肝复康对肝纤维化大鼠肝组织JAK2和STAT3表达的影响
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摘要
目的:观察中药肝复康对肝纤维化大鼠肝组织Janus激酶2(JAK2)及信号转导子和转录激活子3 ( STAT3 )表达的影响,并对其在JAK2/STAT3信号通路上治疗肝纤维化的作用机制进行初步探讨。
方法:将健康SD大鼠随机分为7组,即正常对照组(C)、模型组(M)、模型对照组(MC)、高剂量治疗组(HT)、中剂量治疗组(MT)、低剂量治疗组(LT)和预防组(P)。除正常对照组外,余鼠造模。皮下注射10%四氯化碳,每周两次,共12周。其中预防组于造模同时给予肝复康煎剂灌胃12周。高剂量治疗组、中剂量治疗组、低剂量治疗组则于造模第9周后以肝复康灌胃12周。正常对照组、模型组及模型对照组则以等容量的生理盐水灌胃。第12周末将模型组、预防组大鼠采血处死,迅速取出肝组织。第20周末将其余各组大鼠以同样方法处死。通过测定血清中谷丙转氨酶(alanine aminotransferase, ALT)、谷草转氨酶(aspartate aminotransferase, AST)的活性及白蛋白和总蛋白的含量来反映肝脏功能,HE染色法观察肝组织病理学变化,免疫组织化学方法观察肝组织中金属蛋白酶组织抑制因子-1( tissue inhibitor of metalloproteinase-1, TIMP-1)的表达,逆转录聚合酶链式反应(RT-PCR)观察JAK2和STAT3 mRNA的表达。
结果:(1)血清学指标:20周后,中剂量治疗组与模型组相比,血清ALT降低(P<0.01),AST降低(P<0.01),白蛋白升高(P<0.01),球蛋白降低(P<0.01),白、球比例升高(P<0.01)。(2)肝组织病理学变化:HE染色显示,模型组大鼠肝小叶结构紊乱,大量纤维组织增生。与模型组相比,中剂量治疗组大鼠肝小叶结构明显改善。(3)肝组织TIMP-1蛋白的表达:免疫组织化学染色显示,正常组大鼠肝组织TIMP-1蛋白仅有少量表达,与正常组比较,模型组大鼠肝组织TIMP-1阳性染色明显增多,与模型组相比,中剂量治疗组大鼠肝组织TIMP-1阳性染色明显减少(p<0.01)。(4)肝组织JAK2和STAT3 mRNA的表达:在逆转录聚合酶链式反应中,正常组大鼠肝组织有JAK2和STAT3 mRNA表达,但程度较弱,模型组大鼠肝组织JAK2和STAT3 mRNA表达明显上调,中剂量治疗组大鼠肝组织JAK2和STAT3 mRNA表达较模型组明显下调,且有显著性差异(p<0.01)。
结论:(1)JAK2/STAT3信号通路在肝纤维化的形成过程中起重要作用。(2)肝复康对肝纤维化有疗效,其作用机制可能与降低肝组织JAK2和STAT3的表达,阻断JAK2/STAT3信号通路有关。
Objective: To study the effect of traditional Chinese medicine-Ganfu- kang on the expression of Janus kinase 2(JAK2) and signal transducer and activator of transcription 3(STAT3) in rat with experimental liver fibrosis, and explore its therapy mechanism of action on JAK2/STAT3 pathway.
