Effects Of Paeoniflorin On TGFβ₁-Smads Signalling In Hepatic Fibrosis Of Schistosomiasis Japonicum In Mouse

Schistosomiasis is one of the major tropical diseases of mankind. The primary cause of death in schistosomiasis is the formation of liver egg granulomas and secondary hepatic fibrosis. Studies have showed that granuloma inflammatory reaction and fibrosis in liver tissue would continue to be aggravated, even though efficacious schistosomicides were given. Moreover, once fibrosis has reached a certain level, it will continue to develop, although the inducements are eradicated. So the intervention and control of such aggravation during or before the formation of granuloma or at the early stage of fibrosis become another key therapeutic strategy after efficacious treatment of praziquantel. However, so far, no drug of anti-fibrosis with low toxicity and high efficiency has been applied to prevent or reverse the hepatic fibrosis in schistosomiasis. Hepatic stellate cells (HSCs) play a key role in hepatic fibrosis through secreting extracelluar matrix (ECM). Transforming groeth factor beta1 (TGFβ1) is the most potent fibrogenetic cytokine and can stimulate HSCs activation through paracrine or/and autocrine by mediating TGFβ1-Smads signalling. But the effect of TGFβ1 on hepatic fibrosis of schistosomiasis is controversial at present.Paeony (Paeoniae radix) root is one of the well-known herbs in China and other Asian countries. Paeoniflorin (PAE, C23H28O11) is known to be one of the principal bioactive components of paeony root. PAE has been reported to have immunoregulatory and anti-inflammatory effects. Preparations of many traditional Chinese herbs used in anti-hepatic fibrosis contain paeony root. However, the mechanism by which it elicits anti-hepatic fibrosis in schistosomiasis japonica has not been elucidated.The present study was first designed to investigate the effects of PAE on expression of TGFβ1 in liver tissue, hepatic granuloma and fibrosis of schistosomiasis in mouse induced by S. japonicum cercarie. Then the effect of schistosomal soluble egg antigen (SEA) and PAE on the secretion of TGFβ1 from mouse peritoneal macrophages (PMs) and the effects of TGFβ1 and PAE on the proliferation of HSCs and secretion of collagens were investigated in vitro. Finally, we explored the effects of PAE on TGFβ1-Smads signalling at gene and protein levels in order to realize the partial mechanisms of PAE on hepatic fibrosis of S. japonicum.AIM To study on the effects of PAE on TGFβ1-Smads signalling in hepatic fibrosis of schistosomiasis japonica in mouseMETHODS We first infected mouse via skin with cercarie of S. japonicum, then PAE was orally administrated before and after praziquantel treatmentand and both therapeutics were given simultaneously, with different dose of PAE and at different time after infection. The effects of PAE on formation of hepatic granuloma and fibrosis and expression of TGFβ1 in liver tissue at different time after infection were analyzed by tissue microarray, in combination with immunohistochemistry.We stimulated mouse PMs with SEA, then the effects of SEA and PAE on the production of TGFβ1 from PMs were evaluated by reverse transcriptase polymerase chain reaction (RT-PCR), Western blotting, and enzyme-linked immunosorbent assay (ELISA); The HSCs isolated from health mouse by in situ perfusion and density gradient centrifugation were stimulated by mouse peritoneal macrophage-conditioned medium (PMCM), then the effects of PMCM and PAE on the proliferation of HSCs and collagens secretion from HSCs were detected by methylthiazolyl tetrazolium (MTT) assay and ELISA, respectively. We chose dilution 1:2 as the optimum macrophage-conditioned medium (OPMCM). Then the effects of TGFβ1 on the proliferation of HSCs and collagens secretion from HSCs were detected with OPMCM and anti-TGFβ1 antibodies. Finally, the effects of PAE on Smads and procollagens were assayed by RT-PCR and