Skullcapflavone I from Scutellaria baicalensis induces apoptosis in activated rat hepatic stellate cells

The therapeutic goal in liver fibrosis is the reversal of fibrosis and the selective clearance by apoptosis of hepatic stellate cells (HSCs), which play a central role in liver fibrogenesis. In this study, the apoptotic effect of wogonin, oroxylin A, 2',5,6',7-tetrahydroxyflavone, skullcapflavone I, and baicalein, isolated from the dried root of Scutellaria baicalensis, was investigated in activated rat HSCs, T-HSC/Cl-6 cells transformed with the Simian virus 40. Among the isolated compounds, skullcapflavone I (20 microM for 24 h) significantly induced apoptosis in activated rat HSCs while there was no change in the cell viability of hepatocytes. Skullcapflavone I increased caspase-3 and -9 activities accompanied by the proteolytic cleavage of poly(ADP-ribose) polymerase. Specific inhibitors of caspase-3 and caspase-9 prevented the apoptotic process induced by skullcapflavone I. From these results, skullcapflavone I from S. baicalensis selectively induced apoptosis in T-HSC/Cl-6 cells via caspase-3 and caspase-9 activation.     

A ginsenoside metabolite, 20-O-beta-D-glucopyranosyl-20(S)-protopanaxadiol, triggers apoptosis in activated rat hepatic stellate cells via caspase-3 activation

We investigated the apoptotic effects of the protopanaxadiol ginsenosides, Rb (1) and Rb (2), and their intestinal bacterial metabolite, 20-O-beta-D-glucopyranosyl-20(S)-protopanaxadiol (M1), and of the protopanaxatriol ginsenoside, Rg (1), and its intestinal bacterial metabolite, 20(S)-protopanaxatriol, in activated rat hepatic stellate cells (HSCs) transformed by Simian virus 40 (T-HSC/Cl-6). As HSCs play a central role in liver fibrosis, agents that selectively induce apoptosis of HSCs could be used to treat this disease. Apoptosis was measured using cell viability tests, DNA fragmentation analysis, and immunoblot analysis of poly(ADP-ribose) polymerase cleavage. M1 (40 microM for 24 h) significantly induced apoptosis in activated rat HSCs. M1 induced apoptosis in a dose-dependent manner as shown by DNA fragmentation, an increased population of cells in the sub-G1 phase, and reduced mitochondrial transmembrane potential. M1 induced caspase-3 activity in a dose- and time-dependent manner. A specific inhibitor of caspase-3 prevented induction of apoptosis by M1 as shown by DNA fragmentation analysis. It is concluded that M1 induces apoptosis in T-HSC/Cl-6 cells via caspase-3 activation.     

Apoptotic effect of propyl gallate in activated rat hepatic stellate cells

Hepatic stellate cells (HSCs) play a central role in liver fibrosis. Inhibition of HSC growth and induction of apoptosis have been proposed as therapeutic strategies for the treatment and prevention of liver fibrosis. Propyl gallate (PG) is an antioxidant widely used in processed foods, cosmetics and medicinal preparations. However, the anti-fibrotic effect of PG in liver injury is unclear. In this study, we investigated whether PG could induce apoptosis in activated HSCs. Treatment of activated HSCs with PG inhibited cell viability in a dose- and time-dependent manner. PG induced apoptosis as demonstrated by morphological changes, poly(ADP-ribose) polymerase (PARP) cleavage, caspase-3 cleavage, increased Bad expression, and decreased Bcl-2 protein expression. Through stimulation of the activation of c-Jun NH2-terminal protein kinase (JNK) and p38 mitogen-activated protein kinases (MAPK) by PG treatment, we demonstrated that JNK and p38 MPAK are not involved in PG-induced apoptosis using their specific inhibitors. Taken together, these findings indicate that PG induces apoptosis in activated HSCs. The potential anti-fibrotic effect of PG warrants further evaluation.     

