Anti-fibrotic Effects and Mechanism of Shengmai Injection(生脉注射液)on Human Hepatic Stellate Cells LX-2
|
摘要
Objective: To investigate the effects of Shengmai Injection(生脉注射液, SMI) on the proliferation, apoptosis and N-myc downstream-regulated gene 2(NDRG2, a tumour suppressor gene) expression in varying densities of human hepatic stellate cells LX-2. Methods: LX-2 cells were cultured in vitro. Then, cells were plated in 96-well plates at an approximate density of 2.5×10~4 cells/mL and cultured for 48, 72, 96 or 120 h followed by the application of different concentrations of SMI(0.6, 1.2, 2.4, 4.8 or 6 μL/mL). Cell proliferation was measured after an additional 24 or 48 h using the 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT) assay. The effects of SMI on different cell growth states(cultured for 48, 72, 96, or 120 h) were observed by light microscopy at 24 h after treatment. When the cells reached 80% confluence, apoptosis was detected by flow cytometry after 24 h. Lastly, LX-2 cells were treated with different concentrations of SMI and extracted with protein lysis buffer. The levels of NDRG2 were measured by Western blot. Results: When the LX-2 cells grew for 48, 72, 96 and 120 h, 4.8 and 6 μL/m L of SMI significantly inhibited cell proliferation at 24 and 48 h after treatment(P<0.05). And 2.4 μL/mL of SMI also inhibited cell proliferation at 24 h after treatment when cell growth for 48 h(P<0.05) and at 48 h after treatment when cell growth for 72, 96 and 120 h(P<0.05). The NDRG2 expression level in the LX-2 cell was significantly increased when treated with SMI at concentrations of 1.2, 2.4, 4.8 or 6 μL/mL(P<0.05). Conclusions: The inhibitory effects of SMI on the proliferation of LX-2 cells were related to not only concentration dependent but also cell density. In addition, SMI(2.4, 4.8 and 6 μL/mL) could accelerate apoptosis in LX-2 cells, and the mechanism might be associated with NDRG2 over-expression.
Objective: To investigate the effects of Shengmai Injection(生脉注射液, SMI) on the proliferation, apoptosis and N-myc downstream-regulated gene 2(NDRG2, a tumour suppressor gene) expression in varying densities of human hepatic stellate cells LX-2. Methods: LX-2 cells were cultured in vitro. Then, cells were plated in 96-well plates at an approximate density of 2.5×10~4 cells/mL and cultured for 48, 72, 96 or 120 h followed by the application of different concentrations of SMI(0.6, 1.2, 2.4, 4.8 or 6 μL/mL). Cell proliferation was measured after an additional 24 or 48 h using the 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT) assay. The effects of SMI on different cell growth states(cultured for 48, 72, 96, or 120 h) were observed by light microscopy at 24 h after treatment. When the cells reached 80% confluence, apoptosis was detected by flow cytometry after 24 h. Lastly, LX-2 cells were treated with different concentrations of SMI and extracted with protein lysis buffer. The levels of NDRG2 were measured by Western blot. Results: When the LX-2 cells grew for 48, 72, 96 and 120 h, 4.8 and 6 μL/m L of SMI significantly inhibited cell proliferation at 24 and 48 h after treatment(P<0.05). And 2.4 μL/mL of SMI also inhibited cell proliferation at 24 h after treatment when cell growth for 48 h(P<0.05) and at 48 h after treatment when cell growth for 72, 96 and 120 h(P<0.05). The NDRG2 expression level in the LX-2 cell was significantly increased when treated with SMI at concentrations of 1.2, 2.4, 4.8 or 6 μL/mL(P<0.05). Conclusions: The inhibitory effects of SMI on the proliferation of LX-2 cells were related to not only concentration dependent but also cell density. In addition, SMI(2.4, 4.8 and 6 μL/mL) could accelerate apoptosis in LX-2 cells, and the mechanism might be associated with NDRG2 over-expression.