Western blotting.RESULTS1. PAE early administrated could inhibit formation of hepatic granuloma and fibrosis and expression of TGFβ1 in liver tissue of mouse in schistosomiasis PAE (30,120 mg/kg) administrated at second week (just four weeks before schistosomicides were given) after cercarie attack could obviously decrease the area of hepatic granuloma and the degree of fibrosis, and downregulate the expression of TGFβ1 in liver tissue. However, PAE administrated at sixth week (just at the same time when schistosomicides were given) or at tenth week (just after four weeks when schistosomicides were given) after cercarie attack had no effect on the area of hepatic granuloma or the degree of fibrosis, or the expression of TGFβ1 in liver tissue, suggesting that PAE early given could prevent hepatic granuloma and fibrosis of S. japonicum, which might be related to TGFβ1.2. SEA could promote TGFβ1 secretion from PMs, and PAE could depress TGFβ1 secretionTGFβ1 could be secreted from PMs stimulted by SEA (0,2.5,5,10,20,40 mg/L) and SEA at 10 mg/L could induce the highest production level of TGFβ1 from PMs. Meanwhile, the production of TGFβ1 from PMs could be depressed significantly by PAE (0,7.5,15,30,60,120 mg/L) in a concentration-dependent manner, suggesting that the mechanism by which PAE inhibited the formation of hepatic granuloma and fibrosis might be relate to depressed the secretion of TGFβ1 from PMs.3. PMCM and TGFβ1 could promote the proliferation of HSCs and the secretion of collagens, but PAE could depress the proliferation and secretion at gene and protein levelsPMCM (1:32,1:16,1:8,1:4,1:2,1:0) could promote the proliferation of HSCs and the secretion of collagen type I (Col I) and collagen type III (Col III), moreover PMCM at dilution 1:2 and 1:0 had more significant effects on the proliferation and secretion. But both proliferation and secretion were partly inhibited in the presence of anti-TGFβ1 antibody, suggesting that TGFβ1 could play an important role in the proliferation of HSCs and the sectetion of collagens. Meanwhile our results showed that PAE (0,7.5,15,30,60,120 mg/L) could inhibit the proliferation of HSCs and the production of procollagen, type I, alpha1 (Col Iα1), procollagen, type III, alpha1 (Col IIIα1), Col I and Col III at gene and protein levels.4. PAE could downregulate the Smad3 mRNA transcription and expression levelsPAE (0,7.5,15,30,60,120 mg/L) could downregulte the Smad3 mRNA transcription and expression levels in a concentration-dependent manner, but had no effects on the transcription and expression levels of Smad2, Smad4 and Smad7 mRNA, suggesting that the mechanism by which PAE inhibited the formation of hepatic fibrosis might be relate to downregulating the transcription and expression levels of Smad3 in HSCs.CONCLUSIONS1. TGFβ1 could promote hepatic fibrosis of S. japonicum in mouse;2. PAE early administrated could inhibit formation of hepatic granuloma and fibrosis and expression of TGFβ1 in liver tissue of mouse in schistosomiasis;3. SEA could stimulate PMs to secrete TGFβ1, and the TGFβ1 could promote HSCs proliferation and collagens secretion;4. The anti-fibrotic role of PAE in schistosomiasis might be associated with:a) inhibition of the secretion of TGFβ1 from PMsb) inhibiton of the proliferation of HSCsc) downregulation of Smad3 mRNA transcription and expression which might be clsosely associated with inhibiton of collagens production from HSCs.In conclusion, TGFβ1 could be secreted by PMs stimulated by SEA, and the TGFβ1 could promote the proliferation of HSCs and the secretion of collagens in schistosomiasis. PAE could inhibit the production of collagens by depressing the secretion of TGFβ1 from PMs, reducing the proliferation of HSCs, downregulating the transcription and expression of Smad3 mRNA. Our results might provide the experimental basis for PAE as a natural therapeutic agent to prevent hepatic fibrosis of schistosomiasis japonica.