The Role of PPARγ、KLF6 and the Effects of Rosiglitazone on the Development of Nonalcoholic Steatohepatitis Related Fibrosis in Rats
|School||Third Military Medical University|
|Keywords||Non - alcoholic steatohepatitis Liver fibrosis The Eumenes pomiformis fly -like nuclear transcription factor KLF6 / zinc finger protein 9 Peroxisome proliferator-activated receptor γ Transforming growth factor β1 α smooth muscle actin Rosiglitazone Hepatic stellate cells|
The first part: of PPARγ, KLF6 formed NASH liver fibrosis in rats Expression and significance of the purpose of the process: observation of nuclear transcription factors of PPARγ KLF6 formation process in the expression of liver fibrosis in NASH rats with hepatic stellate cell activation, TGFβ1 expression, and its role in nonalcoholic fatty liver fibrosis. Methods: 1, the NASH liver fibrosis in animal models: choice of 4-week-old male Wistar rats (body weight 170 to 210g) 60 were randomly divided into the basic diet group (control group, C group), high-fat diet group (model group group F), respectively, in feeding 8,12,16,24 weeks 4 time points, weighed and anesthetized rats, abdominal aortic blood specimens from liver tissue specimens, some made of paraffin sections, HE and Masson staining; the portion -70 ℃ storage backup. Dynamic observation groups: ① weight change; ② The HE and Masson staining pathological changes of the liver tissue; ③ serum free fatty acids (free fatty acid, FFA), triglycerides (triglyceride, TG), alanine aminotransferase (alanine aminotrasferase ALT) and aspartate aminotransferase (aspartate aminotrasferase, AST) change; the ④ RIA assay serum hyaluronic acid (hyaluronic, HA), laminin (laminin, LN), C Ⅳ type I collagen (collagen type Ⅳ, C Ⅳ ) level; ⑤ immunohistochemistry assay PPARγ, KLF6, α-SMA positive expression rate. ⑥ RT-PCR was used to detect liver tissue of PPARγ, KLF6, TGFβ1, α-SMA mRNA expression; Results: 1 weight situation: each group of rats weight with the build mode time was increased gradually, but the same time point comparison model group weight were higher than the control group (P lt; 0.05); 2, HE and Masson staining results: C rat liver abnormalities were found; simple fatty liver (F2-3 in the the F rat liver 8 weeks) pathological changes, mixed mainly microvesicular steatosis without inflammation and fibrosis; 12 weeks in addition to steatosis, still visible acini 3 with a small number of balloon-like liver cells, infiltration of inflammatory cells in the lobules point stove necrosis, part of the evolution of non-alcoholic steatohepatitis reached NASH-F (2-4) G (0-1) S0 diagnostic criteria; rat liver tissue at 16 weeks lobular inflammation, further exacerbating Meeting District visible inflammatory cell infiltration, acinar 3 with obvious ballooning hepatocytes, rat liver tissue sinus / cell cycle fibrosis, up to NASH-F (3-4) (1-3) S (0-1 sinus / cell cycle) diagnostic criteria; 24 weeks the rats continued to show - severe fatty liver, but some degree of inflammation in rats has been reduced over 16 weeks, and there are fibrosis still visible obvious bridge-like fibrosis, reached NASH -F (3-4) G (0-2) S (1-3) diagnostic criteria; serological markers: F serum TG and FFA levels eight weeks was significantly higher compared with the control group was statistically significant ( 0.71 ± 0.08 vs 0.48 ± 0.04; 0.54 ± 0.06 vs 0.34 ± 0.04, P lt; 0.05), and with the extension of the high-fat diet increased gradually ; 12 weeks serum AST, ALT also began to rise with the control group difference (116 ± 7.08 vs64.88, ± 8.58; 80.23 ± 3.40 vs 44.35 ± 3.38, P lt; 0.05), 16 weeks reached a peak (148.2 ± 8.33,101.6 ± 6.79, P lt; 0.05), 24 weeks slightly lower (138.4 ± 9.73,90. 4 ± 7.68); the HA content began to increase the changes of serum fibrosis markers: F group at 12 weeks compared with control group was statistically significant (86.45 ± 11.94 vs 63.57 ± 9.49 , P lt; 0.05), and with the extension of the high-fat diet gradually increased; 16 weeks serum LN, C Ⅳ increased compared with control group was statistically significant (94.15 ± 24.43 vs 71.93 ± 11 .84; 59.41 ± 8.89vs 27.87 ± 8.49, P lt; 0.05), 24 weeks increased the most obvious and significant difference compared with the control group (131.67 ± 59.96 VS71.93 ± 11.84; 86.31 ± 32.78 vs 27.87 ± 8.49, P lt; 0.01): 5, immunohistochemical staining: PPARγ in group C liver cell nuclei colored pale, were scattered in distribution, weakly positive expression group F 8 weeks fatty degeneration of the liver nucleus expression began to increase, 12 weeks almost all fatty degeneration of liver cell nuclei are visible brown expression, 16 weeks with the emergence of fatty liver fibrosis, PPARγ positive expression cells begin to reduce over 12 weeks, 24 weeks PPARγ positive cells further reduced staining lighter was a light tan, especially liver fibrosis parts of PPARγ positive staining cells decreased significantly; KLF6 no positive expression in the liver of group C visible part of the positive expression in the nucleus of hepatic steatosis since 12 weeks, 16 and 24 weeks of positive expression gradually increased; C group, α-SMA only a small amount of expression in the