Objective: To investigate the effects of Shengmai Injection(生脉注射液, SMI) on the proliferation, apoptosis and N-myc downstream-regulated gene 2(NDRG2, a tumour suppressor gene) expression in varying densities of human hepatic stellate cells LX-2. Methods: LX-2 cells were cultured in vitro. Then, cells were plated in 96-well plates at an approximate density of 2.5×10~4 cells/mL and cultured for 48, 72, 96 or 120 h followed by the application of different concentrations of SMI(0.6, 1.2, 2.4, 4.8 or 6 μL/mL). Cell proliferation was measured after an additional 24 or 48 h using the 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT) assay. The effects of SMI on different cell growth states(cultured for 48, 72, 96, or 120 h) were observed by light microscopy at 24 h after treatment. When the cells reached 80% confluence, apoptosis was detected by flow cytometry after 24 h. Lastly, LX-2 cells were treated with different concentrations of SMI and extracted with protein lysis buffer. The levels of NDRG2 were measured by Western blot. Results: When the LX-2 cells grew for 48, 72, 96 and 120 h, 4.8 and 6 μL/m L of SMI significantly inhibited cell proliferation at 24 and 48 h after treatment(P<0.05). And 2.4 μL/mL of SMI also inhibited cell proliferation at 24 h after treatment when cell growth for 48 h(P<0.05) and at 48 h after treatment when cell growth for 72, 96 and 120 h(P<0.05). The NDRG2 expression level in the LX-2 cell was significantly increased when treated with SMI at concentrations of 1.2, 2.4, 4.8 or 6 μL/mL(P<0.05). Conclusions: The inhibitory effects of SMI on the proliferation of LX-2 cells were related to not only concentration dependent but also cell density. In addition, SMI(2.4, 4.8 and 6 μL/mL) could accelerate apoptosis in LX-2 cells, and the mechanism might be associated with NDRG2 over-expression.
引文
1.Li F,Ma N,Zhao R,Wu G,Zhang Y,Qiao Y,et al.Overexpression of miR-483-5p/3p cooperate to inhibit mouse liver fibrosis by suppressing the TGF-beta stimulated HSCs in transgenic mice.J Cell Mol Med 2014;18:966-974.
2.Gou X,Tao Q,Feng Q,Peng J,Zhao Y,Dai J,et al.Urine metabolic profile changes of CCl4-liver fibrosis in rats and intervention effects of Yi Guan Jian Decoction using metabonomic approach.BMC Complement Altern Med2013;13:123.
3.D'Argenio G,Amoruso DC,Mazzone G,Vitaglione P,Romano A,Ribecco MT,et al.Garlic extract prevents CCl4-induced liver fibrosis in rats:the role of tissue transglutaminase.Dig Liver Dis 2010;42:571-577.
4.Fan WM,Shi BY.Advances in studies on apoptotic factors of hepatic stellate cell.Med Recapitul(Chin)2012;18:1797-1799.
5.Henderson NC,Mackinnon AC,Farnworth SL,Poirier F,Russo FP,Iredale JP,et al.Galectin-3 regulates myofibroblast activation and hepatic fibrosis.Proc Natl Acad Sci USA 2006;103:5060-5065.
6.Huang CL,Zhao WX.Progress on treating hepatic fibrosis in Chinese medicine.Clin J Chin Med(Chin)2014;2:146-147.
7.Yang Q,Feng Y,Jiang SL.Experiences of Prof.YAO Xixian in treating chronic hepatic fibrosis based on blood stasis theory.Chin J Chin Med Pharm(Chin)2007;22:168-171.
8.Cheng Y,Mai JY,Wang MF,Chen GF,Ping J.Antifibrotic effect of total flavonoids of Astmgali Radix on dimethylnitrosamine-induced liver cirrhosis in rats.Chin JIntegr Med 2017;23:48-54.
