参桃软肝胶囊抗肝癌作用及其机理初探
摘要
目的
本试验目的在于从体内试验、体外实验实验入手,研究参桃软肝胶囊治疗肝癌的机理。
方法
1.体外实验：制备低、中、高剂量含药血清,取处于对数增长期的肿瘤细胞,实验分为6组,即正常对照组,正常血清组,低剂量含药血清组,中剂量含药血清组,高剂量含药血清组和阳性对照组。采用MTT法检测含药血清对于HepG2人肝癌细胞增殖情况的影响；采用流式细胞术检测含药血清对HepG2人肝癌细胞的早期凋亡情况的影响；采用RT-PCR检测含药血清对HepG2人肝癌细胞内bax和bcl-2mRNA水平的影响；采用wb检测含药血清对细胞内bax、bcl-2和活化的caspase-3蛋白水平的影响。
2.体内试验：建立H22荷瘤小鼠模型,将荷瘤小鼠随机分为五组,分别为阴性对照组、低剂量组、中剂量组、高剂量组和阳性对照组,每组8只。阴性对照组(生理盐水),低剂量组(参桃软肝胶囊混悬液0.35g/kg),中剂量组(参桃软肝胶囊混悬液0.7g/kg),高剂量组(参桃软肝胶囊混悬液1.4g/kg),阳性对照组(顺铂注射液2.0mg/kg),每天2次,共3周,停药后24小时取材。观察药物对荷瘤小鼠的抑瘤作用以及促进肿瘤组织内肿瘤细胞凋亡作用；检测荷瘤小鼠血浆及肿瘤组织内TNF-α、IL-1β、IL-6的含量、检测荷瘤小鼠脾脏淋巴细胞对H22细胞的杀伤活性、检测荷瘤小鼠外周血及脾脏淋巴细胞CD4+和CD8+细胞比例,以药物对荷瘤小鼠的免疫功能影响；针对肿瘤组织,采用免疫组化法检测CD34和VEGF水平、采用RT-PCR法检测bcl-2和bax mRNA水平、采用免疫组化检测活化的caspase-3水平、采用western blot方法检测bcl-2,bax和活化的caspase-3的蛋白水平,以探讨参桃软肝胶囊的抗肝癌机制。
结果
1.体外实验
1.1参桃软肝胶囊含药血清作用于HepG,人肝癌细胞后,与用药前相比,形态发生改变,细胞数量减少。
1.2含药血清对HepG2人肝癌细胞增殖的影响,正常大鼠的血清对人肝癌细胞HepG2的增殖没有明显的影响(P>0.05),低、中和高剂量含药血清和顺铂都能够明显抑制肝癌细胞的增殖(P<0.05)。其中,低、中和高剂量含药血清处理组随着血清中中药有效成分浓度的增加,抑制细胞增殖的能力逐渐加强,呈现一定的剂量依赖关系。
1.3流式细胞术结果显示,普通培养的人肝癌细胞HepG2自然凋亡率比较低,约为5%-10%左右,正常的大鼠血清对HepG2细胞的凋亡没有明显的影响,低、中和高剂量含药血清和顺铂都能够明显诱导HepG2细胞的凋亡。
1.4RT-PCR结果显示,正常大鼠的血清对人肝癌细胞HepG2胞内Bax和Bcl-2mRNA水平没有明显的影响(P>0.05),低中高剂量的含药血清和阳性化疗药物都能够明显诱导Bax基因的转录(P<0.01),抑制Bcl-2基因的转录(P<0.01),明显上调Bax/Bcl-2基因的比值。其中低中高剂量含药血清组Bax mRNA水平的上调,Bcl-2mRNA水平的下调,同药物浓度呈现一定的剂量依赖关系。
1.5western blot结果显示,正常大鼠的血清对人肝癌细胞HepG2胞内Bax、Bcl-2和活化的caspase-3蛋白水平没有明显的影响(P>0.05),低、中、高剂量的含药血清和阳性化疗药物都能够明显诱导Bax蛋白的表达,抑制Bcl-2蛋白的表达,明显上调Bax/Bcl-2蛋白的比值,活化caspase-3。其中,低、中、高剂量组中,随着药物浓度的增加,这种作用不断增强。
2.体内实验
2.1模型组肿瘤最大,重量最重；阳性对照组肿瘤最小,肿瘤最轻；低剂量给药组、中剂量给药组和高剂量给药组介于它们之间,随着药物浓度的增加,抑瘤效果不断增强,同模型组相比,各组瘤重明显的降低,具有显著差异(P<0.01)。
2.2模型组小鼠肿瘤组织本身就存在一定的自然凋亡率,在2%-5%左右,低、中、高剂量给药处理和腹腔注射阳性化疗药物,都能够明显的在体内诱导肿瘤细胞的凋亡,细胞的凋亡率明显增加,其中以阳性对照组最为明显(50%-70%),其次为中药高剂量组(40%-60%),接着是中药中剂量组(30%-50%),最次是中药低剂量组(20%-30%)。
2.3荷瘤小鼠血浆中细胞因子的含量明显的低于正常对照组(P<0.01),给予中药处理,随着药物浓度的增加,能够明显的增加血浆内细胞因子的含量,同模型组比,具有明显的差异(P<0.01)；阳性化疗药物处理的小鼠,血浆内这些细胞因子的含量并没有得到改善(P>0.05)。小鼠肿瘤组织内这些细胞因子的含量也有类似于血浆的趋势,中药组均能明显提高局部肿瘤组织内细胞因子的含量,从而改善机体的免疫状态,而阳性化疗药物则没有此作用。
