柴胡皂甙d（SSd）对二乙基亚硝胺（DEN）致大鼠肝癌的抑制作用和免疫功能的影响
摘要
研究背景：
肝肿瘤是人类最常见最难治疗的恶性肿瘤之一，因其病情发展快，死亡率极高，素有“癌中之王”之称。我国每年约有11万人死于肝肿瘤，占全世界肝肿瘤死亡数的55％左右，因此，肝肿瘤的诊治在世界范围内具有举足轻重的地位。一直以来，国内外许多学者都在积极探索肝肿瘤治疗的有效方法，目前药物治疗仍是其重要的治疗手段之一，但迄今为止，尚无实质意义上抗肝肿瘤的高效、低毒、价廉而理想的药物投入临床应用，近年来研究表明，中药治疗原发性肝癌取得了可喜的成果，其在改善患者的生存质量、延长生存期、控制病情发展等方面起到重要作用，日益受到重视和肯定。为此，国内外不少学者将研究重点转向中药抗肝肿瘤的研究。
柴胡皂甙d(SSd)是从传统中药柴胡中分离、提取出来的药理活性最强的成分之一，通常认为具有解热、镇痛和抗炎等药理作用，近年研究表明，SSd通过直接细胞毒作用，影响肿瘤细胞黏附及诱导细胞凋亡等途径，抑制或杀死人肺癌、胃癌、肝癌、白血病细胞和小鼠移植性S-180实体瘤等肿瘤细胞。如：有实验证实SSd具有干扰白血病细胞株HL-60S期DNA合成，干扰蛋白质代谢，抑制细胞增殖，诱导细胞凋亡的作用；还对人肝癌细胞株SMMC-7221具有增殖抑制和凋亡诱导作用等。
由此可见，柴胡皂甙d(SSd)有较强的抗肿瘤活性，加之其低毒、价廉，因此，柴胡皂甙d(SSd)可望做为一种抗肿瘤的良药在临床得到广泛的应用。目前关于SSd抗大鼠肝癌动物实验的研究国内外尚无相关文献报道，本实验采用二乙基亚硝胺(DEN)诱发大鼠肝癌模型，进行SSd抗肝肿瘤的实验研究。
研究目的：
采用二乙基亚硝胺制作大鼠肝癌模型，观察探讨柴胡皂甙d(SSd)对大鼠实验性肝癌的抑制作用和免疫功能的调节，为开发应用柴胡皂甙d(SSd)防治肿瘤提供科学的理论依据。
研究方法：
1、大鼠肝癌模型制备及药物干预：选取清洁级雄性SD大鼠90只，随机分为5组，分别为空白对照组、单纯造模组、SSd大剂量组(2．0 mg／kg)、SSd中剂量组(1．5 mg／kg)、SSd小剂量组(1．0 mg／kg)，模型组、SSd大、中、小剂量组均给予2g／LDEN灌胃，按照10 mg／kg体重给药，5次／周，16周后停药。同时SSd大、中、小剂量组以SSd腹腔注射，分别按2．0 mg／kh、1．5 mg／kg、1．0 mg／kg体重给药，7次／周，16周后停药。正常对照组以等量生理盐水灌胃，5次／周，16周后停药，同时腹腔注射等量蒸馏水，7次／周，16周后停药。
2、血清学指标测定：实验第18周，处死所有大鼠取血清，用自动生化分析仪检测丙氨酸氨基转移酶(ALT)、天门冬氨酸氨基转移酶(AST)、碱性磷酸酶(ALP)、γ-谷氨酰转肽酶(GGT)、α-L-岩藻糖苷酶(AFU)等肝功能指标。
3、大鼠免疫功能测定：应用流式细胞仪测定大鼠外周血T淋巴细胞亚群(CD_3~+CD_4~+％、CD_3~+CD_8~+％及CD_3~+CD_4~+／CD_3~+CD_8~+比值)。
4、肉眼观察肝脏变化，留取肝组织固定包埋，制成切片，HE染色比较各组大鼠肝组织病理变化；同时电镜观察各组肝组织超微结构变化。
5、用免疫组化的方法检测各组大鼠肝组织中IGF-Ⅱ和MMP-2的表达情况。
实验数据应用SPSS13．0统计软件进行统计学处理。
研究结果：
1、实验第18周收集大鼠血清检测肝功指标结果可见，与正常对照组比较，肝癌模型组大鼠的ALT、AST、ALP、GGT、AFU明显升高，差异有统计学意义(P<0．05)；SSd中、小剂量组中各项肝功指标较肝癌模型组降低，差异有统计学意义(P<0．05)，尤其是在SSd大剂量组(P<0．01)。
2、光镜观察病理结果显示，镜下可见正常对照组大鼠无1例出现自发性肝癌，肝小叶结构完整，肝细胞索排列整齐，细胞核清晰；肝癌模型组正常肝小叶结构被破坏，大体观白色结节处为肝癌细胞，根据1954年Edmondson分级法多为Ⅲ级；SSd各组镜下癌细胞分化程度高，异型性低，分级多为Ⅰ-Ⅱ级。
3、肝癌模型组与正常对照组T淋巴细胞亚群比较CD_4~+、CD_4~+／CD_8~+明显下降，CD_8~+上升，差异有统计学意义(P<0．05)；而SSd各组与肝癌模型组T淋巴细胞亚群比较CD_4~+、CD_4~+／CD_8~+明显回升，CD_8~+下降，差异有统计学意义(P<0．05)。
4、电镜观察单纯造模组大鼠出现肝细胞胞浆内肿胀空泡和大量的脂滴，线粒体肿大，粗面内质网扩张，HSC及Kupffer细胞增生活跃、体积增大，部分细胞结构不清晰，SSd各组肝细胞内偶见脂滴，线粒体无明显肿胀，内质网增生，HSC细胞体积不大，Kupffer细胞脂滴不多。
5、正常对照组未见IGF-Ⅱ和MMP-2阳性表达，而肝癌模型组和SSd各治疗组均可见呈棕黄色颗粒的IGF-Ⅱ和MMP-2阳性灶，两种细胞因子均主要定位于细胞浆内，少数细胞出现胞核着色，细胞膜未见着色。检测结果经统计学检验，肝癌模型组的IGF-Ⅱ和MMP-2表达高于SSd各治疗组，而SSd各治疗组间无显著性差异。