9.Liu P.Inhibition of pathological angiogenesis of Chinese medicine against liver fibrosis.Chin J Integr Med2016;22:569-572.
10.Xuan J.Clinical study of Huagan Tongluo Recipe and the mechanism of Th17 cell differentiation involved in hepatic fibrosis.Nanjing:Nanjing University of Chinese Medicine;2018.
11.Deng ZJ,ed.Formulas of Chinese medicine.Beijing:China Press of Traditional Chinese Medicine;2011:152-153.
12.Rong XF,Yao WY.Influence of Shenmai Injection combined with Puerarin Injection on liver fibrosis indexes in patients with cirrhosis.World Chin J Digestol(Chin)2006;14:3326-3329.
13.Wang L,Liu N,Yao L,Li F,Zhang J,Deng Y,et al.NDRG2 is a new HIF-1 target gene necessary for hypoxia-induced apoptosis in A549 cel s.Cel Physiol Biochem 2008;21:239-250.
14.Shen L,Zhao ZY,Wang YZ,Ji SP,Liu XP,Liu XW,et al.Immunohistochemical detection of NDRG2 in the mouse nervous system.Neuroreport 2008;19:927-931.
15.Boulkroun S,Fay M,Zennaro MC,Escoubet B,Jaisser F,Blot-Chabaud M,et al.Characterization of rat NDRG2(N-Myc downstream regulated gene 2),a novel early mineralocorticoidspecific induced gene.J Biol Chem 2002;277:31506-31515.
16.Zheng J,Li Y,Yang J,Liu Q,Shi M,Zhang R,et al.NDRG2inhibits hepatocellular carcinoma adhesion,migration and invasion by regulating CD24 expression.BMC Cancer2011;11:251.
17.Yang J,Zheng J,Wu L,Shi M,Zhang H,Wang X,et al.NDRG2 ameliorates hepatic fibrosis by inhibiting the TGF-beta1/Smad pathway and altering the MMP2/TIMP2 ratio in rats.PLoS One 2011;6:e27710.
18.Yang JD.Expression of NDRG2 in liver injury-repair and preliminary study on its function.Xi'an:The Fourth Military Medical University;2013.
19.Xu L,Hui AY,Albanis E,Arthur MJ,O'Byrne SM,Blaner WS,et al.Human hepatic stellate cell lines,LX-1 and LX-2:new tools for analysis of hepatic fibrosis.Gut 2005;54:142-151.
20.Zhou WC,Zhang QB,Qiao L.Pathogenesis of liver cirrhosis.World J Gastroenterol 2014;20:7312-7324.
21.Duval F,Moreno-Cuevas JE,González-Garza MT,Rodríguez-Montalvo C,Cruz-Vega DE.Protective mechanisms of medicinal plants targeting hepatic stellate cell activation and extracellular matrix deposition in liver fibrosis.Chin Med 2014;9:27.
22.Su TH,Kao JH,Liu CJ.Molecular mechanism and treatment of viral hepatitis-related liver fibrosis.Int J Mol Sci2014;15:10578-10604.
23.Hu W,Fan C,Jiang P,Ma Z,Yan X,Di S,et al.Emerging role of N-myc downstream-regulated gene 2(NDRG2)in cancer.Oncotarget 2016;7:209-223.
24.Yoon SH,Nam YM,Hong JT,Kim SJ,Ko SK.Modification of ginsenoside composition in red ginseng(Panax ginseng)by ultrasonication.J Ginseng Res 2016;40:300-303.
25.Yao C,Shi X,Lin X,Shen L,Xu D,Feng Y.Increased cardiac distribution of mono-PEGylated Radix Ophiopogonis polysaccharide in both myocardial infarction and ischemia/reperfusion rats.Int J Nanomed 2015;10:409-418.
