Molecular Mechanisms Of Effects Of Plumbagin On Human Hepatic Satellite Cells

Hepatic fibrosis is a pathological process of abnormal deposition of fibrous connective tissue in liver resulted from the loss of balance between the proliferation and degradation of extracellular matrix(ECM), like collagen protein, when hepatocytes necrosis and inflammation stimulation occur in liver; it is a reversible lesion. At present, it is considered that the activation and proliferation of hepatic satellite cells and the great production of ECM play key roles in the pathological mechanism of hepatic fibrosis. The activation and proliferation of HSC are subject to the regulation of many cytokines. Therefore, to block the pro-proliferation effect of cytokine shows promise as an effective therapeutic measure for hepatic fibrosis. In recent years, anti-hepatic fibrosis treatment with TCM has achieved greater progress.In the previous work funded by National Natural Science Foundation of China, this topic team has confirmed the anti-hepatic fibrosis effect of Baihuadan (Ceylon Leadword Root or Leaf) and the main active ingredient of Baihuadan (Ceylon Leadword Root or Leaf) for resisting hepatic fibrosis is plumbagin.Plumbagin, as the main active ingredient of Baihuadan, had robust effect on activating blood circulation to dissipate blood stasis. Leptin is a new cytokine with the effect of pro-hepatic fibrosis and recent studies show that leptin can induce the activation and proliferation of HSC.Objective This topic is to thoroughly explore the effects of plumbagin on biological features and the activation of signaling molecule in HSC after stimulation by external leptin at cellular and molecular level, which is mainly focused on its inhibition effect on activation, proliferation, secretion, apoptosis and collagen synthesis with the aim to determine the target sites and potential molecular mechanism of plumbagin in reducing the secretion of collagen by HSC-LX2.The actually mechanism of plumbagin in resisting hepatic fibrosis will be explored at cellular and molecular level, and thus to provide theoretical and experimental evidences for developing ethnic drugs in resisting hepatic fibrosis with characteristics of GuangXi region.Methods 1.To test the effects of plumbagin on the toxicities of HSC-LX2 and human embryonic hepatocyte (L-O2) by MTT method; to determine the content of SOD, MDA and LDH in supernate of HSC-LX2 by ELIASA after action of plumbagin.2. Test the secretion of leptin by ELISA method in culture supernate of HSC-LX2 cells; test the acting timing and concentration choice of leptin when acting with HSC-LX2; test changes of cell cycle by flow cytometry; test the effect of plumbagin on the expression of proteins related to HSC-LX2 cell cycle by Western blot ;test the effects of plumbagin on apoptosis of HSC-LX2 cells by V-FITC and PI double staining; observe the ultrastructure of apoptotic HSC cells by transmission electron microscope; test the expressions of apoptosis-associated proteins p53, Bax and Bcl-2 by cell immunohistochemistry.3. Test the effect of plumbagin on the expression of HSC-LX2TGF-β1 and other cytokines by ELISA, Real-time quantitative PCR and Western blot. 4. Test the secretion of type-1 collagen in supernate of HSC culture by western blot; observe the activation phase of OB-Rb, JAK2, STAT3, ERK1/2 after stimulation by leptin by Western blot; test the effects of ; test the effect of plumbagin on the expression of proteins related to HSC-LX2 signal transduction after stimulation by leptin by Western blot.Results1. The average IC50 of plumbagin to HSC-LX2 is 5.016μg/ml and 18.852μg/ml to human normal embryonic hepatocyte. The toxic selective index (SI) of Plumbagin to plumbagin is 3.76, which indicates a certain selective toxic effect of plumbagin towards HSC-LX2.2. There are no significant differences of the contents of SOD, MDA and LDH in supernate of low-, and middle-dose groups from blank control at 12h and 24h after addition of drug (p>0.05); there are significant differences in supernate of high-dose group from blank control (p<0.05); and there are significant differences in supernate of high-dose group from blank control at 48h after addition of drug (P<0.05 or P<0.01).The results show certain toxicity of plumbagin to human heptocytes L-O2 and the toxicity will be greater with higher concentration of plumbagin and longer acting duration. In light of the toxicity of plumbagin, 2umol/L and 8μmol/L of plumbagin are selected for subsequent experiments.1. The