结论：
1、SSd对DEN大鼠肝癌的肝功能有一定的改善作用。
2、SSd可有效减轻DEN致大鼠肝癌肝脏病理学的改变以及肝脏超微结构的改变。
3、SSd对实验性肝癌大鼠的免疫功能有一定的改善作用。
4、SSd对实验性肝癌大鼠的形成有一定的抑制作用，其机制可能与SSd下调肝癌中IGF-Ⅱ和MMP-2的表达密切相关。
Background
Hepatic tumors is the most common malignant tumor in the human. Because of the rapid development of the illness and the high mortality rate, it has been called the "King of cancer". About 110,000 people die of liver cancer every year, which is account for 55% of the death count in the worldwide, so the diagnosis and treatment of hepatic tumors plays a decisive role in the world. Many domestic and overseas scholars are actively exploring the effective treatment of hepatic tumors.At present drug therapy is still an important means in the treatment, but so far there is no an ideal anti-tumor drug which is efficient, low toxic, cheap into clinical application. The study shows that in recent years the ttherapy of Chinese herbs achieved encouraging results in primary liver cancer, that has played an important role in improving the life quality, extending life span, controling the progression of disease. Chinese medicine has been increasingly received attention and affirmation, so many home and abroard scholars pay attention to the research of Chinese medicine of anti-hepatic tumors.
Saikosaponin-d (SSd) is the strongest pharmacological activity component extracted from the traditional Chinese medicine Bupleurum, that has the function of antipyretic, analgesic and anti-inflammatory, and other pharmacological effects. In recent years the study shows that SSd could inhibit or kill lung cancer, stomach cancer, liver cancer cells in human, and leukemic cells in transplanted S-180 solid tumors of mice and so on through the direct cytotoxic effects and the adhesion and apoptosis of tumor cell. For example, Some experiments have confirmed SSd could interfere S-period of DNA synthesis, protein metabolism, cell proliferation and induce apoptosis in leukemic cell line HL-60 and hepatocellular carcinoma cell line SMMC - 7221 of people.