2.4同正常小鼠比,荷瘤小鼠外周血和脾脏T淋巴细胞中CD4+细胞比例明显降低,CD8+细胞比例明显升高,CD4+/CD8+的比值明显降低。中药低、中、高剂量组均能明显改善小鼠的细胞免疫功能(P<0.01),而阳性化疗药物则没有改善作用(P>0.05)。
2.5同正常小鼠相比,荷瘤小鼠脾脏淋巴细胞对H22细胞的杀伤活性明显减弱,中药低、中、高剂量组能够明显增强荷瘤小鼠脾脏淋巴细胞的杀伤活性(P<0.01),而且杀伤活性均高于正常非荷瘤小鼠。阳性化疗药物对荷瘤小鼠脾脏淋巴细胞的杀伤活性没有改善作用(P>0.05)。
2.6同模型组,阳性对照组、中药低、中、高剂量组MVD计数明显减少,具有明显的统计学意义(P<0.01)；各组之间VEGF表达水平无明显差异。
2.7中药低、中、高剂量组的中药和阳性化疗药物都能够明显诱导Bax基因的转录(P<0.01),抑制Bcl-2基因的转录(P<0.01),明显上调Bax/Bcl-2基因的比值。其中低中高剂量组Bax mRNA水平的上调,Bcl-2mRNA水平的下调,同药物浓度呈现一定的剂量依赖关系。
2.8模型组中,活化的caspase-3日性细胞比例最低,阳性对照组最高,低中高剂量中药处理组介于2者之间,呈现一定的剂量依赖关系。
2.9低中高剂量的中药和阳性化疗药物都能够明显诱导Bax蛋白的表达,抑制Bcl-2蛋白的表达,明显上调Bax/Bcl-2蛋白的比值,其中低中高剂量组中,随着药物浓度的增加,这种作用不断增强。小鼠肿瘤组织内活化的caspase-3蛋白水平表达趋势也与上面免疫组化的结果类似,与模型组相比,低中高剂量组和阳性组小鼠肿瘤组织内活化的caspase-3蛋白水平明显增加,其中低中高剂量组中,活化的casapse-3蛋白水平的增加同剂量呈现一定的依赖关系。
结论
1.参桃软肝胶囊含药血清可能抑制HepG2人肝癌细胞生长,并诱导其凋亡。
2.参桃软肝胶囊可能抑制H22肝癌小鼠肿瘤细胞生长,并诱导凋亡的作用。
3.参桃软肝胶囊可能具有改善机体的免疫功能状态的作用。
4.参桃软肝胶囊可能具有抑制肿瘤血管生成的作用。
5.参桃软肝胶囊可能通过调节Bax/Bcl-2比值,活化casapse-3蛋白来实现抗肿瘤的作用。
Objective
This trial aims to study the therapeutic mechanism of Sen Tao Ruan Gan Capsules (STRGC) for hepatoma from three perspectives, including in vivo, in vitro and Immunohistochemistry.
Methods
1. In vitro study
We prepared low, medium and high dosage of drug serums and tumor cells in increased logarithmic phase. Six groups were established in the trial, including normal control group, normal serum group, three drug serum groups (low, medium and high dosages) and positive control group. With MTT T assay, inhibition ratios of drug serums was determined in the cells of hepatoma HepG2, respectively;with Flow cytometry. effect of drug serum on early apoptosis in human hepatoma HepG2cells; with RT-PCR, effect of drug serum on levels of bax and bcl-2mRNA in human hepatoma HepG2cells; with wb, effect of drug serum on levels of bax, bcl-2and activated caspase-3proteins in cells.