vascular wall, was light brown and yellow staining, F group 12 weeks shows a small amount of positive cells, in addition to the vascular wall visible positive expression, lobular, were scattered in the distribution of light tan coloring, 16 weeks of positive cells began to increase, and deep colored fiber beam deposition site, 24 weeks of positive cells number up, dyed deep brown color, the positive cells were located in the portal area around the liver sinusoids and fibrous septa places; 6, RT-PCR results: F rats 8 weeks liver tissue KLF6, TGFβ1, α-SMA mRNA expression compared with control group was not statistically significant (P gt; 0.05) ,12-24 weeks KLF6 mRNA expression was gradually raised significant difference with the control group (0.62 ± 0.10,1.01 ± 0 the .08,0.96 ± 0.07 vs 0.24 ± 0.05, P lt; 0.05), 16 weeks of TGFβ1, α-SMAmRNA expression began to increase, 24 weeks reached a peak with the control group was statistically significant (0.62 ± 0.19,0.91 ± 0.07 vs 0.36 ± 0.16; 0.77 ± 0.12,1.08 ± 0.19 vs 0.30 ± 0.07, P lt; 0.05); while PPARγmRNA fed with high fat diet for eight weeks significantly increased (0.84 ± 0.07 vs 0.54 ± 0.02, P lt; 0.05)), 12 weeks to express the most obvious ( 1.06 ± 0.14), 16 weeks steatohepatitis obvious expression began to decline (0.73 ± 0.01), 24 weeks down more significantly compared with the control group was statistically significant (0.36 ± 0 .02 vs 0.54 ± 0.02, P lt; 0.05). Pearson linear correlation analysis found: when fat 24 weeks NASH liver fibrosis the KLF6mRNA and of TGFβ1, α-SMA mRNA and liver fibrosis score showed a significant positive correlation (r = 0.848,0.859,0.706 , P lt; 0.01). Conclusion: NASH liver fibrosis model we built to study one of the ideal model of steatohepatitis liver fibrosis pathogenesis; 2, the high-fat diet can induce liver tissue KLF6 TGFβ1, α-SMAmRNA and the protein expression of enhanced and high expression of non-alcoholic fatty liver fibrosis are closely related, and play an important role in the process of simple fatty liver to steatohepatitis, liver fibrosis progression; 3, fat diet induced the NASH rat liver PPARγmRNA and the protein expression first increased and then decreased, suggesting that fat diet induced liver cell injury to fatty degeneration of the liver cells compensatory increase in the synthesis of PPARγ, to inhibit increased variety of pro-inflammatory and promoting fibrogenic factors such as KLF6, TGFβ1 release; injury factors persist, PPARγ synthetic weakened tissue injury homeostasis disorders, KLF6, TGFβ1 pro-fibrogenic factor significantly increased activation of HSC result in the formation of liver fibrosis. The second part: the impact of PPARγ agonist rosiglitazone on rat NASH liver fibrosis formation Objective: To observe Rosiglitazone the NASH liver fibrosis in rats nuclear transcription factor KLF6 signaling pathway to explore rosiglitazone anti-inflammatory, anti-fibrotic effect and mechanism. Methods: 12 male Wistar rats high-fat diet for 8 weeks, giving 4mg/kg/d rosiglitazone (intervention group, T group), while the establishment of a control group (C group) and hyperlipidemia model group (F ), 24 the weekend after weighing all the rats were sacrificed and specimens of serum and liver lipids, liver function, liver fibrosis markers, liver histopathology staining and RT-PCR analysis of liver tissue of PPARγ, KLF6, TGFβ1, α-SMAmRNA and immunohistochemical detection of PPARγ, the KLF6 and α-SMA expression. Results: ① HE and Masson staining showed that the model rats the typical fatty fibrosis treatment group inflammation and fibrosis were significantly reduced and improvement of inflammation, fibrosis, but compared with the control group, there are still differences were seen in 24 weeks ( P lt; 0.01); ② serological indicators suggest that rosiglitazone after 16 weeks of intervention can significantly reduce the high fat diet-induced TG, FFA, AST, ALT, HA, LN, C Ⅳ, increased between the two groups significant difference (P lt; 0.01); ③ RT-PCR display model group KLF6 (0.96 ± 0.08) after 24 weeks of TGFβ1 (0.91 ± 0.07) and α-SMA (1.07 ± 0.19) relative expression were significantly up-regulated, compared with the control group, a statistically significant difference (P lt; 0.01); treatment group of these genes was increased in varying degrees downward, the difference between the two groups was significant (P lt; 0.01). ④ Immunohistochemistry showed that the intervention group KLF6 compared with the model group, the expression of α-SMA significant reduction of PPARγ expression is significantly increased, the difference was statistically significant (P lt; 0.05). Conclusion: 1, rosiglitazone ketone can activate PPARγ, lowered the NASH liver fibrosis in rat liver tissue nuclear transcription factor KLF6 TGFβ1 and its downstream target gene expression, inhibition of HSC activation and prevent liver fibrosis, which may be The play, one of the antifibrotic the mechanism; 2, rosiglitazone on the high-fat diet-induced the NASH liver fibrosis in rats with a certain preventive effect, by lowering blood lipids, improve fatty acid metabolism disorders, reduce inflammation thereby reducing liver fibrosis.