It can be seen that Saikosaponin-d (SSd) has stronger anti-tumor activity, combined with its low toxicity, low price, therefore Saikosaponin-d (SSd) is expected to be as a kind of widely applied anti-tumor drug in clinic. Now there is no research literature in the world about animal experiments of anti-rat hepatoma, this experiment would evaluate the effects of Saikosaponin-d (SSd) on hepatoma in rats induced by diethylnitrosamine (DEN).
Objective
To study the preventive effects and immunoregulation effects of saikosaponin-d on experimental hepatocarcinogenesis in rats.Hepatocarcinogenesis model was induced by low doses of diethylnitrosamine interruptedly.That may be offer new theory and data for further research of SSd.
Methods
1. Establishment of hepatocarcinogenesis rat model and Experimental treatmentsNinety SD male rats, weighting 248.18±12.32g, were divided into 5 groups randomly, control group (10), model group (20) and three treatment groups (20 in each subgroup). Except the control group, other groups are administrated with 0.2% DEN at a dose of 10 mg·kg-1 bodyweight five times one week for sixteenth weeks. At the same time, rats in treatment groups were injected intraperitoneally with SSd at different doses (1.0,1.5 and 2.0 mg/kg) once daily for sixteenth weeks.While the control group received the same administration with the tales doses of normal sodium too.
2. Biochemical parameters of Serum
All rats were killed in the 18th week, the levels of alanine aminotransferase (ALT), aspartateaminotransferase(AST), alkaline phosphatase(ALP),γ-glutamyltransferase (GGT) andalpha-L-fucosidase(AFU) in serum were measured by biochemical examinations.
3.Using flow cytometry to study T lymphocyte sub-group (CD_3~+ CD_4~+%、CD_3~+ CD_8~+%及CD_3~+ CD_4~+/ CD_3~+ CD_8~+ ratio) .
4.The sections were stained with hemotoxylin & eosin (H&E).Hepatic microstructures werestudied under microscope.At the same time,The hepatic ultrastructure of all rats wasobserved by electron microscope.
5.The expressions of Insulin-like growth factor-Ⅱ(IGF-Ⅱ) and Matrix metalloproteinase-2(MMP-2) were detected by immunohistochemistry.
All of measured data were analyzed by statistic software SPSS13.0 for windows.
Results
1.All rats were killed in the 18th week, the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP),γ-glutamyltransferase (GGT) and alpha-L-fucosidase (AFU) in serum were measured by biochemical examinations, The levels of ALT, AST, ALP, GGT, AFU in serum were significantly increased in model group in compared with those in control group(P<0.05), while the treatment with SSd markedly reduced all the above criteria compared with the model group, especial for the High dose group. (162.56±20.70 vs 410.01±93.62, 210.45±22.76 vs 400.25±90.02, 220.62±24.65 vs 410.45±94.85,75.45±24.02 vs 157.84±6.75,126.63±25.13 vs 200.16±7.31, P < 0.01).
2.Histological examination demonstrated that SSd could attenuate the grade of cancer cell differentiation. In model group hepatic lobules structural are destroyed and the endpoint of white nodules could be cancer cells that are most gradeⅢ,while in SSd groups there are more higher cell differentiation and hypo-heteromorphism,most gradeⅠ-Ⅱ.
3. The level of CD_4~+, and CD_4~+/CD_8~+ ratio were significantly decreased in model group compared with those in control group, and the level of CD_8~+ were markly increased. (P < 0.05), while in the treatment with SSd the level of CD_4~+, and CD_4~+/CD_8~+ ratio were significantly increased compared with the model group, and the level of CD_8~+ were markly decreased.(P < 0.05).
4. The change of hepatic ultrastructure could be observed in model group. There were swelling vacuoles and fatty drops in hepatic plasma. SSd would reduce formation of lipid droplet, proliferation of endoplasmic reticulum, swelling of mitochondrion and inhibiting Kupffer cell and HSC activation.
5.The expression of IGF-Ⅱand MMP-2 were lower in the rats treated with SSd comparedwith those of the model group.
Conclusion
Saikosaponin-d could improve hepatic function and the immune function in rats with hepatocarcinoma. Similarly,histological examination and ultrastructural examination demonstrated that SSd could attenuate the grade of cancer cell differentiation and the change of ultrastructure. Moreover, SSd could significantly attenuate DEN-induced hepatocellular carcinoma in rats. Its mechanism of action may be related to inhibiting the expression of IGF-Ⅱand MMP-2.
