金蝉花孢子粉对小鼠肝细胞性肝癌杀伤机制探讨
|
摘要
目的肝细胞性肝癌(hepatocellular carcinoma,HCC)死亡率高,临床干预治疗效果不佳,对新型治疗药物需求迫在眉睫。本研究探讨免疫调节中药金蝉花孢子粉(spore power of vegetable cicadae,SPVC)对HCC的治疗作用及对肿瘤浸润淋巴细胞的影响,为临床治疗提供依据。方法将SPF级C57BL/6J雄性小鼠120只按照体质量均衡随机分为空白对照组、模型组、索拉非尼组和SPVC组,每组30只。模型组、索拉非尼组和SPVC组通过梯度形式皮下注射二乙基亚硝胺(diethylnitrosamine,DEN)50mg/kg,1次/d,连续7周,建立小鼠HCC模型。3组分别给予1mL/kg生理盐水、30mg/kg索拉非尼和300mg/kg SPVC溶液灌胃,1次/d,连续15周。各组小鼠均衡随机分为2个亚组,一亚组观察8周存活率;另一亚组记录小鼠体质量变化,计算肝指数,HE染色分析小鼠肝组织结构变化,比色法检测小鼠血浆ALT和AST含量,ELISA法检测小鼠肝脏匀浆液中IL-4和IFN-γ水平,免疫组化检测肝脏组织CD8和PD-L1含量。结果给药60d,各组小鼠存活率依次为空白对照组100.00%、SPVC组62.77%、索拉非尼组45.09%、模型组20.16%;给药15周,SPVC组小鼠体质量为(49.20±5.24)g,大于模型组(46.10±5.65)g,差异有统计学意义,P=0.032;SPVC组肝脏肿瘤结节数为(4.68±0.99)个,少于索拉非尼组(8.80±1.45)个和模型组(21.33±1.88)个,均P<0.001;SPVC组肝指数为(72.833±16.158)mg/g,低于索拉非尼组(91.384±16.820)mg/g和模型组(113.435±13.139)mg/g,均P<0.05;SPVC组血浆ALT为(89.348±29.847)IU/L,低于索拉非尼组(121.344±30.342)IU/L和模型组(137.583±12.343)IU/L,P值分别为0.047和0.004;SPVC组血浆AST为(84.784±22.372)IU/L,低于索拉非尼组(101.942±24.349)IU/L和模型组(126.985±27.447)IU/L,P值分别为0.048和0.038;SPVC组肝组织IL-4含量为(85.12±12.42)pg/μg,低于索拉非尼组(114.42±10.89pg/μg和模型组(123.55±15.42)pg/μg,P值分别为0.020和0.001;SPVC组肝组织IFN-γ含量为(92.20±15.14)pg/μg,高于索拉非尼组(71.37±12.83)pg/μg和模型组(65.34±13.34)pg/μg,P值分别为0.048和0.045;SPVC组肝组织CD8+T细胞比例为(12.73±2.82)%,高于索拉非尼组(9.91±2.38)%和模型组(7.34±1.58)%,P值分别为0.046和0.021;SPVC组肝组织PD-L1比例为(37.81±12.22)%,低于索拉非尼组(50.38±14.27)%和模型组(72.34±15.34)%,均P<0.05。结论 SPVC通过上调IFN-γ,引起PD-L1下调,升高CD8+T细胞比例,具有免疫靶向杀伤肝癌细胞治疗HCC的作用。
OBJECTIVE The mortality of hepatocellular carcinoma(HCC)is high and the therapeutic effect of clinical intervention is poor.It is urgent to demand new therapeutic drugs.The aim of this study was to investigate the protective effect of spore power of vegetable cicada(SPVC)on HCC and its effect on tumor infiltrating lymphocytes,and to provide evidence for its clinical treatment.METHODS Tatally 120 SPF C57 BL/6 Jmale mice were randomly divided into blank control group,model group,Sorafenib(SO)group and SPVC group,with 30 mice in each group.In the model group,SO group and SPVC group,DEN 50 mg/kg was injected subcutaneous in gradient form once a day for 7 weeks to establish the mouse HCC model.The three groups were given 1 ml/kg normal saline,30 mg/kg sorafenib and 300 mg/kg SPVC solution respectively for 15 weeks.Then the mice in each group were randomly divided into two subgroups.One subgroup was observed the 8-week survival rate,the other subgroup was recorded the weight of the mice,and analyzed the changes of the weight of the mice,and calculated the liver index.The liver tissue structure of mice was analyzed by HE staining.The content of plasma ALT and AST was detected by colorimetry method,the level of IL-4 and IFN-in the liver homogenate of mice was detected by ELISA and the content of CD8 and PD-L1 in liver tissues was detected by immunohistochemistry.RESUITS After 60 days of administration,the survival rates of mice in each group were 100.00%in normal group,62.77%in SPVC group,45.09%in SO group and 20.16%in model group.The body weight of mice in SPVC group(49.20±5.24)g was higher than that in model group(46.10±5.65)g after 15 weeks treatment,the difference was significant,P=0.032.The number of liver tumor nodules in SPVC group(4.68±0.99)was less than that in SO group(8.80±1.45)and model group(21.33±1.88),all P<0.001.The liver index(72.832 8±16.158 2)mg/g in SPVC group was lower than that of SO group(91.384±16.820)mg/g and model group(113.435±13.139)mg/g,all P<0.05.Plasma ALT in SPVC group(89.348±29.847)IU/L was lower than in SO group(121.344±30.342)IU/L and in model group(137.583±12.343)IU/L,Pvalues were 0.047 and 0.004.The plasma AST in SPVC group(84.784±22.372)IU/L was lower than that in SO group(101.942±24.349)IU/L and model group(126.985±27.447)IU/L,Pvalues were 0.048 and 0.038.The liver tissue IL-4 content in SPVC group(85.12±12.42)pg/μg was lower than that in SO group(114.42±10.89)pg/μg and model group(123.55±15.42)pg/μg,Pvalues