Alteration of the ERK5 pathway by hydroxysafflor yellow A blocks expression of MEF2C in activated hepatic stellate cells in vitro: Potential treatment for hepatic fibrogenesis

Context: Hepatic fibrosis ultimately leads to cirrhosis if not treated effectively. Hepatic stellate cells (HSC) are a main mediator of hepatic fibrosis through the accumulation of extracellular matrix proteins. Suppression activation of passaged HSC has been proposed as therapeutic strategies for the treatment and prevention of hepatic fibrosis.

Objective: To evaluate the effect of hydroxysafflor yellow A (HSYA), an active chemical compound derived from the flowers of Carthamus tinctorius L. (Compositae), on HSC inhibition, and to begin elucidating underlying mechanisms.

Materials and methods: Primary HSCs were isolated from rats by in situ pronase/collagenase perfusion. Culture-activated HSCs were treated with or without HSYA at 30?μM in the presence or absence of PD98059 for 48?h, and then cell proliferation was measured by MTS assays. Messenger RNA (mRNA) expression was quantified by polymerase chain reaction, and protein was quantified by Western blots or enzyme-linked immunosorbent assays.

Results: HSYA significantly inhibits culture-activated HSC proliferation in a dose-dependent and time-dependent manner with an IC₅₀ value of 112.79?μM. HSYA (30?μM) induce the suppression of HSC activation, as indicated by decreases in contents of type I alpha collagen in HSC-cultured media and expression of α-smooth muscle actin protein in culture-activated HSC by 55 and 71%, respectively. HSYA (30?μM) also caused significant decreases in mRNA expression of type III alpha collagen in HSC by 28%. HSYA (30?μM) suppresses myocyte enhancer factor 2?C (MEF2C) expression both at its mRNA and protein levels by 60 and 61%, respectively. Further study demonstrated that HSYA (30?μM) caused significant decreases in p-ERK5 by 49%. Blocking extracellular signal-regulated protein kinase 5 (ERK5) activity by XMD 8--92, an ERK5 inhibitor, markedly abrogated the inhibitive effects of HSYA on HSC activation, and blocked the HSYA-mediated MEF2C down-regulation.

Conclusions: HSYA suppress HSC activation by ERK5-mediated MEF2C down-regulation and makes it a potential candidate for prevention and treatment of hepatic fibrogenesis.     

Honokiol induces apoptosis via cytochrome c release and caspase activation in activated rat hepatic stellate cells in vitro

The therapeutic goal in liver fibrosis is the reversal of fibrosis and the selective clearance of activated hepatic stellate cells (HSCs) by inducing apoptosis. Over the past several years, we have screened for natural products that mediate apoptosis in activated HSCs. Among the candidate compounds, honokiol, isolated from Magnoliae cortex, was found to induce apoptotic death in activated rat HSCs, while there was no cell viability change in hepatocytes, at concentrations of 12.5-50 microM. Apoptosis was identified by DNA fragmentation, activation of caspase-3 and -9, and the proteolytic cleavage of poly(ADP-ribose) polymerase, down-regulation of bcl-2 and the release of mitochondrial cytochrome c into the cytoplasm.     

Smad2 increases the apoptosis of activated human hepatic stellate cells induced by TRAIL

