| Liver fibrosis is a pathologic process that leads to deposition of an excess of extra cellular matrix (ECM), which is due to the synthesis of ECM exceeding the degradation of ECM. Pae is a principal active ingredient of total glucosides of paeony. Our previous study showed that TGP has anti-inflammatory, anti-oxidative, immunomodulatory, anti-hepatic injury and anti-immunological hepatofibrotic effects. The protein families of GPCR kinases (GRKs) play apivotal role in the process of desensitization of agonist-activated GPCRs.To further research of the antifibrotic treatment, the present study was designed to investigate the expression of GRK2 on porcine serum-induced liver fibrosis rats in vivo.In addition,the expression of GRK2 on HSC-T6 stimulated with recombinant hunman platelet drived growth factor-BB(rhPDGF-BB) were evaluated in vitro.Meanwhile,the effects of Pae on the expression of GRK2 and trarsmembrane in HSC-T6 stimulated with rhPDGF-BB was also investigated.OBJECTIVE The animal model of porcine serum-induced liver fibrosis was used to evaluate the expression of GRK2 and GRK3 on the rats liver and HSC according to the changes of histopathological examination and immunohistochemistry examination. Effects of Pae on the proliferation of HSC-T6 stimulated with rhPDGF-BB and the expression of GRK2 were observed. The effect of G protein-AC-cAMP signal pathway and the change of G protein expression in HSC-T6 stimulated with rhPDGF-BB were also investigated. To confirm the effect of GRK2 in HSC through signal transduction pathways and the mechanisms of Pae, the effects of Pae on the signal transduction proteins were measured meanwhile.METHODS Rats were intraperitoneally injected with 0.5ml of porcine serum twice a week to establish immunological liver fibrosis model.The rats were randomly divided into normal control group(0wk),liver fibrosis model group(3,6,9,12,16wk).HE stain and Masson stain were used to examine the histopathological change. The expression of GRK2 and GRK3 from rats liver was detected by Western blot analysis and immunohistochemistry staining. Furthmore, the expression of GRK2 from rats HSC by in situ perfusion was detected by Western blot analysis.By using HSC-T6 to establish immunological liver fibrosis model in vitro, the cells were randomly divided into normal control group, rhPDGF-BB ( 50μg·L-1 ) group,GRK2 inhibitor(62.5~1000μmol·L-1)group,Pae(10-4~10-6mol·L-1)group, and the proliferation of HSC was measured by MTT assay and 3H-TdR uptake. Moreover, the cells were randomly divided into normal control group, rhPDGF-BB(50μg·L-1)group,GRK2 inhibitor(250μmol·L-1)group,Pae(10-5mol·L-1)group, and the level of cAMP in HSC-T6 was determined by radioimmunoassay. The expression of GRK2, G protein was detected by Western blot analysis.RESULTS1.The expression of GRK2 in immunological hepatic fibrosis rats induced by porcine serumThe immunological liver fibrosis model was successfully induced at the end of 16th week after the injection of porcine serum. By optical microscope, Sparse connective tissue was slightly observed in the portal zone and the walls of central veins in the normal liver. In the liver of model group, excessive accumulation of connective tissue was observed in centrilobular and periportal zones. Prominent fibrotic septa were observed extending in a radial pattern from central veins and portal zones, resulting in the formation of the pseudolobules. Some hepatocytes surrounded by fibrous connective tissues were observed in centrilobular zones. The expression of GRK2 and GRK3 were significantly decreased by Western blot analysis and immunohistochemistry staining.2. The expression of GRK2 in HSC isolated from porcine serum indeced liver fibrosis ratsHSCs were isolated by in situ perfusion and cultivated at different time course(3,6,9,12 and 16week).Furthermore, the GRK2 expression was detected by Western blot analysis. The expression of GRK2 in HSC isolated from fibrotic rats was gradually decreased.3. Effect of GRK2 inhibitor and Pae on the proliferation of HSC-T6 stimulated with rhPDGF-BBHSC-T6 stimulated with