were 0.020 0 and 0.001 3.The liver tissue IFN-γcontent in SPVC group(92.20±15.14)pg/μg was higher than that in SO group(71.37±12.83)pg/μg and model group(65.34±13.34)pg/μg,Pvalues were 0.048 1 and 0.044 6.The percentage of CD8+T cells in liver tissue of SPVC group(12.73±2.82)% was higher than that of SO group(9.91±2.38)% and model group(7.34±1.58)%,Pvalues were 0.046 2 and 0.021.The proportion of PD-L1 in liver tissue of SPVC group(37.81±12.22)%was lower than that of SO group(50.38±14.27)%and model group(72.34±15.34)%,all P<0.05.CONCLUSION SPVC could treat hepatocellular carcinoma induced by diethylnitrosamine via promoting IFN-γfollow a down-regulation of PD-L1,increase the ratio of CD8~+T cells.
OBJECTIVE The mortality of hepatocellular carcinoma(HCC)is high and the therapeutic effect of clinical intervention is poor.It is urgent to demand new therapeutic drugs.The aim of this study was to investigate the protective effect of spore power of vegetable cicada(SPVC)on HCC and its effect on tumor infiltrating lymphocytes,and to provide evidence for its clinical treatment.METHODS Tatally 120 SPF C57 BL/6 Jmale mice were randomly divided into blank control group,model group,Sorafenib(SO)group and SPVC group,with 30 mice in each group.In the model group,SO group and SPVC group,DEN 50 mg/kg was injected subcutaneous in gradient form once a day for 7 weeks to establish the mouse HCC model.The three groups were given 1 ml/kg normal saline,30 mg/kg sorafenib and 300 mg/kg SPVC solution respectively for 15 weeks.Then the mice in each group were randomly divided into two subgroups.One subgroup was observed the 8-week survival rate,the other subgroup was recorded the weight of the mice,and analyzed the changes of the weight of the mice,and calculated the liver index.The liver tissue structure of mice was analyzed by HE staining.The content of plasma ALT and AST was detected by colorimetry method,the level of IL-4 and IFN-in the liver homogenate of mice was detected by ELISA and the content of CD8 and PD-L1 in liver tissues was detected by immunohistochemistry.RESUITS After 60 days of administration,the survival rates of mice in each group were 100.00%in normal group,62.77%in SPVC group,45.09%in SO group and 20.16%in model group.The body weight of mice in SPVC group(49.20±5.24)g was higher than that in model group(46.10±5.65)g after 15 weeks treatment,the difference was significant,P=0.032.The number of liver tumor nodules in SPVC group(4.68±0.99)was less than that in SO group(8.80±1.45)and model group(21.33±1.88),all P<0.001.The liver index(72.832 8±16.158 2)mg/g in SPVC group was lower than that of SO group(91.384±16.820)mg/g and model group(113.435±13.139)mg/g,all P<0.05.Plasma ALT in SPVC group(89.348±29.847)IU/L was lower than in SO group(121.344±30.342)IU/L and in model group(137.583±12.343)IU/L,Pvalues were 0.047 and 0.004.The plasma AST in SPVC group(84.784±22.372)IU/L was lower than that in SO group(101.942±24.349)IU/L and model group(126.985±27.447)IU/L,Pvalues were 0.048 and 0.038.The liver tissue IL-4 content in SPVC group(85.12±12.42)pg/μg was lower than that in SO group(114.42±10.89)pg/μg and model group(123.55±15.42)pg/μg,Pvalues were 0.020 0 and 0.001 3.The liver tissue IFN-γcontent in SPVC group(92.20±15.14)pg/μg was higher than that in SO group(71.37±12.83)pg/μg and model group(65.34±13.34)pg/μg,Pvalues were 0.048 1 and 0.044 6.The percentage of CD8+T cells in liver tissue of SPVC group(12.73±2.82)% was higher than that of SO group(9.91±2.38)% and model group(7.34±1.58)%,Pvalues were 0.046 2 and 0.021.The proportion of PD-L1 in liver tissue of SPVC group(37.81±12.22)%was lower than that of SO group(50.38±14.27)%and model group(72.34±15.34)%,all P<0.05.CONCLUSION SPVC could treat hepatocellular carcinoma induced by diethylnitrosamine via promoting IFN-γfollow a down-regulation of PD-L1,increase the ratio of CD8~+T cells.