concentration of leptin auto-secreted by HSC is to decrease with time and to the minimum at 24h and recovery of original level at 48h (P>0.05). There is no significant difference between the concentrations at each point, which shows the ambiguity of the change of auto-secretion amount of leptin by HSC.2. Test the DNA content at each phase of cell cycle by PI staining for Flow cytometry: at 24h after addition of plumbagin, the percentage of HSC-LX2 cells at G0/G1 phase is apparent increased and the total percentage of HSC-LX2 cells at S phase+G2/M phase is apparent decreased; and with the increase of dose, the percentage of HSC-LX2 cells at G0/G1 phase becomes higher and the total percentage of HSC-LX2 cells at S phase+G2/M phase becomes lower, indicating dose-dependency. Plumbagin may block the transformation of HSCs from G0/G1 to S, G2/M phase and further inhibit the synthesis of DNA and cell division, which finally slow the cellular cycle progress and block cell proliferation.3. Compared to blank control, the stimulation by leptin may significantly increase the content of HSC Cyclin DI and Cyclin E, and block the expression of inhibiting protein P21; however, plumbagin may significantly decrease the level of Cyclin DI and Cyclin E and increase the expression of p21 protein.4. The results of flow cytometry show significant increased apoptotic rates of HSCs at 24h after addition of plumbagin to HSC-LX2; the apoptotic rates in 2 , 8μmol/L and colchicine groups are(3.07±0.30)%,(5.21±0.41)%,(10.1±1.08)% respectively, which are both significantly higher than blank control (1.40±0.13) %, (P<0.01).The result indicates that plumbagin can promote the apoptosis and necrosis of HSC-LX2.5. The results from electron microscope show that HSCs in blank control group have clear forms, intact nuclear membrane and cell membrane. There is lots of microvillus on cell surface. After addition of plumbagin, a few of necrotic cells and many apoptotic cells of various degrees can be observed in HSC-LX2.6. Small quantities of bax, Bcl-2 and p53 are expressed and a few of brownish particles may be seen after stimulation by leptin; after intervention by 2 and 8μmol/l of plumbagin, the expression of pro-apoptotic genes , Bax and P53, are significantly increased and the expression of anti-apoptotic gene, Bcl-2, is significantly decreased. All these differences are statistically significant compared to leptin-stimulation group (P<0.01).1. Test the secretion of cytokins in supernate by ELISA method. Compared to blank control group, the contents of TGF-β1,α-SMA, TNF-αand PDGF-BB are significantly increased (P<0.01). Compared to leptin group, the contents of TGF-β1,α-SMA, TNF-αand PDGF-BB in each plumbagin groups are significantly decreased (P<0.05 or P<0.01). Additionally, the activity inhibition effect of plumbagin to TGF-β1,α-SMA, TNF-αand PDGF-BB becomes stronger with the increasing concentration of plumbagin.2. Acquire the expression amount of TGF-β1,α-SMA, TNF-αand PDGF-BB relative to leptin group through 2-△△CT formula. The result of real-time PCR shows: certain quantity of expressions of TGF-β1,α-SMA, TNF-α, PDGF-BB mRNAa are observed in HSC of blank control group; at 24h after addition of plumbagin and colchicin, the quantities of TGF-β1,α-SMA, TNF-αand PDGF-BB mRNA in activated HSC-LX2 are all significantly decreased from leptin group(P < 0.05 or P <0.01).3. Result of Western blot shows that, after incubation of HSC-LX2 with leptin for 24h, the expressions of HSC-LX2TGF-β1,α-SMA and PDGF-BB are all apparently increased and the expression of TNF-αis slightly increased. After incubation of HSC-LX2 with plumbagin for 24h, the expressions of HSC-LX2TGF-β1,α-SMA and PDGF-BB are all decreased and the expression of TNF-αis not affected apparently.1. Test the expression level of OB-Rb at different point after addition of leptin of optimal stimulation concentration (100 ng/ml), and the result shows that leptin (100 ng/ml) can induce the expression of HSCOB-Rb with the manner of phase changing. At 6h after leptin stimulation, the expression of OB-Rb is increased without statistical significance; at 24, it reaches to the peak concentration (P<0.01); at 36h, it remains relative high activity (P<0.01); and after 48h, it returns to the initial level. 