Mechanistic Studies On Anti-Liver Fibrosis Of Curcumin Targeting Cellular Senescence

When liver suffer from sustained damage caused by various pathogenic factors will result in chronic liver disease,which in turn lead to hepatic fibrosis.Hepatic fibrosis is a pathological process which the imbalance of the production and degradation of extracellular matrix.And without effective treatment,liver fibrosis will develop into cirrhosis and even liver cancer.As is known to all,activation of hepatic stellate cells(HSCs)and overproduction of extracellular matrix(ECM)are the central events during liver fibrogenesis.Thus inhibition of HSC activation and clearance of activated HSC is an effective strategy for the treatment of liver fibrosis.A lot of researches have shown that regulation of HSC proliferation,autophagy,apoptosis and pyroptosis can affect the fate of HSC.Therefore,the reduction of activated HSCs is critical to treat hepatic fibrosis.Recently,HSC senescence have found in the repair phase of liver fibrosis in hepatitis patients and animal models.Therefore,regulation of HSC senescence is regarded as a novel and probable way to reverse liver fibrosis.Currently,there is no effective chemical antifibrotics for prevention and treatment of hepatic fibrosis.More and more basic and clinical studies have shown that traditional Chinese medicine has the exact effect in improvement of liver fibrosis.In recent years,the active ingredients of traditional Chinese medicine have become the main source of anti-liver fibrosis medicine.Our research group has committed to the studies of natural product curcumin over the long-term.Prior studies focused on the antifibrotic effect and the molecular mechanism of curcumin,which is the primary bioactive component of Curcuma longa L.,have indicated that curcumin can inhibit the proliferation of activated HSC,epithelial-mesenchymal transition and induce autophagy and apoptosis of activated HSC.By vitue of the excellent anti-fibrotic effect of curcumin,we propose a scientific hypothesis that curcumin may ameliorate hepatic fibrosis through stimulating the senescence of activated HSC.We first observed the phenomenon of HSC senescence during the development and repair of liver fibrosis.We used CCl4 to build the model of hepatic fibrosis for four weeks in mice.From the fifth week,no intraperitoneal injection of CCl4 allowed for a natural repair for four weeks in mice.At the end of experiments,the liver of mice was used for histopathological studies and detection of a series of markers of HSC activation and senescence.H&E and sirius red staining showed that with the extension of CCl4 modeling time,liver injury aggravated and collagen deposition increased.However,liver injury reduced and collagen deposition decreased with the extension of repair period.Immunofluorescence staining and western blot analyses were used to detect the expression of activated HSC markers of al(Ⅰ)-procollagen,α-SMA and senescence markers of p16,p21 and Hmgal.The results indicated that activated HSCs are gradually increased and senescent HSCs are not change during CCl4 modeling period.However,senescent HSCs are gradually increased and activated HSCs are gradually decreased during repair period of liver fibrosis.These data collectively suggested that the existence of senescent HSC in the repair period of liver fibrosis.And induction of senescence of activated HSC may be an effective way to ameliorate liver fibrosis.The subsequent work was aimed to evaluate the effect of curcumin on senescence of activated HSCs and to elucidate the underlying mechanisms.We used CCl4 to build the model of hepatic fibrosis and given curcumin treatment in rat.Curcumin inhibited the expression of activated HSC marker α-SMA and promoted the expression of senescence marker Hmgal in rat fibrotic liver.In addition,curcumin increased the number of senescence-associated β-galactosidase-positive HSCs in vitro.At the same time,curcumin induced HSC senescence by elevating the expression of senescence markers p16,p21 and Hmgal,concomitant with reduced abundance of HSC activation markers α-smooth muscle actin and α1(Ⅰ)-procollagen in cultured HSCs.Moreover,curcumin affected the cell cycle and telomerase activity.We further demonstrated that p53 pharmacological inhibitor pifithrin-a(PFT-α)or transfection with p53 siRNA abrogated the curcumin induced HSC senescence in vitro.Meanwhile,curcumin disruption of p53 leading to increased senescence of activated HSCs was further verified in vivo.Further studies indicated that curcumin promoted the expression of p53 through a PPARγ activation dependent mechanism.Moreover,promoting PPARγ transactivating activity by a PPARγ agonist 15d-PGJ2 markedly enhanced curcumin induction of senescence of activated HSCs.However,the PPARγ antagonist PD68235 eliminated curcumin induction of HSC senescence.Taken together,our results provided a novel insight into the mechanisms underlying curcumin inhibition of HSC activation through inducing senescence.We need deep investigated the whereabouts of senescent HSC in the next stage.Senescent cell could be killed by immune cell when the cell happened to senesce,such as senescent hepatocytes are easily cleared by lymphoid T cells.It has been shown that senescent HSCs are easily killed by NK cells.We investigated how NK cells target senescent HSC and assessed the effect of this process on liver fibrosis.In order to realize how NK cells target senescent cells,we performed a cytotoxicity assay whereby normal and senescent cells were co-cultured with the human NK cell line(NK-92)in vitro.We found that senescent HSC induced by curcumin are susceptible to NK cells killing,due to the increased expression of NK cell activating ligand major histocompatibility complex class I chain-related genes A(MICA)and UL16-binding proteins 2(ULBP2),but not Poliovirus Receptor(PVR).Further studies displayed that the interaction between NK cells and senescent LX2 cells stimulated granule exocytosis.Moreover,the inhibition of granule exocytosis weakened the cytotoxicity of NK cells and promoted the accumulation of senescent LX2 cells.Therefore,these aggregated data indicated that NK cells mediated clearance of senescent LX2 cells and granule exocytosis could play a protective role in the improvement of liver fibrosis.Based on the above findings,we also explored the effect of curcumin on the senescence of hepatoma cell.We detected cellular senescence by SA-β-gal staining after curcumin administration in cultured HepG2 cells.The results illustrated curcumin induced the senescence of HepG2 cells.Western blot was used to test the expression of H3K9me3,γ-H2AX,p16,p21 and Hmgal in HepG2 cells.The results showed that the expression of senescent markers significantly increased under curcumin treatment.Flow cytometry showed that curcumin blocked the cell cycle to G1 phase in HepG2 cells.Real-time PCR and western blot showed that curcumin inhibited the expression of telomerase reverse transcriptase subunit TERT and induced the expression of telomere-related protein TRF1,but no significant changes in TRF2 mRNA and protein levels.Immunofluorescence staining showed that curcumin induced heterochromatin formation in HepG2 cells.These results initially indicated that curcumin reduced the number of hepatoma cell by promoting the senescence of hepatoma cell.It therefore could be inhibit the formation of liver cancer.In summary,the current study clarified the regulatory mechanism and molecular targets of curcumin-induced activated HSC senescence,which will contribute to reveal the important role of HSC senescence in anti-liver fibrosis.On this basis,the mechanism of NK cell clearance of curcumin-induced senescent HSC was confirmed.In addition,we also used the vitro experiments to reveal the role of curcumin in regulation of the senescence of hepatoma cell.Our results provide a deeper theoretical and experimental basis for revealing the role of curcumin,an active ingredient of traditional Chinese medicine,in treatment of liver fibrosis and hepatic carcinoma and making it as a potential anti-hepatic fibrosis medicine.