Effect of adiponectin on hepatic stellate cell contraction induced by endothelin-1 and its mechanism of action

Objective To observe the effect of exogenous adiponectin on hepatic stellate cell( HSC) contraction induced by endothelin- 1( ET- 1),and to investigate the possible mechanism of action of adiponectin in this process. Methods Collagen lattice was applied to observe the effects of adiponectin( 0. 25,0. 5 μg / ml) alone and adiponectin( 0. 5 μg / ml) combined with nitro- L- arginine methyl ester( L- NAME) on the contraction of HSC- T6 cells induced by ET- 1. Quantitative real- time PCR and Western blot were applied to measure the mRNA and protein expression levels of ET- 1 in HSC- T6 cells after the action of adiponectin( 0. 5 μg / ml),and enzyme- linked immunosorbent assay was applied to measure the protein expression level of ET- 1 in HSC- T6 cell culture medium after the action of adiponectin( 0. 5 μg / ml). Quantitative real- time PCR and Western blot were also applied to measure the mRNA and protein expression levels of inducible nitric oxide synthase( i NOS) in HSC- T6 cells after the combined action of adiponectin( 0. 25,0. 5 μg / ml) and ET- 1,as well as the protein expression levels of AMPK,p- AMPK,Akt,and p- Akt in HSC- T6 cells. Analysis of variance was applied for comparison of continuous data between groups and pairwise comparision between any two groups was made by Dunnett test. Results The collagen lattice experiments showed that compared with the blank control group,the ET- 1 group had a significantly reduced gel area [( 24. 8 ± 7. 3) % vs( 71. 9± 4. 1) %,P < 0. 01]; after the treatment with adiponectin( 0. 5 μg / ml),the contraction induced by ET- 1 was significantly inhibited in the ET- 1 group [( 52. 7 ± 20. 6) % vs( 24. 8 ± 7. 3) %,P < 0. 05]; L- NAME( 5 mmol / L) partially counteracted the inhibitory effect of adiponectin( P > 0. 05). Adiponectin( 0. 5 μg / ml) inhibited the mRNA and protein expression levels of ET- 1 in HSC- T6 cells( P < 0. 05)and the protein expression level of ET- 1 in cell supernatants; after L- NAME was added,the above inhibitory effect of adiponectin was partially reversed. The mRNA expression of i NOS was detected in HSC- T6 cells,and the mRNA expression level of i NOS was significantly reduced after ET- 1 was added( P < 0. 01); after addition of ET- 1 and adiponectin,the mRNA expression level of i NOS was significantly increased compared with the ET- 1 group,and tended to increase with the increasing concentration of adiponectin; the protein expression level of i NOS had a similar trend. At 24 hours after ET- 1 treatment,the expression level of p- AMPK in HSC- T6 cells decreased significantly,and after adiponectin was added,the expression level of p- AMPK increased significantly with the increasing concentration of adiponectin; at 24 hours after ET- 1 treatment,the expression level of p- Akt in HSC- T6 cells increased significantly,and after adiponectin was added,the expression level of p- Akt decreased gradually with the reduction in the concentration of adiponectin. Conclusion Adiponectin can inhibit the HSC contraction induced by ET- 1,and its mechanism of action is to activate AMPK,increase the synthesis of nitric oxide,reduce the synthesis and secretion of ET- 1,and block the Akt signaling pathway. The inhibitory effect of adiponectin on HSC contraction may be one of its anti- fibrosis mechanisms.