2. In vivo study
The tumor-bearing mouse models were randomized into five groups,8in each, with the aim to explore anti-hepatoma mechanism of STRGC. The negative control group was assigned to saline; low, medium and high dosage groups to STRGC Suspension0.35g/kg,0.7g/kg and1.4g/kg, respectively; positive control group to Cisplatin Injection2.Omg/kg; twice daily, three weeks in total. The samples were harvested24hours after the withdrawal. We observed the drug effect on tumor inhibition and apoptosis in tumor cells in mice and determine the contents of TNF-a, IL-1β and IL-6in serums and tumor tissues of mice as well as the killing quality of splenic lymphocytes on H22cells, the proportion of CD4+and CD8+in peripheral blood and splenic lymphocytes and drug effect on mice's immunity. In tumor tissues, with immunohistochemistry, the levels of CD34and VEGF and activated caspase-3were determined; with RT-PCR, the levels of bcl-2and bax-RNA; wi th western blot, the levels of bcl-2, activated caspase-3proteins.
Results
1. In vitro study
1.1After the administration of STRGC-containing serum, morphology change was noted in the human hepatoma HepG2cells as well as a decline in cell number.
1.2The role of drug serum in hepatoma cell proliferation:the normal mouse serum plays'no role in the hepatoma cell proliferation (P>0.05). Low, medium and high dosage of drug serum and Cisplatin showed significant inhibitory action (P<0.05). The inhibitory action grew with the increased concentration of STRGC in serum in three drug serum groups (low, medium and high dosages) and had dose-dependent quality.
1.3Flow cytometry revealed that:normally cultured human hepatcma hepG2cells have5%-10%natural apoptosis; normal mouse serum has no remarkable effect on apoptosis of hepG2cells; low, medium and high dosage of drug serum and Cisplatin had significant role in inducing apoptosis of hepG2cells.
1.4RT-PCR showed that:normal mouse serum had no significant effect on the levels of Bax and Bcl-2mRNA in human hepatoma HepG2cells (P>0.05). Low, medium and high dosage drug serums and positive chemo drug played a significant role in inducing the transcription of Bax gene (P<0.01), inhibiting the transcription of Bcl-2gene (P<0.01) and elevating the value of Bax/Bcl-2. The increase in the Bax mRNA level and decrease in Bcl-2mRNA level in low, medium and high dosage drug serum groups were to some extent dependent upon the STRGC concentration.
1.5western blot suggested that:normal mouse serum had no significant effect on the levels of Bax, Bcl-2and caspase-3proteins in human hepatoma HepG2cells (P>0.05). Low, medium and high dosage drug serums and positive chemo drug played a significant role in inducing the expression of Bax protein, inhibiting the expression of Bcl-2protein, elevating the ratio of Bax/Bcl-2and activating caspase-3. This action grew with the increased concentration of STRGC in low, medium and high dosage groups.
2. In vivo study
2.1The maximum tumor size and weight were noted in model group while the minimum in positive control group with the low, medium and high dosage group in between. The anti-tumor effect grew with the increased concentration of drug and compared to the model group, a significant reduction was noted in tumor weight in all other groups, which is significantly different (P<0.01).
2.2The mouse tumor tissue in model group has2%-5%natural apoptosis. The low, medium and high dosage of STRGC and peritoneal injection of positive chemo drug played a significant role in inducing the apoptosis of tumor cells in vivo. The apoptosis ratio was significantly elevated and the highest of which was noted in positive control group (50%-70%), followed successively by high dosage group (40%-60%), medium dosage group (30%-50%), and the low dosage group (20%-30%).
2.3The content of cytokine in serum of tumor-bearing mouse, which was obviously lower than that in the normal control group (P<0.01), was significantly elevated with the increased concentration of STRGC, which is significantly different (P<0.01), compared to the model group. No improvement was made in the cytokine content in serum of mouse managed with positive chemo drug (P>0.05). The cytokine content in the tumor tissue of mouse held the trend that was similar to blood serum. The STRGC was able to significantly elevate the content of cytokine in local tumor tissue, thus improving the immunity, but not the positive chemo drug.
2.4Compared with the normal mouse, a reduction was noted in CD4+cells and an increase in CD8+cells in the peripheral blood and splenic T lymphocytes, resulting in a reduction in the value of CD4+/CD8+.The low, medium and high dosage of STRGC was capable of significantly improving the cellular immunity in mice (P<0.01), but not the positive chemo drug (P>0.05).