引文
[1]Rawat D,Shrivastava S,Naik RA,et al.An overview of natural plant products in the treatment of hepatocellular carcinoma[J].Anticancer Agents Med Chem,2018,45(7):750-761.
[2]Zhang B,Zhang B,Zhang Z,et al.42,573cases of hepatectomy in China:a multicenter retrospective investigation[J].Sci China Life Sci,2018,61(6):660-670.
[3]Ponzetto A,Figura N.Risk of hepatocellular carcinoma after cure of hepatitis C virus[J]Gastroenterology,2018,154(5):1549-1562.
[4]Yoo J,Hann HW,Coben R,et al.Update treatment for HBV infection and persistent risk for hepatocellular carcinoma:prospect for an HBV cure[J].Diseases,2018,6(2):65-79.
[5]Jung H,Km BI,Cho YK,et al.Complete cure of advanced hepatocellular carcinoma with right adrenal gland metastasis and portal vein thrombosis by multiple applications of an interdisciplinary therapy:case report with 8-year follow up[J].Clin Mol Hepatol,2017,11(22):496-512
[6]Wang H,Wang J,Shi X,et al.Genetically engineered bone marrow-derived mesenchymal stem cells co-expressing IFN-gamma and IL-10inhibit hepatocellular carcinoma by modulating MAPKpathway[J]J BUON,2017,22(6):1517-1524.
[7]D’Ambrosio R,Aghemo A,Rumi MG,et al.Persistence of hepatocellular carcinoma risk in hepatitis C patients with a response to IFN and cirrhosis regression[J].Liver Int,2018,38(8):1459-1467.
[8]Sun Y,Wink M,Wang P,et al.Biological characteristics,bioactive components and antineoplastic properties of sporoderm-broken spores from wild Cordyceps cicadae[J].Phytomedicine,2017,36(3):217-228.
[9]Chu ZB,Chang J,Zhu Y,et al.Chemical constituents of cordyceps cicadae[J].Nat Prod Commun,2015,10(12):2145-2146.
[10]Santos NP,Colaco AA,Oliveira PA.Animal models as a tool in hepatocellular carcinoma research:a review[J].Tumour Biol,2017,39(3):65-86.
[11]Heindryckx F,Colle I,Van Vlierberghe H.Experimental mouse models for hepatocellular carcinoma research[J].Int J Exp Pathol,2009,90(4):367-386.
[12]Zhang Q,Olatunji OJ,Chen H,et al.Evaluation of the anti-diabetic activity of polysaccharide from cordyceps cicadae in experimental diabetic rats[J].Chem Biodivers,2018,15(8):180-195.
[13]陆雷,王轩,张斌,等.索拉非尼治疗肝细胞性肝癌肝移植后的复发[J].中国组织工程研究,2012,25(31):5706-5710.
[14]Wang H,Zhang J,Sit WH,et al.Cordyceps cicadae induces G2/M cell cycle arrest in MHCC97H human hepatocellular carcinoma cells:a proteomic study[J].Chin Med,2014,9(5):15-22.
[15]Weng SC,Chou CJ,Lin LC,et al.Immunomodulatory functions of extracts from the Chinese medicinal fungus Cordyceps cicadae[J].J Ethnopharmacol,2002,83(1-2):79-85.