The activation of hepatic stellate cells (HSCs) plays a critical role in the development of liver fibrosis. The induction of apoptosis in activated HSCs during the recovery phase of hepatic fibrosis represents a potential anti-fibrotic therapy. We have previously shown that Smad2 protects against hepatic fibrogenesis; however, the role of Smad2 in the regulation of activated HSC apoptosis remains unknown. We hypothesized that Smad2 regulates the apoptosis of activated HSCs, leading to the resolution of liver fibrosis. To test this hypothesis, the livers of rats were harvested at 0 and 4 weeks after hepatic fibrosis was established by CCl₄ injection. Furthermore, TGF-β1-activated HSCs were treated with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) following the silencing or overexpression of Smad2. Both the phosphorylation of Smad2 and TRAIL were detected in fibrotic liver tissues. The results of TUNEL and α-SMA double-staining showed an increase in the apoptosis of activated HSCs during the spontaneous recovery phase. The knockdown of Smad2 reduced TRAIL-induced apoptosis in TGF-β1-activated human LX-2 cells and resulted in an increased expression of α-SMA and collagen I (Col. I). In contrast, the overexpression of Smad2 increased TRAIL-induced HSC apoptosis and reduced the expression of α-SMA and Col. I. The mechanisms underlying these findings were associated with the Smad2-mediated down-regulation of X-linked inhibitor of apoptosis protein (XIAP), resulting in enhanced caspase-3 activity and apoptosis. In conclusion, Smad2 enhances TRAIL-induced apoptosis in activated HSCs, which facilitates the resolution of hepatic fibrosis.     

Quercetin stimulates mitochondrial apoptosis dependent on activation of endoplasmic reticulum stress in hepatic stellate cells

Context: Activation of hepatic stellate cells (HSCs) is a hallmark of liver fibrosis. Quercetin has benefits for liver fibrosis, but the mechanisms are unknown.

Objective: We investigated the quercetin effect on HSC survival and the role of endoplasmic reticulum stress (ERS).

Materials and methods: Rat HSCs and LO2 hepatocytes were treated with quercetin (0.5–120?μM) for 24?h. Quercetin (10–40?μM) effects on apoptosis for 24?h were analyzed by flow cytometry and TUNEL staining. Quercetin (10–40?μM) effects on the expression of Bcl-2, caspase-9, caspase-3, PARP-1, PERK, IRE1, ATF6, calnexin and CHOP for 24?h were analyzed by Western blot. Quercetin (10–40?μM) effects on mRNA expression of calnexin and CHOP for 24?h were analyzed by Real-time PCR.

Results: Quercetin at concentrations greater than 20?μM significantly inhibited HSC proliferation (IC₅₀ 27.2?μM), but did not affect hepatocyte growth until 80?μM (IC₅₀ 68.5?μM). Quercetin stimulated HSC apoptosis and the apoptotic rate reached 40% at a concentration of 40?μM (EC₅₀ 51.6?μM). Quercetin induced downregulation of Bcl-2 and upregulation of Bax, and increased cytochrome C in the cytoplasm in HSCs. The cleaved forms of caspase-9, caspase-3 and PARP-1 were also increased by quercetin. Furthermore, quercetin elevated mRNA and protein expression of calnexin and CHOP in HSCs but not in hepatocytes. Quercetin also increased phosphorylation of PERK and IRE1 and ATF6 cleavage. However, ERS inhibitor salubrinal significantly abrogated quercetin induction of HSC apoptosis.

Conclusion: Quercetin activated ERS pathway in HSCs leading to apoptosis. We characterized an ERS-mediated mechanism for quercetin as a promising antifibrotic agent.     

Cucurbitacin E induces apoptosis and attenuates activation of hepatic stellate cells via PI_3K/Akt-AMPK-mTOR signaling pathway