2.5Compared with the normal mouse, the ability of splenic lymphocytes to kill the H22cells in tumor-bearing mouse is significantly weakened. The low, medium and high dosage of STRGC can significantly enhance the killing ability of splenic lymphocytes (P<0.01) which is stronger than that in normal mouse, but not the positive chemo drug (P>0.05).
2.6Compared with the model group, a significant reduction in the MVD counts was noted in the positive control group, low, medium and high dosage group, which is statistically significant (P<0.01). There's no definite difference in VEGF expression level between groups.
2.7STRGC in low, medium and high dosage groups and positive chemo drug played a significant role in inducing the transcription of Bax gene (P<0.01), inhibiting the transcription of Bcl-2gene (P<0.01) and elevating the value of Bax/Bcl-2. The increase in the Bax mRNA level and decrease in Bcl-2mRNA level in low, medium and high dosage drug serum groups were to some extent dependent upon the STRGC concentration.
2.8The minimum proportion of activated caspase-3cell was noted in model group while the maximum in positive group, with low, medium and high dosage group in between, which was to some extent dependent upon the dosage.
2.9Low, medium and high dosage of STRGC and positive chemo drug played a role in inducing the expression of Bax protein, inhibiting the expression of Bcl-2protein and elevating the value of Bax/Bcl-2. In low, medium and high dosage groups, these effects grew with the increased concentration of STRGC. The tread of expression of activated caspase-3protein in tumor tissues of mice was similar to that in the immunohistochemistry study and compared with the model group, a significant increase in the level of activated caspase-3protein was noted in the tumor tissues of mice in the low, medium and high dosage groups (depending on the dosage) and positive group.
Conclusions
1. STRGC-containing serum could play a role in inhibition and apoptosis of the human hepatoma HepG2cells.
2. STRGC could play a role in tumor growth inhibition and apoptosis of tumor cells in H22hepatoma-bearing mouse.
3. STRGC could contribute to improving the immunity.
4. STRGC could play a role in inhibition of tumor angiogenesis.
5. STRGC could play a role in activating caspase-3protein for suppress tumor by modulating the ratio of Bax/Bcl-2.
本试验目的在于从体内试验、体外实验实验入手,研究参桃软肝胶囊治疗肝癌的机理。
方法
