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To explore the mechanism of Ezhu-containing serum in inhibiting the expression of sonic hedgehog(Shh) and glioma-associated oncogene homolog-1(Gli1) in hepatic stellate cells(HSCs) induced by leptin. Twenty sprague-dawley (SD) rats were randomly divided into 2 groups (n=10), and given Ezhu-decoction and physiological saline by gavage for 10 days to prepare drug-containing serums. The HSCs during the exponential growth phase were divided into 7 groups： blank control group, model group, hedgehog pathway inhibitor(cyclopamine) group, Ezhu group, Ezhu and cyclopamine group, hedgehog pathway agonost(pumorphamine) group, Ezhu and purmorphamine group. HSCs were cultured in vitro and induced with 100 μg•L ⁻¹ leptin(except for the blank control group), then treated separately with the corresponding drugs for 24 hours. After the cells were collected, HSCs proliferation was detected using MTT colorimetric assay; the expressions of Shh and Gli1 were determined by PT-PCR, Western blot and immunofluorescence, respectively. The expressions of Shh and Gli1 were significantly increased after the HSCs of rats were induced by leptin (compared with the blank control group, P<0.01). After being interfered with Hh pathway inhibitor (cyclopamine) and Ezhu-containing serum, the expressions of Shh and Gli1 were decreased significantly(compared with the model group, P<0.01). After Ezhu-containing serum was used to interfere the Hh pathway inhibitor group, the mRNA and protein expressions of Shh and Gli1 were decreased significantly(compared with the model group, P<0.01). After Ezhu-containing serum was used to interfere the purmorphamine group, the mRNA and protein expressions of Shh and Gli1 decreased significantly(compared with the purmorphamine group, P<0.01). Ezhu-containing serum plays an important role in inhibiting HSCs activation by taking part in hedgehog signaling pathway, so as to regulate the expression of Shh and Gli1 in leptin-induced HSCs and then inhibit liver fibrosis., Competing Interests: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose., (Copyright© by the Chinese Pharmaceutical Association.)

To observe the effects of Danshen-containing serum on SuFu and DYRK2 expression in the HSCs stimulated by leptin. SD rats (n = 60) were used to make danshen-containing serum by gastric perfusion for ten days with Danshen water decoction, normal saline and colchicine. The HSCs that were cultured in vitro would be stimulated for 24 hours by leptin (100 μg x L(-1)) except blank control group, after being intervened, the drug serum in each group would be cultured at 37 degrees C in 5% incubator. The cells would be collected after 24 hours, then the effects of danshen-containing serum on the proliferation of HSCs were detected by MTT, the expression of SuFu mRNA and DYRK2 mRNA were detected by RT-PCR, the expression of SuFu and DYRK2 proteins were tested by Western blot. Compared with blank control group, the expression of DYRK2 mRNA and DYRK2 proteins were enhanced obviously after stimulated the HSCs of rats by leptin (P < 0.01), but the expression of SuFu mRNA and SuFu proteins were decreased significantly (P < 0.01). Compared with the model group, after cyclopamine group (Hh pathway inhibitor), Danshen-containing serum and colchicine were interfered, the expression of DYRK2 mRNA and DYRK2 proteins were decreased clearly (P < 0.01), but the expression of SuFu mRNA and SuFu proteins were increased significantly (P < 0.01 or P < 0.05). Compared with model group, adding purmorphamine (Hh pathway agonist) to model group and making it activate could increase the expression of DYRK2 mRNA and DYRK2 proteins, but the expression of SuFu mRNA and SuFu proteins were decreased significantly (P < 0.01). Compared with the model group, using the Danshen-containing serum to interfere the purmorphamine group could make the expression of DYRK2 mRNA and DYRK2 proteins decrease and the expression of SuFu mRNA and SuFu proteins increase significantly (P < 0.01). Danshen-containing serum would inhibition the activation and increment of HSCs by interfering the expression of SuFu and DYRK2 which were induced by leptin.

To observe the effect of Ligusticum wallichii-containing serum on the expressions of Toll-like receptor 4 and myeloid differentiation factor 88 in hepatic stellate cells. Clean-grade SD rats were randomly divided into 5 groups and orally given L. wallichii decoction, colchicine and normal saline for 7 d to prepare L. wallichii-containing serums. Except for the blank group, all of the remaining groups were stimulated with LPS 1 mg x L(-1) for 24 h. After being intervened, the L. wallichii-containing serums were cultured in 5% CO2 incubator at 37 degrees C for 24 hours. The expression of TLR4 and MyD88 were detected by RT-PCR and Western blot. After HSC was stimulated with LPS, TLR4 and MyD88 mRNA and protein expressions were significantly higher than the blank control group (P < 0.01). After being intervened with L. wallichii-containing serum, TLR4 and MyD88 mRNA and protein expressions were notably lower than the model group (P < 0.05 or P < 0.01). In conclusion, L. wallichii-containing serum could regulate the TLR4 signaling pathway and show the anti-fibrosis effect by inhibiting the expression of TLR4 and MyD88 in LPS-induced HSCs.