Methods: SD rats were randomly divided into normal control group(C), model group(M), model control group(MC), high dose treatment group(HT), middle dose treatment group(MT), low dose treatment group(LT) and me- dicine prevention group(P). The hepatic fibrosis in rats except normal con- trol group were injected by 10% CCl4 for 12 weeks. P group rats were trea- ted with Ganfukang and CCl4 simultaneously for 12 weeks. HT, MT and LT group were treated with Ganfukang from 9th week to 20th week. C group, M group and MC group were treated with the same capacity of sodium chloride. M and P group rats were killed at the end of 12th week and dislodge liver tissues quickly. The remaining rats were killed at the end of 20th week by the same way. The levels of serum alanine aminotransferase(ALT), aspar- tate aminotransferase(AST), albumin, total protein were measured. The pathological changes of hepatic tissues were observed under light mic- roscopy by HE staining. The tissue inhibitor of metalloproteinase -1 (TIMP -1) of hepatic tissues were measured by immunohistochemistry staining. Hepatic Janus kinase 2(JAK2)and signal transducer and activator of transcription 3(STAT3) were detected by reverse polymerase chain reaction (RT-PCR).
Results: (1) Serum index: At the end of 20th week, compared with M group, the levels of serum ALT decreased(P<0.01), AST decreased(P<0.01), albumin increased(P<0.01), globulin decreased(P<0.01), the ratio of albu- min and globulin increased(P<0.01) in MT group. (2) The pathological changes of hepatic tissues: HE staining displayed the structure of hepatic lobules in M group disordered and fibrilla increased. The structure of hepatic lobules in the MT group improved obviously compared with those in M group. (3) Expression of TIMP-1: In the hepatic tissue of rats in C group, few TIMP-1 positive staining cells were observed by immunohistochemistry staining. The quantity of TIMP-1 positive staining cells of liver tissue in M group increased obviously comparison with those in C group. TIMP-1 positive staining cells in MT group were less than those in M group evi- dently (p<0.01). (4)Expression of JAK2 and STAT3 mRNA: RT-PCR analysis showed that liver tissue of C group had low but detectable levels of JAK2 and STAT3 mRNA. Rats in M group showed higher JAK2 and STAT3 levels in liver tissue. The levels decreased in MT group comparison with those in M group, the difference was significant (p<0.01).
Conclusion: (1)JAK2/STAT3 signal transduction pathway played an important role in hepatic fibrosis. (2)Ganfukang had therapeutic effect on hepatic fibrosis. Its mechanism of action may be related to decrease the expression of JAK2 and STAT3 of hepatic tissue and block JAK2/STAT3 signal transduction pathway.
方法:将健康SD大鼠随机分为7组,即正常对照组(C)、模型组(M)、模型对照组(MC)、高剂量治疗组(HT)、中剂量治疗组(MT)、低剂量治疗组(LT)和预防组(P)。除正常对照组外,余鼠造模。皮下注射10%四氯化碳,每周两次,共12周。其中预防组于造模同时给予肝复康煎剂灌胃12周。高剂量治疗组、中剂量治疗组、低剂量治疗组则于造模第9周后以肝复康灌胃12周。正常对照组、模型组及模型对照组则以等容量的生理盐水灌胃。第12周末将模型组、预防组大鼠采血处死,迅速取出肝组织。第20周末将其余各组大鼠以同样方法处死。通过测定血清中谷丙转氨酶(alanine aminotransferase, ALT)、谷草转氨酶(aspartate aminotransferase, AST)的活性及白蛋白和总蛋白的含量来反映肝脏功能,HE染色法观察肝组织病理学变化,免疫组织化学方法观察肝组织中金属蛋白酶组织抑制因子-1( tissue inhibitor of metalloproteinase-1, TIMP-1)的表达,逆转录聚合酶链式反应(RT-PCR)观察JAK2和STAT3 mRNA的表达。
结果:(1)血清学指标:20周后,中剂量治疗组与模型组相比,血清ALT降低(P<0.01),AST降低(P<0.01),白蛋白升高(P<0.01),球蛋白降低(P<0.01),白、球比例升高(P<0.01)。(2)肝组织病理学变化:HE染色显示,模型组大鼠肝小叶结构紊乱,大量纤维组织增生。与模型组相比,中剂量治疗组大鼠肝小叶结构明显改善。(3)肝组织TIMP-1蛋白的表达:免疫组织化学染色显示,正常组大鼠肝组织TIMP-1蛋白仅有少量表达,与正常组比较,模型组大鼠肝组织TIMP-1阳性染色明显增多,与模型组相比,中剂量治疗组大鼠肝组织TIMP-1阳性染色明显减少(p<0.01)。(4)肝组织JAK2和STAT3 mRNA的表达:在逆转录聚合酶链式反应中,正常组大鼠肝组织有JAK2和STAT3 mRNA表达,但程度较弱,模型组大鼠肝组织JAK2和STAT3 mRNA表达明显上调,中剂量治疗组大鼠肝组织JAK2和STAT3 mRNA表达较模型组明显下调,且有显著性差异(p<0.01)。
结论:(1)JAK2/STAT3信号通路在肝纤维化的形成过程中起重要作用。(2)肝复康对肝纤维化有疗效,其作用机制可能与降低肝组织JAK2和STAT3的表达,阻断JAK2/STAT3信号通路有关。
Objective: To study the effect of traditional Chinese medicine-Ganfu- kang on the expression of Janus kinase 2(JAK2) and signal transducer and activator of transcription 3(STAT3) in rat with experimental liver fibrosis, and explore its therapy mechanism of action on JAK2/STAT3 pathway.
