| Backgrounds: Hepatic cirrhosis is the end stage of various kinds of chronic liver diseases.There are no effective drugs in anti-fibrotic treatments clinically and liver transplantation remains the final method for the patients.It has been proved that liver fibrosis is a reversible process in the repair of liver damage.This provides the theoretical basis for the treatment of liver cirrhosis.There are many cells and cytokines involved in liver fibrosis process,they cause excessive extracellular matrix(ECM)accumulation and deposition.Tremendous ECM separates normal liver into nodules,and eventually lead to liver cirrhosis.Hepatic stellate cells(HSCs)are nonparenchymal cells living in the space of Disse.In normal liver,HSCs are vital to various kinds of liver function.Hepatic stellate cells are activated and transformed into myofibroblasts in liver damage.They synthesize and secrete large amounts of extracellular matrix,and inhibit the activity of metalloproteinases to degrade extracellular matrix,eventually lead to the accumulation of ECM.Hence,HSCs could be a target of the therapy for hepatic liver fibrosis.Transforming growth factor-?1(TGF-?1)is a major cytokine to induce HSCs activation.It has been proved that the interference of TGF-?1/Smad signaling pathway could ameliorate liver fibrosis.Melatonin(MEL)is a kind of hormone secreted by the pineal gland and other organs or tissues.Melatonin is well known as mediating circadian rhythm,sleep and aging.It has been confirmed that melatonin has the property to protective liver fibrosis.The hepatic protective ability of melatonin may be related to its antioxidant,anti-inflammatory and anti-fibrotic effects,and melatonin may also be involved in the regulation of apoptosis signal and inhibition of hepatic stellate cells.In our preliminary experiment,melatonin could alleviate carbon tetrachloride-induced hepatic fibrosis in rats,and it may be related to its antioxidant effect and blocking TGF-?1/Smad signaling pathway.However,most previous studies were in vivo experiments,and the effect of melatonin on TGF-?1/Smad signaling pathway has not been confirmed in hepatic stellate cells.In our experiment,we speculated that melatonin could inhibit the activation of HSCs,which might be related to TGF-?1/Smad signaling pathway.Methods: The HSC-T6 cells were divided into five groups: control group,model group and three experimental groups.After cells attachment,they were cultured in FBS-free medium and treated with transforming growth factor-?1(TGF-?1,5ng/ml)excepted the control group,and melatonin was added immediately with different concentrations(1nmol /L,1?mol/L,0.1mmol/L)in three experimental groups.After drugs incubation for 48 h,MTT assay was performed to assess the proliferation of cells,immunocytochemistry and western blot were used to assess the expression levels of a-SMA and Smads in TGF-?1/ Smad pathway.Results: Compared to the control group,the expression of a-SMA in TGF-?1-treated group was dramatically elevated,and the proliferation of HSC-T6 cells were significantly stimulated by TGF-?1(p <0.05).After being treated with TGF-?1 and melatonin,the cells proliferation was obviously inhibited by melatonin(p <0.05).The results of immunocytochemistry showed that ?-SMA and Smad2/3 were mainly located in the cytoplasm,and p-Smad2/3 was located almost in the nucleus.Melatonin could significantly attenuated the expression of a-SMA,Smad2/3 and p-Smad2/3 compared to the model group(p <0.05).The expressions of Smad2/3 and p-Smad2/3 analyzed by western blot were consistent with immunocytochemistry.Western blot analysis was performed to assess the expressions of Smad7,the result showed that melatonin significantly elevated the expression levels of Smad7(p <0.05).Conclusions:(1)The proliferation of HSC-T6 cells and the expression of a-SMA were significantly stimulated by TGF-?1,hence TGF-?1 is the activator of HSCs.(2)Melatonin could directly inhibit the HSC-T6 cells activation and proliferation,and it might be related to blocking TGF-?1/Smad signaling pathway. |
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