26.Szopa A,Ekiert R,Ekiert H.Current knowledge of Schisandra chinensis(Turcz.)Baill.(Chinese magnolia vine)as a medicinal plant species:a review on the bioactive components,pharmacological properties,analytical and biotechnological studies.Phytochem Rev 2017;16:195-218.
27.Liu X,Tan W,Yang F,Wang Y,Yue S,Wang T,et al.Shengmai Injection reduces apoptosis and enhances angiogenesis after myocardial ischaemia and reperfusion injury in rats.Biomed Pharmacother 2018;104:629-636.
28.Yang H,Li L,Zhou K,Wang Y,Guan T,Chai C,et al.Shengmai Injection attenuates the cerebral ischemia/reperfusion induced autophagy via modulation of the AMPK,mTOR and JNK pathways.Pharm Biol 2016;10:2288-2297.
29.Duan B,Xie J,Rui Q,Zhang W,Xi Z.Effects of Shengmai Injection add-on therapy to chemotherapy in patients with non-small cell lung cancer:a meta-analysis.Support Care Cancer 2018;26:2103-2111.
2.Gou X,Tao Q,Feng Q,Peng J,Zhao Y,Dai J,et al.Urine metabolic profile changes of CCl4-liver fibrosis in rats and intervention effects of Yi Guan Jian Decoction using metabonomic approach.BMC Complement Altern Med2013;13:123.
3.D'Argenio G,Amoruso DC,Mazzone G,Vitaglione P,Romano A,Ribecco MT,et al.Garlic extract prevents CCl4-induced liver fibrosis in rats:the role of tissue transglutaminase.Dig Liver Dis 2010;42:571-577.
4.Fan WM,Shi BY.Advances in studies on apoptotic factors of hepatic stellate cell.Med Recapitul(Chin)2012;18:1797-1799.
5.Henderson NC,Mackinnon AC,Farnworth SL,Poirier F,Russo FP,Iredale JP,et al.Galectin-3 regulates myofibroblast activation and hepatic fibrosis.Proc Natl Acad Sci USA 2006;103:5060-5065.
6.Huang CL,Zhao WX.Progress on treating hepatic fibrosis in Chinese medicine.Clin J Chin Med(Chin)2014;2:146-147.
7.Yang Q,Feng Y,Jiang SL.Experiences of Prof.YAO Xixian in treating chronic hepatic fibrosis based on blood stasis theory.Chin J Chin Med Pharm(Chin)2007;22:168-171.
8.Cheng Y,Mai JY,Wang MF,Chen GF,Ping J.Antifibrotic effect of total flavonoids of Astmgali Radix on dimethylnitrosamine-induced liver cirrhosis in rats.Chin JIntegr Med 2017;23:48-54.
9.Liu P.Inhibition of pathological angiogenesis of Chinese medicine against liver fibrosis.Chin J Integr Med2016;22:569-572.
10.Xuan J.Clinical study of Huagan Tongluo Recipe and the mechanism of Th17 cell differentiation involved in hepatic fibrosis.Nanjing:Nanjing University of Chinese Medicine;2018.
11.Deng ZJ,ed.Formulas of Chinese medicine.Beijing:China Press of Traditional Chinese Medicine;2011:152-153.
12.Rong XF,Yao WY.Influence of Shenmai Injection combined with Puerarin Injection on liver fibrosis indexes in patients with cirrhosis.World Chin J Digestol(Chin)2006;14:3326-3329.
13.Wang L,Liu N,Yao L,Li F,Zhang J,Deng Y,et al.NDRG2 is a new HIF-1 target gene necessary for hypoxia-induced apoptosis in A549 cel s.Cel Physiol Biochem 2008;21:239-250.
14.Shen L,Zhao ZY,Wang YZ,Ji SP,Liu XP,Liu XW,et al.Immunohistochemical detection of NDRG2 in the mouse nervous system.Neuroreport 2008;19:927-931.
