【作者】 李睿；

【导师】 魏伟；

【作者基本信息】 安徽医科大学 ， 药理学， 2010， 硕士

【摘要】 肝纤维化是由于细胞外基质(extracellular matrix,ECM)的合成大于降解导致过度沉积,是纤维增生与纤维分解不平衡而引起的病理过程。芍药苷(paeoniflorin,Pae)是白芍总苷(total glucosides of paeony,TGP)中的主要有效成分,本课题组以往研究发现,TGP具有抗炎、抗氧化、免疫调节、抗肝损伤和肝纤维化等作用。G蛋白偶联受体激酶(G-protein-coupled receptors kinases,GRKs)是一簇与G蛋白偶联受体(G protein-coupled receptors,GPCRs)快速脱敏相关的激酶,在调节GPCRs信号中起到至关重要的作用。本实验在以往研究基础上,采用猪血清诱导的免疫性肝纤维化模型,首先从整体水平考察了免疫性肝纤维化过程中大鼠肝脏与肝星状细胞(hepatic stellate cell,HSC)中G蛋白偶联受体激酶2(G-protein-coupled receptors kinase 2,GRK2)表达水平的变化;体外选用HSC-T6细胞株,从细胞和分子水平探讨GRK2在HSC表达的变化,以进一步探讨其调节HSC G蛋白偶联信号转导通路以及抑制HSC异常增殖的作用机制,并观察Pae对HSC GRK2表达及转膜的影响,目的:采用猪血清诱导的免疫性肝纤维化模型,从病理形态学、免疫组织化学等方面,观察免疫性肝纤维化大鼠肝脏及HSC中GRK2与G蛋白偶联受体激酶3(G-protein-coupled receptors kinase 3,GRK3)的表达情况;以肝纤维化过程中的关键细胞HSC为突破口,观察Pae对rhPDGF-BB刺激HSC-T6增殖的影响以及GRK2在HSC-T6增殖中的作用;探讨G蛋白偶联的信号转导通路在rhPDGF-BB刺激HSC-T6增殖中的作用和G蛋白表达的改变,部分阐明GRK2在HSC信号转导中的调节作用及Pae抑制HSC增殖的分子机制。方法:Wistar大鼠腹腔注射猪血清建立免疫性肝纤维化模型,设立正常对照组(0周)、模型组(3、6、9、12、16周),采用HE染色和Masson染色对肝脏组织作病理检查;免疫组织化学染色法、蛋白免疫印迹法检测肝脏中GRK2与GRK3表达水平的变化。原位灌流分离肝纤维化大鼠HSC,Western blot法检测HSC中GRK2表达水平的变化。体外以HSC-T6为模型,设立正常对照组、rhPDGF-BB(50μg·L-1)刺激组、GRK2抑制剂(62.5~1000μmol·L-1)组、Pae(10-4~10-6mol·L-1)组,采用MTT法检测HSC增殖情况;3H-TdR法检测HSC增殖情况;另外,设立正常对照组、rhPDGF-BB(50μg·L-1)刺激组、GRK2抑制剂(250μmol·L-1)组、Pae(10-5mol·L-1)组,采用放射性免疫法测定HSC-T6 cAMP水平;采用Western blot法检测HSC-T6中GRK2、Gαi-1、Gαi-2、Gαs蛋白的表达水平的变化。结果:1.免疫性肝纤维化大鼠肝脏中GRK2与GRK3的表达情况Wistar大鼠腹腔注射猪血清在16周时可以成功建立免疫性肝纤维化模型,在光镜下可见中央静脉和汇管区结缔组织变厚,增生的胶原纤维从汇管区延伸至小叶间,并向邻近的肝小叶伸出纤维索,形成粗大的纤维间隔,并将增生的肝细胞团包绕分隔成大小不等的假小叶。免疫组织化学染色与Western-blot法检测发现免疫性肝纤维化大鼠肝脏中GRK2与GRK3的表达较正常组明显降低,并且伴随造模时间的延长,表达量有下降趋势。