肝肿瘤是人类最常见最难治疗的恶性肿瘤之一，因其病情发展快，死亡率极高，素有“癌中之王”之称。我国每年约有11万人死于肝肿瘤，占全世界肝肿瘤死亡数的55％左右，因此，肝肿瘤的诊治在世界范围内具有举足轻重的地位。一直以来，国内外许多学者都在积极探索肝肿瘤治疗的有效方法，目前药物治疗仍是其重要的治疗手段之一，但迄今为止，尚无实质意义上抗肝肿瘤的高效、低毒、价廉而理想的药物投入临床应用，近年来研究表明，中药治疗原发性肝癌取得了可喜的成果，其在改善患者的生存质量、延长生存期、控制病情发展等方面起到重要作用，日益受到重视和肯定。为此，国内外不少学者将研究重点转向中药抗肝肿瘤的研究。
柴胡皂甙d(SSd)是从传统中药柴胡中分离、提取出来的药理活性最强的成分之一，通常认为具有解热、镇痛和抗炎等药理作用，近年研究表明，SSd通过直接细胞毒作用，影响肿瘤细胞黏附及诱导细胞凋亡等途径，抑制或杀死人肺癌、胃癌、肝癌、白血病细胞和小鼠移植性S-180实体瘤等肿瘤细胞。如：有实验证实SSd具有干扰白血病细胞株HL-60S期DNA合成，干扰蛋白质代谢，抑制细胞增殖，诱导细胞凋亡的作用；还对人肝癌细胞株SMMC-7221具有增殖抑制和凋亡诱导作用等。
由此可见，柴胡皂甙d(SSd)有较强的抗肿瘤活性，加之其低毒、价廉，因此，柴胡皂甙d(SSd)可望做为一种抗肿瘤的良药在临床得到广泛的应用。目前关于SSd抗大鼠肝癌动物实验的研究国内外尚无相关文献报道，本实验采用二乙基亚硝胺(DEN)诱发大鼠肝癌模型，进行SSd抗肝肿瘤的实验研究。
研究目的：
采用二乙基亚硝胺制作大鼠肝癌模型，观察探讨柴胡皂甙d(SSd)对大鼠实验性肝癌的抑制作用和免疫功能的调节，为开发应用柴胡皂甙d(SSd)防治肿瘤提供科学的理论依据。
研究方法：
1、大鼠肝癌模型制备及药物干预：选取清洁级雄性SD大鼠90只，随机分为5组，分别为空白对照组、单纯造模组、SSd大剂量组(2．0 mg／kg)、SSd中剂量组(1．5 mg／kg)、SSd小剂量组(1．0 mg／kg)，模型组、SSd大、中、小剂量组均给予2g／LDEN灌胃，按照10 mg／kg体重给药，5次／周，16周后停药。同时SSd大、中、小剂量组以SSd腹腔注射，分别按2．0 mg／kh、1．5 mg／kg、1．0 mg／kg体重给药，7次／周，16周后停药。正常对照组以等量生理盐水灌胃，5次／周，16周后停药，同时腹腔注射等量蒸馏水，7次／周，16周后停药。
2、血清学指标测定：实验第18周，处死所有大鼠取血清，用自动生化分析仪检测丙氨酸氨基转移酶(ALT)、天门冬氨酸氨基转移酶(AST)、碱性磷酸酶(ALP)、γ-谷氨酰转肽酶(GGT)、α-L-岩藻糖苷酶(AFU)等肝功能指标。
3、大鼠免疫功能测定：应用流式细胞仪测定大鼠外周血T淋巴细胞亚群(CD_3~+CD_4~+％、CD_3~+CD_8~+％及CD_3~+CD_4~+／CD_3~+CD_8~+比值)。
4、肉眼观察肝脏变化，留取肝组织固定包埋，制成切片，HE染色比较各组大鼠肝组织病理变化；同时电镜观察各组肝组织超微结构变化。
5、用免疫组化的方法检测各组大鼠肝组织中IGF-Ⅱ和MMP-2的表达情况。
实验数据应用SPSS13．0统计软件进行统计学处理。
研究结果：
1、实验第18周收集大鼠血清检测肝功指标结果可见，与正常对照组比较，肝癌模型组大鼠的ALT、AST、ALP、GGT、AFU明显升高，差异有统计学意义(P<0．05)；SSd中、小剂量组中各项肝功指标较肝癌模型组降低，差异有统计学意义(P<0．05)，尤其是在SSd大剂量组(P<0．01)。
2、光镜观察病理结果显示，镜下可见正常对照组大鼠无1例出现自发性肝癌，肝小叶结构完整，肝细胞索排列整齐，细胞核清晰；肝癌模型组正常肝小叶结构被破坏，大体观白色结节处为肝癌细胞，根据1954年Edmondson分级法多为Ⅲ级；SSd各组镜下癌细胞分化程度高，异型性低，分级多为Ⅰ-Ⅱ级。
3、肝癌模型组与正常对照组T淋巴细胞亚群比较CD_4~+、CD_4~+／CD_8~+明显下降，CD_8~+上升，差异有统计学意义(P<0．05)；而SSd各组与肝癌模型组T淋巴细胞亚群比较CD_4~+、CD_4~+／CD_8~+明显回升，CD_8~+下降，差异有统计学意义(P<0．05)。
4、电镜观察单纯造模组大鼠出现肝细胞胞浆内肿胀空泡和大量的脂滴，线粒体肿大，粗面内质网扩张，HSC及Kupffer细胞增生活跃、体积增大，部分细胞结构不清晰，SSd各组肝细胞内偶见脂滴，线粒体无明显肿胀，内质网增生，HSC细胞体积不大，Kupffer细胞脂滴不多。
5、正常对照组未见IGF-Ⅱ和MMP-2阳性表达，而肝癌模型组和SSd各治疗组均可见呈棕黄色颗粒的IGF-Ⅱ和MMP-2阳性灶，两种细胞因子均主要定位于细胞浆内，少数细胞出现胞核着色，细胞膜未见着色。检测结果经统计学检验，肝癌模型组的IGF-Ⅱ和MMP-2表达高于SSd各治疗组，而SSd各治疗组间无显著性差异。