15.Boulkroun S,Fay M,Zennaro MC,Escoubet B,Jaisser F,Blot-Chabaud M,et al.Characterization of rat NDRG2(N-Myc downstream regulated gene 2),a novel early mineralocorticoidspecific induced gene.J Biol Chem 2002;277:31506-31515.
16.Zheng J,Li Y,Yang J,Liu Q,Shi M,Zhang R,et al.NDRG2inhibits hepatocellular carcinoma adhesion,migration and invasion by regulating CD24 expression.BMC Cancer2011;11:251.
17.Yang J,Zheng J,Wu L,Shi M,Zhang H,Wang X,et al.NDRG2 ameliorates hepatic fibrosis by inhibiting the TGF-beta1/Smad pathway and altering the MMP2/TIMP2 ratio in rats.PLoS One 2011;6:e27710.
18.Yang JD.Expression of NDRG2 in liver injury-repair and preliminary study on its function.Xi'an:The Fourth Military Medical University;2013.
19.Xu L,Hui AY,Albanis E,Arthur MJ,O'Byrne SM,Blaner WS,et al.Human hepatic stellate cell lines,LX-1 and LX-2:new tools for analysis of hepatic fibrosis.Gut 2005;54:142-151.
20.Zhou WC,Zhang QB,Qiao L.Pathogenesis of liver cirrhosis.World J Gastroenterol 2014;20:7312-7324.
21.Duval F,Moreno-Cuevas JE,González-Garza MT,Rodríguez-Montalvo C,Cruz-Vega DE.Protective mechanisms of medicinal plants targeting hepatic stellate cell activation and extracellular matrix deposition in liver fibrosis.Chin Med 2014;9:27.
22.Su TH,Kao JH,Liu CJ.Molecular mechanism and treatment of viral hepatitis-related liver fibrosis.Int J Mol Sci2014;15:10578-10604.
23.Hu W,Fan C,Jiang P,Ma Z,Yan X,Di S,et al.Emerging role of N-myc downstream-regulated gene 2(NDRG2)in cancer.Oncotarget 2016;7:209-223.
24.Yoon SH,Nam YM,Hong JT,Kim SJ,Ko SK.Modification of ginsenoside composition in red ginseng(Panax ginseng)by ultrasonication.J Ginseng Res 2016;40:300-303.
25.Yao C,Shi X,Lin X,Shen L,Xu D,Feng Y.Increased cardiac distribution of mono-PEGylated Radix Ophiopogonis polysaccharide in both myocardial infarction and ischemia/reperfusion rats.Int J Nanomed 2015;10:409-418.
26.Szopa A,Ekiert R,Ekiert H.Current knowledge of Schisandra chinensis(Turcz.)Baill.(Chinese magnolia vine)as a medicinal plant species:a review on the bioactive components,pharmacological properties,analytical and biotechnological studies.Phytochem Rev 2017;16:195-218.
27.Liu X,Tan W,Yang F,Wang Y,Yue S,Wang T,et al.Shengmai Injection reduces apoptosis and enhances angiogenesis after myocardial ischaemia and reperfusion injury in rats.Biomed Pharmacother 2018;104:629-636.
28.Yang H,Li L,Zhou K,Wang Y,Guan T,Chai C,et al.Shengmai Injection attenuates the cerebral ischemia/reperfusion induced autophagy via modulation of the AMPK,mTOR and JNK pathways.Pharm Biol 2016;10:2288-2297.
29.Duan B,Xie J,Rui Q,Zhang W,Xi Z.Effects of Shengmai Injection add-on therapy to chemotherapy in patients with non-small cell lung cancer:a meta-analysis.Support Care Cancer 2018;26:2103-2111.
© 2004-2018 中国地质图书馆版权所有 京ICP备05064691号 京公网安备11010802017129号 地址：北京市海淀区学院路29号 邮编：100083 电话：办公室：(+86 10)66554848；文献借阅、咨询服务、科技查新：66554700 |