Methods: SD rats were randomly divided into normal control group(C), model group(M), model control group(MC), high dose treatment group(HT), middle dose treatment group(MT), low dose treatment group(LT) and me- dicine prevention group(P). The hepatic fibrosis in rats except normal con- trol group were injected by 10% CCl4 for 12 weeks. P group rats were trea- ted with Ganfukang and CCl4 simultaneously for 12 weeks. HT, MT and LT group were treated with Ganfukang from 9th week to 20th week. C group, M group and MC group were treated with the same capacity of sodium chloride. M and P group rats were killed at the end of 12th week and dislodge liver tissues quickly. The remaining rats were killed at the end of 20th week by the same way. The levels of serum alanine aminotransferase(ALT), aspar- tate aminotransferase(AST), albumin, total protein were measured. The pathological changes of hepatic tissues were observed under light mic- roscopy by HE staining. The tissue inhibitor of metalloproteinase -1 (TIMP -1) of hepatic tissues were measured by immunohistochemistry staining. Hepatic Janus kinase 2(JAK2)and signal transducer and activator of transcription 3(STAT3) were detected by reverse polymerase chain reaction (RT-PCR).
Results: (1) Serum index: At the end of 20th week, compared with M group, the levels of serum ALT decreased(P<0.01), AST decreased(P<0.01), albumin increased(P<0.01), globulin decreased(P<0.01), the ratio of albu- min and globulin increased(P<0.01) in MT group. (2) The pathological changes of hepatic tissues: HE staining displayed the structure of hepatic lobules in M group disordered and fibrilla increased. The structure of hepatic lobules in the MT group improved obviously compared with those in M group. (3) Expression of TIMP-1: In the hepatic tissue of rats in C group, few TIMP-1 positive staining cells were observed by immunohistochemistry staining. The quantity of TIMP-1 positive staining cells of liver tissue in M group increased obviously comparison with those in C group. TIMP-1 positive staining cells in MT group were less than those in M group evi- dently (p<0.01). (4)Expression of JAK2 and STAT3 mRNA: RT-PCR analysis showed that liver tissue of C group had low but detectable levels of JAK2 and STAT3 mRNA. Rats in M group showed higher JAK2 and STAT3 levels in liver tissue. The levels decreased in MT group comparison with those in M group, the difference was significant (p<0.01).
Conclusion: (1)JAK2/STAT3 signal transduction pathway played an important role in hepatic fibrosis. (2)Ganfukang had therapeutic effect on hepatic fibrosis. Its mechanism of action may be related to decrease the expression of JAK2 and STAT3 of hepatic tissue and block JAK2/STAT3 signal transduction pathway.
