Paeoniflorin Ameliorates Schistosomiasis Liver Fibrosis Through Regulating IL-13 And Its Signaling Molecules In Mice

OBJECTIVE To study the effects of paeoniflorin (PAE) on IL-13-STAT6 signalling pathway in hepatic fibrosis of Schistosoma japonica. in mice.METHODS Mice were percutaneously infected with cercariae of S.japonicum. The infected animals were divided into 3 groups randomly: group 1 were control mice (normal group), group 2 were infected/untreated mice (model group), and group 3 were infected/PAE-treated mice (PAE-treated group). In order to decrease the mortality of infected mice, each group, except for the control, was orally given praziquantel (PZQ, 500 mg/kg/day) on the 42nd day (2 days of successive administration) after infection. In the PAE treated group, the PAE (30 mg/kg/day) was given orally on the 12th day (30 days of successive administration) after infection; the mice in the model group were synchronously given the same volume of solvent only. On the 102nd day following infection, the livers of mice were obtained under ether anaesthesia. Liver tissues were preserved for histological analysis in 4% paraformaldehyde (including the effects of PAE on formation of hepatic granuloma and fibrosis and expression ofα-SMA and ColI by HE & MASSON straing, in combination with immunohistochemistry in liver tissues), the remaining tissues were homogenized for analyses of hydroxyproline, ColI,ColШ, IL-13, and IL-13Rα2; The hepatic stellate cells (HSCs) isolated from normal mice by in situ perfusion and density gradient centrifugation were stimulated by recombinant IL-13 and PAE, then the effects of PAE on the proliferation of HSCs and collagens production from HSCs were detected by methylthiazolyl tetrazolium (MTT)assay, reverse transcriptase polymerase chain reaction (RT-PCR),Western blotting, and enzyme-linked immunosorbent assay (ELISA), respectively. Finally, the effects of PAE on IL-13 receptors and signal molecules of IL-13-STAT6 signalling (including STAT6, p-STAT6 and SOCS-1) in HSCs were assayed by RT-PCR and Western blotting.RESULTS1. PAE early administrated could inhibit formation of hepatic granuloma and fibrosis in liver tissues of mice with schistosomiasis.PAE (30 mg/kg/day) was administrated orally on the 12th day (30 days of successive administration) after infection. On the 102nd day following infection, the livers were obtained from mice under ether anaesthesia. Liver tissues were detected as follows: 1) Effect of PAE on liver histopathologyLiver tissues were fixed in 4% (v/v) paraformaldehyde in PBS and embedded in paraffin, and then processed for histology. Masson trichrome and H&E staining revealed that in the PAE-treatment group,the mean area of granuloma and degree of fibrosis were reduced by nearly 53.4% and 38.5% compared with the model group.2) Effect of PAE on the expression of a-SMA and Col I in the liver tissue.Immunohistochemistry(IHC) staining demonstrated that the content of a-SMA and Col I in the model group was dramatically increased compared with the normal mice. In the PAE-treatment group, the degree ofα-SMA and Col I expression were markedly decreased by 30.3% and 46.6% compared with the model group.3) Effect of PAE on production of hydroxyproline in liverAnalysis of the hydroxyproline content in BALB/c mice with hepatic fibrosis was carried out as an index of total collagen. Elevated hydroxyproline levels were measured in the model group in comparison to normal controls. However, after treatment with PAE, the level of hepatic hydroxyproline in BALB/c mice was significantly reduced (by 54.7%) compared with the model group.4) Effect of PAE on Col I /Col III in liverLiver collagen I and III content was significantly increased in the model mice compared with normal group, but obviously decreased in PAE-treated mice with respect to model mice.2. Effect of PAE on IL-13 /IL-13Rα2 in the liver of miceIn normal controls, the levels of IL-13 and IL-13Rα2 were lower than that of the model group. Treatment with PAE significantly suppressed the IL-13 level (by nearly 43%), but significantly elevated the IL-13Rα2 level in the liver when compared with model mice (by about 37.7 %).3. Effect of PAE on HSC proliferation stimulated by IL-13.MTT showed that (i) co-culture of HSCs with IL-13 induced a significant proliferative response; (ii) pre-incubation with increasing concentrations of PAE significantly reduced the proliferative response in a dose-dependent manner;4. PAE