结论：
1、SSd对DEN大鼠肝癌的肝功能有一定的改善作用。
2、SSd可有效减轻DEN致大鼠肝癌肝脏病理学的改变以及肝脏超微结构的改变。
3、SSd对实验性肝癌大鼠的免疫功能有一定的改善作用。
4、SSd对实验性肝癌大鼠的形成有一定的抑制作用，其机制可能与SSd下调肝癌中IGF-Ⅱ和MMP-2的表达密切相关。
Background
Hepatic tumors is the most common malignant tumor in the human. Because of the rapid development of the illness and the high mortality rate, it has been called the "King of cancer". About 110,000 people die of liver cancer every year, which is account for 55% of the death count in the worldwide, so the diagnosis and treatment of hepatic tumors plays a decisive role in the world. Many domestic and overseas scholars are actively exploring the effective treatment of hepatic tumors.At present drug therapy is still an important means in the treatment, but so far there is no an ideal anti-tumor drug which is efficient, low toxic, cheap into clinical application. The study shows that in recent years the ttherapy of Chinese herbs achieved encouraging results in primary liver cancer, that has played an important role in improving the life quality, extending life span, controling the progression of disease. Chinese medicine has been increasingly received attention and affirmation, so many home and abroard scholars pay attention to the research of Chinese medicine of anti-hepatic tumors.
Saikosaponin-d (SSd) is the strongest pharmacological activity component extracted from the traditional Chinese medicine Bupleurum, that has the function of antipyretic, analgesic and anti-inflammatory, and other pharmacological effects. In recent years the study shows that SSd could inhibit or kill lung cancer, stomach cancer, liver cancer cells in human, and leukemic cells in transplanted S-180 solid tumors of mice and so on through the direct cytotoxic effects and the adhesion and apoptosis of tumor cell. For example, Some experiments have confirmed SSd could interfere S-period of DNA synthesis, protein metabolism, cell proliferation and induce apoptosis in leukemic cell line HL-60 and hepatocellular carcinoma cell line SMMC - 7221 of people.