2. GRK2在免疫性肝纤维化大鼠HSC中的表达在造模第3,6,9,12,16周原位灌流法分离、培养大鼠HSC, Western-blot法检测发现,免疫性肝纤维化大鼠HSC中GRK2的表达较正常组明显降低,并且伴随造模时间的延长,表达量有下降趋势。3. GRK2抑制剂与Pae对rhPDGF-BB刺激HSC-T6增殖的影响体外建立rhPDGF-BB诱导的HSC-T6增殖模型,结果表明Pae在10-4~10-6mol·L-1浓度范围内明显抑制HSC-T6增殖。当加入GRK2抑制剂(5-甲基[2-(5-硝基-2-呋喃基)乙烯基]-2-糠酸酯,C12H9NO6)后,GRK2抑制剂(62.5~1000μmol·L-1)可以明显抑制HSC-T6增殖。4. rhPDGF-BB刺激下HSC-T6 G蛋白-AC-cAMP通路的改变分离HSC-T6胞浆、胞膜蛋白,应用Western-blot方法,检测rhPDGF-BB(50μg·L-1)刺激HSC-T6中G蛋白-AC-cAMP通路的改变。结果发现,rhPDGF-BB可明显促进HSC-T6胞浆蛋白中GRK2的表达升高,同时降低Gαi-1和Gαi-2的表达,但对Gαs的表达无明显影响。而在HSC-T6胞膜蛋白中,rhPDGF-BB可明显降低GRK2的表达,同时促进Gαi-1和Gαi-2的表达升高。但对Gαs的表达无明显影响。同时rhPDGF-BB可以降低细胞内cAMP水平,促进HSC-T6增殖。5. rhPDGF-BB刺激的HSC-T6 G蛋白-AC-cAMP信号通路与GRK2的关系及Pae的作用在rhPDGF-BB刺激的HSC-T6中加入GRK2抑制剂(终浓度250μmol·L-1)后,Western blot检测发现,HSC-T6胞浆蛋白中GRK2表达降低,Gαi-1和Gαi-2表达升高,而在胞膜蛋白中GRK2表达升高,Gαi-1和Gαi-2表达降低,同时Gαs的表达均无明显改变。此外,GRK2抑制剂可以明显提高由rhPDGF-BB诱导的HSC-T6 cAMP水平降低,抑制细胞增殖。而在HSC-T6中加入GRK2抑制剂(终浓度250μmol·L-1)与Pae(10-5mol·L-1)共培养后,结果发现,HSC-T6胞浆蛋白中Pae可升高GRK2抑制剂诱导的GRK2表达减少,同时促进了Gαi-1和Gαi-2的表达的下降,而在胞膜蛋白中Pae可降低GRK2抑制剂诱导的GRK2表达升高,同时促进了Gαi-1和Gαi-2的表达的增加,而Gαs的表达均无明显改变。结论:1.猪血清诱导的免疫性肝纤维化大鼠肝脏组织和HSC中GRK2表达水平随造模时间的延长有下降趋势,提示GRK2可能是导致免疫性肝纤维化大鼠HSC异常增殖的重要分子。2.rhPDGF-BB可以明显促进HSC-T6胞浆蛋白中GRK2的表达,降低Gαi-1和Gαi-2的表达,而在胞膜蛋白中,rhPDGF-BB降低GRK2的表达,促进Gαi-1和Gαi-2的表达。同时rhPDGF-BB可以降低细胞内cAMP水平,促进HSC-T6增殖。提示rhPDGF-BB可能改变HSC-T6胞浆胞膜上GRK2蛋白的分布,使HSC-T6 Gαi蛋白偶联的信号通路激活,从而刺激了HSC-T6过度增殖。3. GRK2抑制剂可明显抑制rhPDGF-BB诱导的HSC-T6胞浆蛋白中GRK2表达增加,促进Gαi-1和Gαi-2的表达的升高,而在胞膜蛋白中正好与此相反,同时可以升高细胞内cAMP水平,而Pae可能通过改变GRK2的表达和活性,恢复了G蛋白-AC-cAMP信号通路的正常转导,从而抑制HSC-T6增殖、维持其正常功能。更多还原

【Abstract】 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.更多还原