OBJECTIVE Hepatic fibrosis is a wound-healing response for injury.Activated hepatic stellate cells(HSCs)are the preferred targets of anti-hepatic fibrotic therapies.cucurbitacin E(CuE)is,one well-known natural compound derived from traditional Chinese medicine,used in Asian countries for the prevention and treatment of hepatic disease.Therefore,the present study elucidated the mechanism of CuE on inducing apoptosis and attenuating hepatic fibrosis towards activated HSCs.METHODS The murine HSC(tHSC/Cl-6)cell line were incubated in 96-well plates and treated with TNF-αand CuE at various concentrations and indicated times.Cell viability was assessed with MTT assay.Another,t-HSC/Cl-6 were incubated in 6-well plates and also treated with TNF-α,CuE,AICAR or metformin for the indicated time and concentration.Cell protein and mRNA were prepared using kit and relevant signaling were detected by Western blotting and RT-PCR.RESULTS CuE inhibited cell proliferation of activated HSC/T-6cells in concentration-and time-dependent manner.CuE triggered the activation of caspase-3,cleaved the PARP,ration of bcl-2/bax,and cytochrom c protein in a time-and concentration-dependent manner.CuE decreased p-Erk/MAPK without effects on p-p38 and p-JNK.CuE inhibited the protein and mRNA expressions ofα-SMA,TIMP-1 and collagenⅠ in activated HSC-T6.CuE broadly blocked p-PI3 K,p-Akt,p-mTOR and p-p70S6 Kexpressions.CuE significantly increased phosphorylated AMPK expression as well as AICAR and metoformin.And metformin showed significantly higher activation of AMPK than AICAR.Ability of CuE on activation of AMPK was between AICAR and metformin.It′s also found that CuE significantly decreased p-mTOR as well as AICAR and metformin.CONCLUSION CuE could modulate HSC survival and activation as a potential anti-fibrotic agent for liver fibrosis treatment.The findings demonstrate that CuE induced HSC apoptosis via ERK/MAPK and PI3K/Akt-AMPK-mTOR signaling.     

2',4',6'-Tris(methoxymethoxy) chalcone induces apoptosis by enhancing Fas-ligand in activated hepatic stellate cells

Suppression of hepatic stellate cell (HSC) activation and proliferation, and induction of apoptosis in activated HSCs have been proposed as therapeutic strategies for the treatment and prevention of the hepatic fibrosis. We previously showed that 2′,4′,6′-tris(methoxymethoxy) chalcone (TMMC), a synthesized chalcone derivative, inhibits platelet-derived growth factor-induced HSC proliferation at 5–20 μM. Here, we showed that TMMC induces apoptosis in activated HSCs at higher concentrations (30–50 μM), but is not cytotoxic to primary hepatocytes. Moreover, TMMC induces hyperacetylation of histone by inhibiting histone deacetylase (HDAC) in activated HSCs. Interestingly, TMMC treatment remarkably increased Fas-ligand (FasL) mRNA expression in a dose-dependent manner. Cycloheximide treatment reversed the induction of TMMC on apoptosis, indicating that de novo protein synthesis was required for TMMC-induced apoptosis in activated HSCs. In addition, FasL synthesis by TMMC is closely associated with maximal procaspase-3 proteolytic processing. In vivo, TMMC reduced activated HSCs in CCl₄-intoxicated rats during liver injury recovery, as demonstrated by α-smooth muscle actin expression in rat liver. TMMC treatment also resulted in apoptosis, as demonstrated by cleavage of poly(ADP-ribose) polymerase in rat liver. In conclusion, TMMC may have therapeutic potential by inducing HSC apoptosis for the treatment of hepatic fibrosis.     

Molecular mechanisms of ZD1839 （Iressa）-induced apoptosis in human leukemic U937 cells

Aim： To investigate the molecular mechanisms of ZD 1839-induced apoptosis in human leukemic U937 cells. Methods： The inhibition of human leukemic U937 cell growth was assessed by 3-（4,5-dimethyl-2-thiazolyl）-2,5-diphnyl-2H-tetrazolim bromide （MTT） assays, lactate dehydrogenase （LDH） release, and cell cycle distribution. The expression of anti- and pro-apoptotic proteins was detected by Western blot analysis. Results： This study demonstrated that ZD1839 induced apoptosis in leukemic U937 cells by the downregulation of Bcl-2, caspase activation and subsequent apoptotic features. Cotreatment with ZD 1839 and the caspase- 3 inhibitor z-DEVD-fmk blocked apoptosis, indicating that caspase-3 activation is at least partially responsible for ZD 1839-induced apoptosis. The ectopic expression of Bcl-2 attenuated caspase-3 activation, PARP cleavage, and subsequent indicators of apoptosis, including sub-G1 DNA content and LDH release. These results indicate that the downregulation of Bcl-2 plays a major role in the initiation of ZD1839-induced apoptosis, and that the activation of a caspase cascade is involved in the execution of apoptosis. Furthermore, ZD1839 treatment triggered the activation of p38 mitogen-activated protein kinase （MAPK） and the downregulation of c-Jun-N-terminal kinase （JNK）, extracellular signal-regulated kinase （ERK） and phosphatidyl inositol 3-kinase （PI3K）/Akt. The inhibition of the ERK and PI3K/Akt pathways also significantly increased cellular death. Conclusion： ZD 1839 activated caspase-3 and the inhibited Bcl-2 in human leukemic U937 cells through the downregulation of the ERK and PI3K/Akt pathways.     

