibrosis model is divided into primary sensitization followed by immunological attack. At the same time, all groups were given corresponding medicine; in addition to the control group and model group were lavaged with distilled water, the other groups were set according to the dose of intragastric administration of drugs; daily dosing rats continued for four weeks after the experimental period. The serum samples, liver and spleen tissues of rats were collected. The serum alanine aminotransferase(GPT), aspartic acid transaminase(GOT), alkaline phosphatase(ALP), total protein(TP), albumin(ALB), serum total bilirubin(TB) and serum direct bilirubin(DB) index were measured using automatic biochemical analyzer. The serum fibrosis markers of serum hyaluronic acid(HA), laminin(LN), procollagen III(PC III) and type IV collagen(IV-C) were determined by radioimmunoassay; serum TGF β1 levels were measured by ELISA kit. Tissues were stained with conventional H&E and Masson’s trichrome staining to observe histopathological changes under a light microscope. Expression of collagen type I, collagen type III, and TGF-β1 in liver tissues was analyzed by immunohistochemical staining. 2)HSC cells were treated with CPh Gs at different concentrations(0, 3.91, 7.81, 15.63, 31.25, 62.50, 125.00, 250.00, and 500.00 μg/m L) for 48?h. The proliferation of CPh Gs and the half inhibition rate(IC50) values were obtained by MTT. HSC cells and liver cells(HL7702) were exposed and incubated to different concentrations of CPh Gs(25, 50, 100 μg/ m L) for 48 h. The cell proliferation inhibition effects and the level of cell viability(based on the measurement of LDH activity released from damaged cells into the supernatant) were determined by an MTT assay. Flow cytometry was used to detect the CPh Gs(25, 50, 100 μg/m L) for the apoptotic effect of HSC. Using real-time quantitative PCR(RT-PCR) to detect the m RNA expression of α-SMA、NF-k B p65 and caspase3. Further using western blot method to detect different subjects of effects of drugs on apoptosis protein Bcl-2, Bax expression. 3)The anti-proliferative effects of CPh Gs(25, 50, 100 μg /m L) on HSC activated by TGF-β1 were determined by an MTT assay. Using RT-PCR to detect the m RNA expression of TGF β1/smad pathway in smad 2, smad 3 and smad 7. Using western blot to detect the protein expression of CPh Gs of TGF β1/smad pathway in smad 2, smad 3, p-smad 2, p-smad 3 and smad 7. The anti-proliferative effects of CPh Gs(25, 50, 100 μg /m L) on HSC activated by rr PDGF-BB were determined by an MTT assay. Using RT-PCR to detect the m RNA expression of CPh Gs PDGF /ERK1/2 pathway in type collagen I, c-jun, c-fos, Erk1/2, α-SMA. Then using western blot to detect the difference expression of CPh Gs PDGF /ERK1/2 pathway protein type collagen I, Erk1/2 and p-Erk1/2.ResμLts 1) The liver and spleen indices of the CPhGs at different dose groups were significantly reduced compared with the model group(P<0.05 or P<0.01). In addition to the low dose group TB and DB, various dose groups of CPh G significantly reduced the BSA-induced elevated GPT, GOT, ALP, TP and ALB serum levels(P<0.05), decreased Hy P content(P<0.01), and reduced serum TGF β1 content(P<0.01). The results of HE staining and Masson staining showed that there were reduction in inflammatory cell infiltration in the different CPh Gs groups compared with the model group. Also observed were less necrosis and fatty degeneration of liver cells as well as alleviation of fibrosis. CPh Gs significantly mitigated the pathology of BSA-induced hepatic fibrosis in rats, alleviated the swelling of liver cells, and effectively prevented hepatocyte necrosis and inflammatory cells infiltration(P<0.01). TGF β1, type collagen I and type collagen III expressions of the CPh Gs at different dose groups were also inhibited significantly(P<0.05 or P<0.01). 2) With the increase of the concentration of the drug, the inhibition rate of CPh Gs on HSC was gradually increased, the IC50 values of CPh Gs was 119.125 μg/m L; HL7702 with different concentrations of CPh Gs had slight proliferation effects, the difference was not statistically significant. The results of flow cytometry showed that the apoptosis rate of CPh Gs 100 μg/m L concentration group was 20.8%. Compared with the blank control group, the difference was statistically significant(P<0.01). RT-PCR showed that, compared with the blank control group, the different concentrations of the CPh Gs group of HSC can significantly inhibit m RNA levels of NF-k B p65 and α-SMA and promote the m RNA level of caspase 3(P<0.05). Western Blot showed that, compared with the blank control group, the different concentrations of the CPh Gs group of HSC were significantly decreased in the Bcl-2/Bax(P<0.05). 3)(50- 100) μg/m L CPh Gs can inhibit the proliferation of HSC(P<0.05) induced by TGF β1,(25- 100) μg/m L CPh Gs has no obvious toxic effect on HSC cells(P>0.05);(25- 100) μg/m L dose group of CPh Gs can inhibit the m RNA and protein expression of smad2 and smad3, and significantly inhibited the phosphorylated protein of smad2 and smad3, and promoted the m RNA and protein expression of smad 7. 4)(50- 100) μg/m L CPh Gs can inhibit the proliferation of HSC induced by rr PDGF-BB(P<0.05),(25- 100) μg/m L CPh Gs has no obvious toxic effect on HSC cells(P>0.05);(25- 100)μg/m L dose group CPh Gs can inhibit m RNA levels of type collagen I, c-jun, c-fos, Erk1/2 and α-SMA. Western Blot showed that CPh Gs could significantly inhibit the protein expression of type collagen I, Erk1/2 and p-Erk1/2.Conclusion 1) CPh Gs has a better protective effect on immune liver fibrosis rats which induced by BSA,The mechanism may be related to reducing the expression of TGF β1, type collagen I, type collagen III, which can inhibit the activation of hepatic stellate cells and reduce the formation of collagen. 2) CPh Gs can obviously inhibit the proliferation of HSC cell and induce its apoptosis. CPh Gs has a positive regulatory effect on the growth of HL7702; the mechanism of apoptosis of HSC may be related to the decrease of Bcl-2 protein expression and the increase of Bax protein expression and the decrease the ratio of Bcl-2 /Bax.3) The anti hepatic fibrosis effect of CPh Gs may be related to the blocking of the TGF β1 /smad pathway and the PDGF /Erk1/2 pathway, thus blocking the activation and proliferation of HSC. |
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