It can be seen that Saikosaponin-d (SSd) has stronger anti-tumor activity, combined with its low toxicity, low price, therefore Saikosaponin-d (SSd) is expected to be as a kind of widely applied anti-tumor drug in clinic. Now there is no research literature in the world about animal experiments of anti-rat hepatoma, this experiment would evaluate the effects of Saikosaponin-d (SSd) on hepatoma in rats induced by diethylnitrosamine (DEN).
Objective
To study the preventive effects and immunoregulation effects of saikosaponin-d on experimental hepatocarcinogenesis in rats.Hepatocarcinogenesis model was induced by low doses of diethylnitrosamine interruptedly.That may be offer new theory and data for further research of SSd.
Methods
1. Establishment of hepatocarcinogenesis rat model and Experimental treatmentsNinety SD male rats, weighting 248.18±12.32g, were divided into 5 groups randomly, control group (10), model group (20) and three treatment groups (20 in each subgroup). Except the control group, other groups are administrated with 0.2% DEN at a dose of 10 mg·kg-1 bodyweight five times one week for sixteenth weeks. At the same time, rats in treatment groups were injected intraperitoneally with SSd at different doses (1.0,1.5 and 2.0 mg/kg) once daily for sixteenth weeks.While the control group received the same administration with the tales doses of normal sodium too.
2. Biochemical parameters of Serum
All rats were killed in the 18th week, the levels of alanine aminotransferase (ALT), aspartateaminotransferase(AST), alkaline phosphatase(ALP),γ-glutamyltransferase (GGT) andalpha-L-fucosidase(AFU) in serum were measured by biochemical examinations.
3.Using flow cytometry to study T lymphocyte sub-group (CD_3~+ CD_4~+%、CD_3~+ CD_8~+%及CD_3~+ CD_4~+/ CD_3~+ CD_8~+ ratio) .
4.The sections were stained with hemotoxylin & eosin (H&E).Hepatic microstructures werestudied under microscope.At the same time,The hepatic ultrastructure of all rats wasobserved by electron microscope.
5.The expressions of Insulin-like growth factor-Ⅱ(IGF-Ⅱ) and Matrix metalloproteinase-2(MMP-2) were detected by immunohistochemistry.
All of measured data were analyzed by statistic software SPSS13.0 for windows.
Results
1.All rats were killed in the 18th week, the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP),γ-glutamyltransferase (GGT) and alpha-L-fucosidase (AFU) in serum were measured by biochemical examinations, The levels of ALT, AST, ALP, GGT, AFU in serum were significantly increased in model group in compared with those in control group(P<0.05), while the treatment with SSd markedly reduced all the above criteria compared with the model group, especial for the High dose group. (162.56±20.70 vs 410.01±93.62, 210.45±22.76 vs 400.25±90.02, 220.62±24.65 vs 410.45±94.85,75.45±24.02 vs 157.84±6.75,126.63±25.13 vs 200.16±7.31, P < 0.01).
2.Histological examination demonstrated that SSd could attenuate the grade of cancer cell differentiation. In model group hepatic lobules structural are destroyed and the endpoint of white nodules could be cancer cells that are most gradeⅢ,while in SSd groups there are more higher cell differentiation and hypo-heteromorphism,most gradeⅠ-Ⅱ.
3. The level of CD_4~+, and CD_4~+/CD_8~+ ratio were significantly decreased in model group compared with those in control group, and the level of CD_8~+ were markly increased. (P < 0.05), while in the treatment with SSd the level of CD_4~+, and CD_4~+/CD_8~+ ratio were significantly increased compared with the model group, and the level of CD_8~+ were markly decreased.(P < 0.05).
4. The change of hepatic ultrastructure could be observed in model group. There were swelling vacuoles and fatty drops in hepatic plasma. SSd would reduce formation of lipid droplet, proliferation of endoplasmic reticulum, swelling of mitochondrion and inhibiting Kupffer cell and HSC activation.
5.The expression of IGF-Ⅱand MMP-2 were lower in the rats treated with SSd comparedwith those of the model group.
Conclusion
Saikosaponin-d could improve hepatic function and the immune function in rats with hepatocarcinoma. Similarly,histological examination and ultrastructural examination demonstrated that SSd could attenuate the grade of cancer cell differentiation and the change of ultrastructure. Moreover, SSd could significantly attenuate DEN-induced hepatocellular carcinoma in rats. Its mechanism of action may be related to inhibiting the expression of IGF-Ⅱand MMP-2.
引文
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