PD98059 triggers G1 arrest and apoptosis in human leukemic U937 cells through downregulation of Akt signal pathway

MEK/ERK pathways are frequently activated in acute myelogenous leukemia, and this signal pathway's inhibitor has made it an interesting candidate for cancer chemotherapy. Little is known, however, about the effects of cellular and molecular mechanisms on human leukemic U937 cells. In the present study, we found that treatment with PD98059 significantly arrests the G1 phase through up-regulation of cyclin-dependent kinase (Cdk) inhibitor, and produces morphological features of apoptosis in U937 cells, which were associated with poly(ADP-ribose)polymerase (PARP) cleavage and PLC-gamma1 degradation. PD98059 also decreased the Cdk-2, Cdk-4, cyclin D1, and cyclin E expression, and increased high levels of the mitotic inhibitors p16(INIa), p21(Waf1), and p27(Kip1). Also, Bcl-2's overexpression and a caspase-3 inhibitor z-DEVD-fmk significantly attenuated PD98059-induced apoptosis through the down-regulation of caspase-3 activity, but did not attenuate G1 phase arrest. Moreover, PD98059 down-regulated Akt phosphorylation and produced a synergy effect of apoptosis with LY294002 co-treatment. Thus, our results imply that PD98059-induced apoptosis is significantly involved in down-regulation of Bcl-2, caspase-3 activity, the Akt pathway, and some of the biological functions in U937 cells.     

Tanshinone II A Induces Apoptosis and S Phase Cell Cycle Arrest in Activated Rat Hepatic Stellate Cells

Abstract: Tanshinone IIA, a major component extracted from the traditional herbal medicine, Salvia miltiorrhiza Bunge, improves blood circulation and treats chronic hepatitis and hepatic fibrosis. Activation of hepatic stellate cells (HSCs) is the predominant event in liver fibrosis. The therapeutic goal in liver fibrosis is the reversal of fibrosis and selective clearance of activated HSCs. We used rat HSCs transformed by Simian virus 40 (t‐HSC/Cl‐6) to overcome the limitations inherent in studying subcultures of HSCs. Treatment of t‐HSC/Cl‐6 cells with tanshinone IIA inhibited cell viability in a dose‐ and time‐dependent manner. Tanshinone IIA induced apoptosis as demonstrated by DNA fragmentation, poly(ADP‐ribose) polymerase and caspase‐3 cleavage, increased Bax/Bcl‐2 protein ratio, and depolarization of mitochondrial membranes to facilitate cytochrome c release into the cytosol. Furthermore, this compound markedly induced S phase cell cycle arrest, and down‐regulated cyclins A and E, and cdk2. Thus, tanshinone IIA induces apoptosis and S phase cell cycle arrest in rat HSCs in vitro.     

