| ObjectiveTo investigate the effect of putative receptor protein related to the angiotensinreceptor AT1(APJ) antagonist Apelin 13(F13A) on the hepatic fibrosis and to explore the possible mechanism from the perspective of autophagy in rats with hepatic fibrosis. MethodsThe Sprague-Dawley(S-D) male rats were randomly divided into the control group, hepatic fibrosis model group and Apelin 13(F13A) treatment group. The rat model of hepatic fibrosis was induced by intraperitoneal injection with 3 m L/kg 40% carbon tetrachloride(CCl4) 2 times a week for 12 weeks. Apelin 13(F13A)(100 μg/kg) was administered by intraperitoneal injection once a day for 12 weeks. After 12 weeks, the indexs reflected the liver metabolism and synthesis of protein function including the globin(GLB), albumin(ALB) and total protein(STP) and the indexs reflected the hepatocellular damage including the cholinesterase(CHE), aspartate transaminase(AST) and alanine transaminase(ALT) were measured by automatic biochemistry analyzer. The levels of serum procollagen(PcⅢ Ⅲ), collagen IV(IV-C), hyaluronic acid(HA) and 1aminin(LN) were tested by ELISA assay. The levels of serum Apelin-36 and α-Smooth muscle actin(α-SMA) by ELISA assay. The pathological structure of liver was observated by Hematoxylin-Eosin(H-E) staining. The ultrastructural of hepatic stellate cells was observated by transmission electron microscope. The m RNA and protein expressions of Apelin-36, APJ and α-SMA in liver were measured by Real-time PCR and Western Blot respectively. The expressions of autophagy related gene Beclin-1, LC3 and p62/SQSTM1 in liver were measured by Western Blot. Results(1) At the end of the experiment after 12 weeks, all the rats were survival and no death in control group, the five rats were dead in hepatic fibrosis model group and one rat was dead in the Apelin 13(F13A) group. The hair was brown and dull, spirits was drooping, the activity was significantly decreased, the response to external stimuli was slow and the weight was significantly decreased in the rats of hepatic fibrosis model group. The rats moved freely, above-mentioned phenomenons did not appear in the control group. The above-mentioned phenomenons were significantly reduced, the activity was improved in the Apelin 13(F13A) group.(2) Compared to the control group, the level of serum Apelin-36, m RNA and protein expressions of Apelin-36 and APJ in the liver were significantly increased in the hepatic fibrosis model group(all P<0.05). Apelin 13(F13A) did not affect the serum Apelin-36, m RNA and protein expressions of Apelin-36 and APJ in the liver compared to the hepatic fibrosis model group(all P>0.05).(3) Compared to the control group, the levels of serum STP, ALB and CHE were significantly decreased(all P<0.05), the levels of serum GLB, ALT and AST were significantly increased in the hepatic fibrosis model group(all P<0.05). Compared to the model group, the levels of serum STP, ALB and CHE were significantly increased(all P<0.05), the levels of serum GLB, ALT and AST were significantly decreased in the Apelin 13(F13A) group(all P<0.05).(4) Compared to the control group, the levels of serum HA, LN, PCⅢ and IV-C were significantly increased in the hepatic fibrosis model group(all P<0.05). Compared to the model group, the levels of serum HA, LN, PC Ⅲand IV-C were significantly decreased in the Apelin 13(F13A) group(all P<0.05).(5) The lobules of liver of rats was integrate, hepatic cord arranged regularity, the structure of hepatic cord was integrate, the hepatic cords and sinusoids with central vein were arranged radially, therr was no obvious hyperplasia of collagen fiber and the degree of liver fibrosis was 0 in the control group. The lobules of liver was destroyed severely, hepatic cord arranged turbidly, there were many inflammatory cells and necrotic cells and fibrous connective tissue hyperplasia in the hepatic portal area, the false lobule was formatted and the degree of liver fibrosis was 3-4 in the hepatic fibrosis model group. Compared to the model group, the amount of degenerating cells and necrotic cells was significantly decreased, the degree of liver fibrosis was significantly reduced and the degree of liver fibrosis was 1-2 in the Apelin 13(F13A) group(P<0.05).(6) Compared to the control group, the levels of serum α-SMA and m RNA and protein expressions of TGF-β1 and α-SMA in the liver were significantly increased in the hepatic fibrosis model group(all P<0.05). Compared to the model group, the levels of serum α-SMA and m RNA and protein expressions of α-SMA in the liver were significantly decreased in the Apelin 13(F13A) group(all P<0.05).(7) Cell morphology of hepatic stellate cells(HSCs) in the control group was normal. The nuclear membrane and nucleolus of HSCs were clear. The autophagic vacuole and autophagy in the cytoplasm of HSCs were occasionally observated in the control group. The nucleus of HSCs was irregular in the hepatic fibrosis model group. The chromatin of nucleus of HSCs was partly pyknotic. The autophagy and autolysosome in the cytoplasm of HSCs were obviously observated in the hepatic fibrosis model group. The number of autophagy and autolysosome in the cytoplasm of HSCs was significantly increased compared with the control group in the Apelin 13(F13A) group. The chromatin of nucleus of HSCs was still partly pyknotic in the Apelin 13(F13A) group. The number of autophagy and autolysosome in the cytoplasm of HSCs was significantly decreased. Compared with the control group, the percentage of autophagic vacuole area and total area of cytoplasm in the hepatic fibrosis model group was significantly increased(P<0.05). Compared with the hepatic fibrosis model group, the percentage of autophagic vacuole area and total area of cytoplasm in the Apelin 13(F13A) group was significantly decreased(P<0.05).(8) Compared with the control group, the expressions of Beclin-1 and LC3-Ⅱ and the ratio of LC3-Ⅱ/LC3-Ⅰwere significantly increased, and the expression of p62 was significantly decreased in the model group(all P<0.05). Compared with the model group, the expressions of Beclin-1 and LC3 were significantly decreased and the expression of p62 was significantly increased in the Apelin 13(F13A) group(all P<0.05). ConclusionsAPJ antagonist Apelin 13(F13A) inhibits the hepatic fibrosis in rats. This mechanism may be related with inhibittion of autophagy and inhibition of activation of hepatic stellate cells. |
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