rhPDGF-BB(50μg·L-1) was used as in vitro model to evaluate the effect of GRK2 inhibitor and Pae. Pae at concentration of 10-4~10-6mol·L-1 could significantly inhibit the proliferation of HSC-T6 stimulated with rhPDGF-BB. While incorporating GRK2 inhibitor (62.5~1000μmol·L-1), the proliferation of HSC-T6 stimulated with rhPDGF-BB was inhibited.4. Changes of G protein-AC-cAMP pathways in HSC-T6 stimulated with rhPDGF-BBThe membrane fractions and cytosolic fractions of HSC were obtained by differential centrifugation approach. Then, the change of the expression of G-protein in membrane and cytosolic fractions of HSC-T6 stimulated with rhPDGF-BB(50μg·L-1)were detected by Western-blot.The results showed that the expression of GRK2 was remarkably increased, while Gαi-1 and Gαi-2 were decreased in cytosolic fractions of HSC-T6 stimulated by rhPDGF-BB, but the expression of Gαs had no significant change. On the contrary, the expression of GRK2 was remarkably decreased, while Gαi-1 and Gαi-2 were increased in membrane fractions of HSC-T6 stimulated by rhPDGF-BB, but the expression of Gαs had no significant change. Meanwhile, the level of cAMP in HSC-T6 was obviously decreased after addition of rhPDGF-BB and the proliferation of HSC-T6 was promoted.5. Role of GRK2 in G protein-AC-cAMP pathways in HSC-T6 stimulated with rhPDGF-BB and the effect of PaeThe change of the expression of G-protein in membrane and cytosolic fractions of HSC-T6 stimulated with rhPDGF-BB(50μg·L-1)were detected by Western-blot after addition of GRK2 inhibitor(250μmol·L-1). The results showed that the expression of GRK2 was remarkably decreased, while Gαi-1 and Gαi-2 were increased in cytosolic fractions of HSC-T6 stimulated by rhPDGF-BB and GRK2 inhibitor. On the contrary, the expression of GRK2 was remarkably increased, while Gαi-1 and Gαi-2 were decreased in membrane fractions of HSC-T6 stimulated by rhPDGF-BB and GRK2 inhibitor, but the expression of Gαs had no significant change. Meanwhile, the level of cAMP in HSC-T6 was obviously increased after addition of rhPDGF-BB and GRK2 inhibitor and the proliferation of HSC-T6 is inhibited. When GRK2 inhibitor (250μmol·L-1) and Pae(10-5mol·L-1) were given simultaneously, the expression of GRK2 was remarkably increased and Gαi-1 and Gαi-2 were decreased in cytosolic fractions of HSC-T6 stimulated by rhPDGF-BB. On the contrary, the expression of GRK2 was remarkably decreased and Gαi-1 and Gαi-2 were increased in membrane fractions of HSC-T6 stimulated by rhPDGF-BB, but the expression of Gαs had no significant change. Meanwhile, the level of cAMP in HSC-T6 was decreased after addition of GRK2 inhibitor and Pae.CONCLUSIONS1. The expression of GRK2 in immunological hepatic fibrosis rats induced by porcine serum was gradually decreased. This suggested that the down-regulation of GRK2 may play an important role in liver fibrosis. 2. The expression of GRK2 was remarkably increased and Gαi-1 and Gαi-2 were decreased in cytosolic fractions of HSC-T6 stimulated by rhPDGF-BB. On the contrary, the expression of GRK2 was remarkably decreased and Gαi-1 and Gαi-2 were increased in membrane fractions of HSC-T6 stimulated by rhPDGF-BB. Meanwhile, the level of cAMP in HSC-T6 was obviously decreased after addition of rhPDGF-BB and the proliferation of HSC-T6 was promoted. This suggested that rhPDGF-BB induced GRK2 transmembrane from membrane to endochylema and then promote the proliferation of HSC-T6 induced by rhPDGF-BB via Gi-AC-cAMP pathway.3. GRK2 inhibitor can inhibit the increased expression of GRK2 in cytosolic fractions of HSC-T6 stimulated by rhPDGF-BB, while promote the expression of Gαi-1 and Gαi-2. Meanwhile, the results was opposite in membrane fractions of HSC-T6 stimulated by rhPDGF-BB. The level of cAMP in HSC-T6 was obviously increased after addition of GRK2 inhibitor and the proliferation of HSC-T6 was inhibited. In addition, Pae can regulate G protein-AC-cAMP signal transduction pathway of HSC-T6, which may be one of the important mechanisms for the effects of Pae on HSC-T6 stimulated by rhPDGF-BB. |
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