Antiproliferative effect of salvianolic acid A on rat hepatic stellate cells

Suppression of activation or proliferation, or induction of apoptosis in hepatic stellate cells (HSCs) have been proposed as therapeutic strategies against liver fibrosis. Salvia miltiorrhiza has been reported to exert antifibrotic effects in rats with hepatic fibrosis, but its mechanisms of action remain to be clarified. We have investigated the effects of salvianolic acid A (Sal A), an active principle from S. miltiorrhiza, on the proliferation-related biomarkers in a cell line of rat HSCs (HSC-T6) stimulated with platelet-derived growth factor-BB homodimer (PDGF-BB). DNA synthesis (bromodeoxyuridine (BrdU) incorporation), cell cycle related proteins and apoptosis markers were determined to evaluate the inhibitory effects of Sal A. The results showed that Sal A (1-10 microM) concentration-dependently attenuated PDGF-BB-stimulated proliferation (BrdU incorporation) in HSC-T6 cells. Sal A at 10 microM induced cell apoptosis in PDGF-BB-incubated HSCs, together with a reduction of Bcl-2 protein expression, induction of cell cycle inhibitory proteins p21 and p27, and down-regulation of cyclins D1 and E, suppression of Akt phosphorylation, reduction in PDGF receptor phosphorylation, and an increase in caspase-3 activity. Sal A exerted no direct cytotoxicity on primary hepatocytes and HSC-T6 cells under experimental concentrations. Our results suggested that Sal A inhibited PDGF-BB-activated HSC proliferation, partially through apoptosis induction.     

Methyl helicterte ameliorates liver fibrosis by regulating miR-21-mediated ERK and TGF-β1/Smads pathways

Methyl helicterate (MH) has been reported to have protective effects against CCl₄-induced hepatic injury and fibrosis in rats, but its protective mechanism, especially on hepatic stallete cells (HSCs), remains unclear. Recently, our pilot experiment showed that MH could inhibit miR-21 expression in HSC-T6 cells, suggesting that miR-21 may be one of the targets of MH to intervene liver fibrosis. To verify the hypothesis, the present study would focus on the regulatory effect of MH on the miR-21-mediated ERK and TGF-β1/Smads pathways. Briefly, rats were intraperitoneally injected with 0.5 ml porcine serum (PS) twice a week for 24 weeks to induce liver fibrosis, and meanwhile, the rats were treated with MH from weeks 16 to 24. In vitro experiment, miR-21 expression in HSC-T6 cells was up- or down-regulated using lentiviral transfection assay. Collagen accumulation, inflammatory cytokines, cell apoptosis, miR-21 expression, and activation of the ERK and TGF-β1/smad2/3 pathways were then assessed. The results showed that MH treatment markedly alleviated PS-induced liver injury, as evidenced by the attenuation of histopathological changes and the decrease in serum alanine and aspartate aminotransferases activity. MH significantly decreased the content of inflammatory cytokines and recruited the anti-oxidative defense system. Moreover, MH treatment significantly decreased miR-21 expression and inhibited the activation of the ERK and TGF-β1/smad2/3 pathways in liver tissues. In vitro experiments showed that MH strongly inhibited HSC-T6 cell activation and reduced collagen accumulation. Interestingly, miR-21 overexpression significantly promoted HSC-T6 cell proliferation, reduced HSC apoptosis, and increased collagenation, while these abnormal changes induced by miR-21overexpression were significantly reversed by MH treatment. Furthermore, miR-21 overexpression notably activated the ERK and TGF-β1/Smads pathways via repressing SPRY2 and Smad7 expression respectively, however, these effects were largely abolished by MH treatment. In conclusion, our study demonstrates that MH significantly alleviates PS-induced liver injury and fibrosis by inhibiting miR-21-mediated ERK and TGF-β1/Smads pathways.     