2. The study on the regulation effect of plumbagin on the expression of OB-Rb after stimulation by leptin through Western blot shows that leptin can significantly stimulate the expression of OB-Rb receptor protein and the treatment by plumbagin can significantly decrease the expression of OB-Rb receptor protein.3. The study on the effects of plumbagin on the expression of JAK2 and PJAK2 through western blot, leptin can significantly increase the expression of JAK2 and pJAK2 compared to blank control group, however, the treatment by plumbagin can apparently decrease the expression level of pJAK2 (P<0.01) and has no significant effect on the expression of JAK2 (P<0.05).4. The study on the regulation effect of plumbagin on the expression of STAT3 and pSTAT3 after stimulation of HSC-LX2 by leptin through Western blot, leptin can significantly increase the expression of STAT3 and pSTAT3 compared to blank control group (P<0.01), however, the treatment by plumbagin can apparently decrease the expression level of STAT3 and pSTAT3 receptor proteins (P<0.05).5. The study on the regulation effect of plumbagin on the expression of ERK1/2 and pERK1/2 after stimulation of HSC-LX2 by leptin through Western blot, the result of western blot shows that leptin can significantly increase the abundances of ERK1/2 and pERK1/2 compared to blank control group(P<0.01 or P<0.05). However, the treatment by plumbagin can apparently decrease the expression level of ERK1/2 and pERK1/2 proteins (P<0.01 or P<0.05).6. The study on the effect of plumbagin on the expressions of HSC-LX2 MMP-1 and MMP-13 after stimulation by leptin through Western blot, the analysis result shows that leptin can significantly decrease the expression of HSC-LX2MMP-1 and has no effect on the expression of MMP-13 compared to blank control group(P<0.05). However, after addition of plumbagin for 24h, the expression level of MMP-1 protein is increased and the expression of MMP-13 has no significantly alteration (P>0.05).7. The study on the effect of plumbagin on the expression of HSC-LX2 type-1 collagen after stimulation by leptin through ELISA, the result shows that the content of type-1 collagen in HSC culture supernate is significantly increased compared to blank control group (P<0.01). However, the content of type-1 collagen in HSC culture supernate in each concentration group of plumbagin is apparently decreased compared to leptin group and the difference is of statistical significance(P<0.05).8. In the test of the effect of plumbagin on the expression of HSC-LX2 type-1 collagen after stimulation by leptin through western blot, the result shows that leptin can increase the proliferation and expression of type-I collagen compared to control group (100 ng/ml) and the proliferation and expression of type-I collagen in HSCs are significantly inhibited in plumbagin treatment group.Conclusions1. Plumbagin shows some toxicity to human normal hepatocytes L-O2 and the toxicity will be greater with higher concentration of plumbagin and longer acting duration.2. External leptin can promote the proliferation and activation of HSC with dose- and time-dependent manner. Plumbagin can inhibit the stimulatory effect of external lepton on the proliferation of HSC-LX2, which indicates that plumbagin can decrease the inflammation amplification effect and such effect may inhibit the development of hepatic fibrosis.3. Plumbagin can block HSC-LX2 entering S phase from HSC-LX2 and in this way to inhibit the proliferation induced by leptin. Its mechanism may be related to the decrease of the expressions of cyclin D1 and cyclin E1 and the increase of P21 protein.4. Plumbagin shows certain functions in promotions of apoptosis and necrosis of HSC-LX2. The actual mechanism of plumbagin in induction of HSC apoptosis relies on up-regulating the expressions of p53 and bax and down-regulating the expression of Bcl-2.5. Plumbagin can inhibit the stimulation of external leptin on the secretions of HSC-LX2 TGF-β1,α-SMA, PDGF-BB at the level of gene and protein; it also can inhibit the expression of TNF-αat the level of gene.6. Plumbagin can inhibit the proliferation of HSC induced by external leptin at the level of membrane receptor and decrease the expression of HSC-LX2 OB-Rb protein.7. Plumbagin can decrease the protein phosphorylation level of JAK2 and STAT3, and inhibit the proliferation of HSC-LX2 induced by external leptin through blocking the signal transduction of JAK2-STAT3 in HSC-LX2.8. Plumbagin also can block the protein phosphorylation of the protein factor, ERK, of the leptin-dependent MAPK signal transduction, and by this way, it can significantly inhibit the proliferation of HSC induced by HSC MAPK signal transduction.9. Plumbagin can inhibit the expressions of type-I collagen and MMP-1.