inhibits IL-13-induced collagen production in HSCs.RT-PCR, Western blotting, and ELISA analysis demonstrated that PAE led to the expression of Col I mRNA and protein. Furthermore, when HSCs were cultured with various concentrations of PAE, Col I secretion and expression by IL-13-stimulated HSCs was inhibited significantly. In summary, Col I produced by IL-13-stimulated HSCs was suppressed by PAE at the gene and protein level in a concentration-dependent manner.5. Analysis of IL-4/IL-13 receptor components on HSCs.By using the RT-PCR method, the transcripts of IL-4Rαand IL-13Rα1 on HSCs were induced by treatment with IL-13 whereas IL-13Rα2, a critical downregulatory factor of IL-13–mediated tissue fibrosis induced by S. mansoni. and S. japonicum., was not detectable. Furthermore, the expression of IL-4Rαand IL-13Rα1 mRNA did not change in HSCs treated with increasing concentrations of PAE. The results suggested that IL-13 could stimulate the expressions of IL-4Rαand IL-13Rα1 mRNA, but not IL-13Rα2. So, we presumed that IL-13 induces the expression of Col I mainly through the IL-4Rα/IL-13Rα1 signal transduction pathway and PAE inhibits the production of collagens by interfering with the IL-13 signalling pathway.6. PAE inhibits IL-13-induced STAT6 phosphorylation.we examined the expression of STAT6 and p-STAT6 by use of Western blotting. IL-13-induced STAT6 phosphorylation, and this effect did not peak until 2h but was significantly attenuated at 4h after addition of IL-13, and IL-13 had no effect on STAT6 protein expression in HSCs. PAE, however, effectively reduced phosphorylation of STAT6, but had no effect on STAT6 protein in HSCs.Furthermore, levels of STAT6 phosphorylation were markedly down-regulated by PAE in a concentration-dependent manner, and AG490, a inhibitor of JAKs, was shown to inhibit phosphorylation of STAT6, but had no effect on STAT6 protein. This suggested that supression of STAT6 activation probably induce inhibition of collagen production.7. PAE elevated the expression of SOCS-1RT-PCR and Western blotting analysis demonstrated that SOCS-1 mRNA levels peaked after 1h in comparison with the control. After a long incubation of the cells, SOCS-1 mRNA and protein expression were still at elevated levels. Moreover, levels of SOCS-1 mRNA and protein were up-regulated following PAE treatment relative to IL-13 treatment alone. Additionally, levels of SOCS-1 mRNA protein were up-regulated by PAE in a concentration-dependent manner.CONCLUSIONS1. PAE, if early administrated, could inhibit formation of hepatic granuloma and fibrosis in liver tissue of mouse in schistosomiasis.2. One of the mechanisms for the decline in liver fibrosis brought about by PAE may be the decrease of IL-13 level in liver. Meanwhile, the enhanced level of IL-13Rα2 is correlated with attenuation of hepatic fibrosis.3. Pretreatment with PAE led to a dose-dependent suppression of Col I protein which stimulated by rIL-13. PAE could inhibit the HSC proliferation induced by IL-13, suggesting that PAE may decrease collagen production by suppressing HSC activation.4. Neither IL-13 nor PAE treatment induced the expression of the decoy receptor IL-13Rα2 and, thus, this receptor does not seem to contribute to the inhibitory effects of PAE. The IL-4Rα/IL-13Rα1 complex responded to IL-13 in HSCs and was probably linked to intracellular signalling pathways.5. Pre-treatment with PAE decreased the amount of total p-STAT6 observed in response to IL-13, without altering the total level of STAT6 protein in the cells. AG490 inhibit phosphorylation of STAT6, but had no effect on STAT6 protein also. Meanwhile, PAE pre-treatment induces higher levels of SOCS-1 which induce by rIL-13 in HSCs in a concentration-dependent manner, and the elevated expression of this protein coincides with decreased STAT6 phosphorylation.This suggested that the mechanisms of PAE's suppressing STAT6 activation is as follows: (i) PAE could directly inhibit STAT6 activation, or simultaneously inhibiting Jak lead to decrease the level of p-STAT6 indirectly. (ii)PAE's attenuating impact on the level of p-STAT6 protein might be partly through the elevation of SOCS-1 protein expression. In conclusion, PAE could inhibit the production of collagens by depressing the proliferation of HSCs, downregulating the activation of STAT6. Our results might provide the experimental basis for PAE as a natural therapeutic agent to prevent hepatic fibrosis of schistosomiasis japonica.