Oridonin ameliorates carbon tetrachloride-induced liver fibrosis in mice through inhibition of the NLRP3 inflammasome

Liver fibrosis is characterized by the activation of hepatic stellate cells (HSCs) and accumulation of the extracellular matrix. There are limitations in the current therapies for liver fibrosis. Recently, oridonin was shown to induce apoptosis in HSCs. Thus, we aimed to determine the roles of oridonin in chronic liver injury and fibrosis. Liver fibrosis was induced by CCl₄ in mice injected intraperitoneally with oridonin for 6 weeks. The administration of oridonin significantly attenuated liver injury and reduced ALT levels. In addition, Sirius Red staining and the expression of α-smooth muscle actin (α-SMA) were significantly reduced by oridonin in murine livers with fibrosis. The expression of NLRP3, caspase-1, and IL-1β was downregulated with the oridonin treatment. Furthermore, the expression of F4/80 in liver tissues was also decreased by oridonin treatment. These results demonstrate that oridonin ameliorates chronic liver injury and fibrosis. Mechanically, oridonin may inhibit the activity of the NLRP3 inflammasome and inflammation in the liver. These results highlight the potential of oridonin as a therapeutic agent for liver fibrosis.     

Hydroxysafflor yellow A protects against chronic carbon tetrachloride-induced liver fibrosis

Hydroxysafflor yellow A (HSYA) was isolated from the dried flower of Carthamus tinctorius L. which was extensively used in traditional Chinese medicine to treat cirrhosis. However, the potential protective effect of HSYA in liver fibrosis is still unknown. In the present study, we investigated the effects of HSYA in rats with carbon tetrachloride (CCl4)-induced liver fibrosis. Sprague-Dawley (SD) rats were subjected to biweekly CCl4 injections over 12 weeks, while controls were given isovolumetric injections of olive oil. HSYA was given in a daily dose of 5 mg/kg by means of intraperitoneal concurrent with CCl4. Hepatic fibrosis was quantified by digital analysis of Masson's trichrome stained slides and hydroxyproline content. mRNA expression was quantified by real-time polymerase chain reaction (PCR), and protein was quantified by western blot or enzyme-linked immunosorbent assay (ELISA). CCl4 treatment induced micronodular liver fibrosis with a pronounced deposition of collagen fibers. HSYA significantly reduced liver fibrosis. HSYA down regulates α-smooth muscle actin (SMA), collagen α type I, matrix metalloproteinases (MMP)-9, and tissue inhibitors of metalloproteinases (TIMP)-1 gene expression. This was accompanied by a decreased expression of transforming growth factor (TGF)-β1 and phosphorylation of Smad4. These results indicate that HSYA might be a promising antifibrotic agent in chronic liver disease.     

Dracorhodin perchlorate induces apoptosis in HL-60 cells

Dracorhodin perchlorate, an anthocyanin red pigment, induces human premyelocytic leukemia HL-60 cell death through apoptotic pathway. Caspase -1, -3, -8, -9, and -10 inhibitors partially reversed the cell death induced by dracorhodin perchlorate. Caspase-3 and -8 were activated followed to the degradation of caspase-3 substrates, inhibitor of caspase-activated DNase (ICAD) and poly-(ADP-ribose) polymerase (PARP). Dracorhodin perchlorate up-regulated the expression ratio of mitochondrial proteins, Bax/Bcl-X_L. The cell death was accompanied with phosphorylation of ERK, JNK and p38 MAPK and partially reduced by MEK inhibitor (PD98059), JNK MAPK inhibitor (SP600125) and p38 MAPK inhibitor (SB 203580). Taken together, dracorhodin perchlorate-induced apoptosis in HL-60 cells via up-regulation of Bax, activation of caspases and ERK/p38/JNK MAPKs.     