引文
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12. Indira N, Zhimin L, Nagadhara D, et al. An Essential Role of the Jak-2/STAT-3/Cytosolic Phospholipase A2 Axis in Platelet-derived Growth Factor BB- induced Vascular Smooth Muscle Cell Motility. The journal of biological chemi- stry, 2004; 279(44): 46122-46128.
13. Simon AR, Takahashi Satoe, Fanburg BL, et al. Role of the JAK-STAT pathway in PDGF-stimulated proliferation of human airway smooth muscle cells. Am J Physiol Lung Cell Mol Physiol, 2002; 282: L1296-L1304.
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2. Tsukamoto H. Cytokine regulation of hepatic stellate cells in liver fibrosis. Alcohol Clin Exp Res, 1999; 23: 911-916.
3. Halaas JL, Gajiwala KS, Maffei M, et a1. Weight-reducing efects of the plasma protein encoded by the obese gene. Science, 1995; 269: 543-546.
4. Ahima RS, Elier JS. Leptin. Annu Rev Phsiol, 2000; 62: 413-437.
5. Kanoski SE, Walls EK, Davidson TL. Interoceptive "satiety" signals produced by leptin and CCK. Peptides, 2007; 28(5): 988-1002.
6. Tartaglia LA, Dembski M, Weng X, et al. Identification and expression cloning of a leptin receptor, OB-R. Cel1, 1995; 83: 1263-1271.
7. Peelman F, Waelput W, Iserentant H, et al. Leptin linking adipocyte metabolism with cardiovascular and autoimmune diseases. Prog Lipid Res, 2004; 43(4): 283- 301.
8. Flynn MC, Plata-Salaman CR. Leptin(OB protein) and meal size. Nutrition, 1999; 15(6): 508-509.
9. Somasundar P, McFadden DW, Hileman SM, et al. Leptin is a growth factor in cancer. J Surg Res, 2004; 116(2): 337-349.
10. Potter JJ, Womack L, Mezey E, et al. Transdifferentiation of rat hepatic stellate cells results in leptin expression. Biochem Biophys Res Commun, 1998; 244: 178-182.
11. Wellhoener P, Fruehwald-Schultes B, Kern W, et al. Glucose metabolism rather than insulin is a main determinant of leptin secretion in humans. J Clin End- ocrunol Metab, 2000; 85: 1267-1271.
12. Ihle JN. STATs: Signal transducers and activator of transcription. Cell, 1996; 84 (3): 331-334.
13. Saxena NK, Titus MA, Ding X, et al. Leptin as a novel profibrogenic cytokine in hepatic stellate cells: mitogenesis and inhibition of apoptosis mediated by extracellular regulated kinase (Erk) and Akt phosphorylation. FASEB J, 2004; 18(13): 1612-1614.
14. Cao Q, Mak KM, Ren C, et al. Leptin Stimulates Tissue Inhibitor of Metallopr- oteinase-1 in Human Hepatic Stellate Cells. The journal of biological chemistry,2004; 279(6): 4292-4304.
15. Cao Q, Mak KM, Charles S. Leptin represses matrix metalloproteinase-1 gene expression in LX2 human hepatic stellate cells. Journal of Hepatology, 2007; 46: 124-133.
16. Hoch RV, Soriano P. Roles of PDGF in animal development. Development, 2003; 130(20): 4769-4784.
17. Pinzani M, Milani S, Herbst H, et al. Expression of platelet-derived growth factor and its receptor in normal human liver and during active hepatic fibro- genesis. Am J Pathol, 1996; 148(3): 785-800.
18. Di Sario A, Bendia E, Svegliati-Baroni G, et al. Rear-rangement of the cyto- skeletal network induced by platelet-derived growth factor in rat hepatic stellate cells: role of different intracellular signaling pathways. J Hepatology, 2002; 36(2): 179-190.
19. Carloni V, Romanelli RG, Pinzani M, et al. Focal adhesion kinase and phos- pholipase C gamma involvement in adhesion and migration of human hepatic stellate cells. Gastroenterology, 1997; 112(2): 522-531.
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