[16]Kuo YC,Weng SC,Chou CJ,et al.Activation and proliferation signals in primary human T lymphocytes inhibited by ergosterol peroxide isolated from Cordyceps cicadae[J].Br J Pharmacol,2003,140(5):895-906.
[17]Yee D,Shah KM,Coles MC,et al.MicroRNA-155induction via TNF-alpha and IFN-gamma suppresses expression of programmed death ligand-1(PD-L1)in human primary cells[J].JBiol Chem,2017,292(50):20683-20693.
[18]Ke BJ,Lee CL.Cordyceps cicadae NTTU 868mycelium prevents CCl4-induced hepatic fibrosis in BALB/c mice via inhibiting the expression of pro-inflammatory and pro-fibrotic cytokines[J].JFunct Foods,2018,43(2):214-223.
[2]Zhang B,Zhang B,Zhang Z,et al.42,573cases of hepatectomy in China:a multicenter retrospective investigation[J].Sci China Life Sci,2018,61(6):660-670.
[3]Ponzetto A,Figura N.Risk of hepatocellular carcinoma after cure of hepatitis C virus[J]Gastroenterology,2018,154(5):1549-1562.
[4]Yoo J,Hann HW,Coben R,et al.Update treatment for HBV infection and persistent risk for hepatocellular carcinoma:prospect for an HBV cure[J].Diseases,2018,6(2):65-79.
[5]Jung H,Km BI,Cho YK,et al.Complete cure of advanced hepatocellular carcinoma with right adrenal gland metastasis and portal vein thrombosis by multiple applications of an interdisciplinary therapy:case report with 8-year follow up[J].Clin Mol Hepatol,2017,11(22):496-512
[6]Wang H,Wang J,Shi X,et al.Genetically engineered bone marrow-derived mesenchymal stem cells co-expressing IFN-gamma and IL-10inhibit hepatocellular carcinoma by modulating MAPKpathway[J]J BUON,2017,22(6):1517-1524.
[7]D’Ambrosio R,Aghemo A,Rumi MG,et al.Persistence of hepatocellular carcinoma risk in hepatitis C patients with a response to IFN and cirrhosis regression[J].Liver Int,2018,38(8):1459-1467.
[8]Sun Y,Wink M,Wang P,et al.Biological characteristics,bioactive components and antineoplastic properties of sporoderm-broken spores from wild Cordyceps cicadae[J].Phytomedicine,2017,36(3):217-228.
[9]Chu ZB,Chang J,Zhu Y,et al.Chemical constituents of cordyceps cicadae[J].Nat Prod Commun,2015,10(12):2145-2146.
[10]Santos NP,Colaco AA,Oliveira PA.Animal models as a tool in hepatocellular carcinoma research:a review[J].Tumour Biol,2017,39(3):65-86.
[11]Heindryckx F,Colle I,Van Vlierberghe H.Experimental mouse models for hepatocellular carcinoma research[J].Int J Exp Pathol,2009,90(4):367-386.
[12]Zhang Q,Olatunji OJ,Chen H,et al.Evaluation of the anti-diabetic activity of polysaccharide from cordyceps cicadae in experimental diabetic rats[J].Chem Biodivers,2018,15(8):180-195.
[13]陆雷,王轩,张斌,等.索拉非尼治疗肝细胞性肝癌肝移植后的复发[J].中国组织工程研究,2012,25(31):5706-5710.
[14]Wang H,Zhang J,Sit WH,et al.Cordyceps cicadae induces G2/M cell cycle arrest in MHCC97H human hepatocellular carcinoma cells:a proteomic study[J].Chin Med,2014,9(5):15-22.
[15]Weng SC,Chou CJ,Lin LC,et al.Immunomodulatory functions of extracts from the Chinese medicinal fungus Cordyceps cicadae[J].J Ethnopharmacol,2002,83(1-2):79-85.
[16]Kuo YC,Weng SC,Chou CJ,et al.Activation and proliferation signals in primary human T lymphocytes inhibited by ergosterol peroxide isolated from Cordyceps cicadae[J].Br J Pharmacol,2003,140(5):895-906.
[17]Yee D,Shah KM,Coles MC,et al.MicroRNA-155induction via TNF-alpha and IFN-gamma suppresses expression of programmed death ligand-1(PD-L1)in human primary cells[J].JBiol Chem,2017,292(50):20683-20693.
[18]Ke BJ,Lee CL.Cordyceps cicadae NTTU 868mycelium prevents CCl4-induced hepatic fibrosis in BALB/c mice via inhibiting the expression of pro-inflammatory and pro-fibrotic cytokines[J].JFunct Foods,2018,43(2):214-223.