Inhibition of P2X7R modulates ECM deposition in HSCs and crosstalk with macrophages in liver fibrosis

OBJECTIVE Modulation of immune response and reduction of extracellular matrix(ECM)deposition are both essential in the therapy of liver fibrosis.Regulating P2 X7 R might be a potential therapeutic strategy to treat liver fibrosis, we investigated whether the blockade of P2 X7 R could reverse liver fibrosis and how P2 X7 R is involved in fibrogenesis during hepatic stellate cells(HSCs) and macrophages crosstalk. METHODS In vivo,liver fibrosis model was established by thioacetamide(TAA) intraperitoneal administration in male C57 BL/6 mice. In vitro, LX-2 cells were treated with TGF-β and LPS/ATP respectively. Supernatant from LPS/ATP stimulated THP-1 macrophages were supplemented to LX-2 cells to mimic cellular crosstalk between HSCs and macrophages. RESULTS Blockade of P2 X7 R with its selective antagonist A438079, not only decreased liver injury and ECM deposition but also ameliorated inflammation by inhibiting NLRP3 inflammasome, NF-κB activation and IL-1β production in TAA-induced liver fibrosis. And the recruitment of macrophages, monocytes and granulocytes were also inhibited. In TGF-β-stimulated LX-2 cells,ECM deposition was reduced by the inhibiting of P2 X7 RTLR4-NLRP3 axis. Protein synthesis and cleavage of IL-1β and its m RNA level was dramatically increased by LPS 4 h combined with ATP 30 min than those in HSC treated with LPS or ATP alone. Additionally, LX-2 cells primed with LPS/ATP greatly increased m RNA and protein expression of caspase-1, NLRP3 and P2 x7 R, as well as liver fibrosis markers, α-SMA and typeⅠcollagen.These events were remarkably suppressed by A438079 pretreatment. si RNA against P2 x7 R reduced protein expression of NLRP3 and α-SMA, and suppressed deposition and secretion of type Ⅰ collagen induced by LPS/ATP. Ectopic overexpression of P2 X7 R reduced the threshold of ECM deposition in HSCs induced by TGF-β.Inhibiting upstream receptors of NLRP3 inflammasome,P2 X7 receptor-selective antagonist(A438079), TLR4 inhibitor(CLI-095) reduced the fibrotic markers in both models of TGF-β and LPS/ATP-activated HSCs. The cultured medium of the THP-1 macrophages by LPS/ATP aggravated ECM deposition in LX-2 cells. The decreased IL-1β by the pharmacological inhibitors of P2 X7 R,caspase-1 and TLR4 treated to THP-1 macrophages attenuates ECM deposition in LX-2 cells. CONCLUSION Both ECM producing in HSCs and inflammatory cytokines secreting from macrophages were regulated by P2 X7 R, suggesting a therapeutic utility of P2 x7 R blockade in liver fibrosis treatment.     

S-adenosylmethionine inhibits the activated phenotype of human hepatic stellate cells via Rac1 and Matrix metalloproteinases

ObjectiveTo investigate the effects of S-adenosylmethionine (SAM) on the proliferation, adhesion, migration, invasion and apoptosis of activated human hepatic stellate cells (HSCs) and to explore the relevant potential mechanisms.MethodsHuman HSCs LX-2 were cultured with SAM. The proliferation and adhesion were detected by CCK-8. Cell apoptosis rate were analyzed by flow cytometry, and cell migration and invasion were tested by the transwell assay. The expression of Rac1 and MMP-2 was identified by real-time PCR or Western blotting, and activated Rac1 was detected by GST pull-down assay. The activity of MMP-2 and MMP-9 was analyzed by substrate zymography.ResultsThe proliferation of LX-2 cells was inhibited by SAM, exhibiting a dose-dependent manner. Cell apoptosis rate induced by SAM was remarkably increased. SAM decreased the adhesion, migration and invasion of LX-2 cells. The expression and activation of Rac1 in LX-2 cells were significantly suppressed by SAM. In contrast, the activity of MMP-2 and MMP-9 was enhanced by SAM. SAM attenuated the up-regulated expression of Smad3/4 and Rac1 induced by TGF-β1.ConclusionSAM inhibits the proliferation, adhesion, migration and invasion of LX-2 cells in vitro probably via attenuating the expression and activation of Rac1 and up-regulating MMP-2 and MMP-9 expression, which possibly provide a molecular basis for potential application of SAM in the therapy of liver fibrosis.