1.体外实验：制备低、中、高剂量含药血清,取处于对数增长期的肿瘤细胞,实验分为6组,即正常对照组,正常血清组,低剂量含药血清组,中剂量含药血清组,高剂量含药血清组和阳性对照组。采用MTT法检测含药血清对于HepG2人肝癌细胞增殖情况的影响；采用流式细胞术检测含药血清对HepG2人肝癌细胞的早期凋亡情况的影响；采用RT-PCR检测含药血清对HepG2人肝癌细胞内bax和bcl-2mRNA水平的影响；采用wb检测含药血清对细胞内bax、bcl-2和活化的caspase-3蛋白水平的影响。
2.体内试验：建立H22荷瘤小鼠模型,将荷瘤小鼠随机分为五组,分别为阴性对照组、低剂量组、中剂量组、高剂量组和阳性对照组,每组8只。阴性对照组(生理盐水),低剂量组(参桃软肝胶囊混悬液0.35g/kg),中剂量组(参桃软肝胶囊混悬液0.7g/kg),高剂量组(参桃软肝胶囊混悬液1.4g/kg),阳性对照组(顺铂注射液2.0mg/kg),每天2次,共3周,停药后24小时取材。观察药物对荷瘤小鼠的抑瘤作用以及促进肿瘤组织内肿瘤细胞凋亡作用；检测荷瘤小鼠血浆及肿瘤组织内TNF-α、IL-1β、IL-6的含量、检测荷瘤小鼠脾脏淋巴细胞对H22细胞的杀伤活性、检测荷瘤小鼠外周血及脾脏淋巴细胞CD4+和CD8+细胞比例,以药物对荷瘤小鼠的免疫功能影响；针对肿瘤组织,采用免疫组化法检测CD34和VEGF水平、采用RT-PCR法检测bcl-2和bax mRNA水平、采用免疫组化检测活化的caspase-3水平、采用western blot方法检测bcl-2,bax和活化的caspase-3的蛋白水平,以探讨参桃软肝胶囊的抗肝癌机制。
结果
1.体外实验
1.1参桃软肝胶囊含药血清作用于HepG,人肝癌细胞后,与用药前相比,形态发生改变,细胞数量减少。
1.2含药血清对HepG2人肝癌细胞增殖的影响,正常大鼠的血清对人肝癌细胞HepG2的增殖没有明显的影响(P>0.05),低、中和高剂量含药血清和顺铂都能够明显抑制肝癌细胞的增殖(P<0.05)。其中,低、中和高剂量含药血清处理组随着血清中中药有效成分浓度的增加,抑制细胞增殖的能力逐渐加强,呈现一定的剂量依赖关系。
1.3流式细胞术结果显示,普通培养的人肝癌细胞HepG2自然凋亡率比较低,约为5%-10%左右,正常的大鼠血清对HepG2细胞的凋亡没有明显的影响,低、中和高剂量含药血清和顺铂都能够明显诱导HepG2细胞的凋亡。
1.4RT-PCR结果显示,正常大鼠的血清对人肝癌细胞HepG2胞内Bax和Bcl-2mRNA水平没有明显的影响(P>0.05),低中高剂量的含药血清和阳性化疗药物都能够明显诱导Bax基因的转录(P<0.01),抑制Bcl-2基因的转录(P<0.01),明显上调Bax/Bcl-2基因的比值。其中低中高剂量含药血清组Bax mRNA水平的上调,Bcl-2mRNA水平的下调,同药物浓度呈现一定的剂量依赖关系。
1.5western blot结果显示,正常大鼠的血清对人肝癌细胞HepG2胞内Bax、Bcl-2和活化的caspase-3蛋白水平没有明显的影响(P>0.05),低、中、高剂量的含药血清和阳性化疗药物都能够明显诱导Bax蛋白的表达,抑制Bcl-2蛋白的表达,明显上调Bax/Bcl-2蛋白的比值,活化caspase-3。其中,低、中、高剂量组中,随着药物浓度的增加,这种作用不断增强。
2.体内实验
2.1模型组肿瘤最大,重量最重；阳性对照组肿瘤最小,肿瘤最轻；低剂量给药组、中剂量给药组和高剂量给药组介于它们之间,随着药物浓度的增加,抑瘤效果不断增强,同模型组相比,各组瘤重明显的降低,具有显著差异(P<0.01)。
2.2模型组小鼠肿瘤组织本身就存在一定的自然凋亡率,在2%-5%左右,低、中、高剂量给药处理和腹腔注射阳性化疗药物,都能够明显的在体内诱导肿瘤细胞的凋亡,细胞的凋亡率明显增加,其中以阳性对照组最为明显(50%-70%),其次为中药高剂量组(40%-60%),接着是中药中剂量组(30%-50%),最次是中药低剂量组(20%-30%)。
2.3荷瘤小鼠血浆中细胞因子的含量明显的低于正常对照组(P<0.01),给予中药处理,随着药物浓度的增加,能够明显的增加血浆内细胞因子的含量,同模型组比,具有明显的差异(P<0.01)；阳性化疗药物处理的小鼠,血浆内这些细胞因子的含量并没有得到改善(P>0.05)。小鼠肿瘤组织内这些细胞因子的含量也有类似于血浆的趋势,中药组均能明显提高局部肿瘤组织内细胞因子的含量,从而改善机体的免疫状态,而阳性化疗药物则没有此作用。
2.4同正常小鼠比,荷瘤小鼠外周血和脾脏T淋巴细胞中CD4+细胞比例明显降低,CD8+细胞比例明显升高,CD4+/CD8+的比值明显降低。中药低、中、高剂量组均能明显改善小鼠的细胞免疫功能(P<0.01),而阳性化疗药物则没有改善作用(P>0.05)。
2.5同正常小鼠相比,荷瘤小鼠脾脏淋巴细胞对H22细胞的杀伤活性明显减弱,中药低、中、高剂量组能够明显增强荷瘤小鼠脾脏淋巴细胞的杀伤活性(P<0.01),而且杀伤活性均高于正常非荷瘤小鼠。阳性化疗药物对荷瘤小鼠脾脏淋巴细胞的杀伤活性没有改善作用(P>0.05)。
2.6同模型组,阳性对照组、中药低、中、高剂量组MVD计数明显减少,具有明显的统计学意义(P<0.01)；各组之间VEGF表达水平无明显差异。
2.7中药低、中、高剂量组的中药和阳性化疗药物都能够明显诱导Bax基因的转录(P<0.01),抑制Bcl-2基因的转录(P<0.01),明显上调Bax/Bcl-2基因的比值。其中低中高剂量组Bax mRNA水平的上调,Bcl-2mRNA水平的下调,同药物浓度呈现一定的剂量依赖关系。
2.8模型组中,活化的caspase-3日性细胞比例最低,阳性对照组最高,低中高剂量中药处理组介于2者之间,呈现一定的剂量依赖关系。
2.9低中高剂量的中药和阳性化疗药物都能够明显诱导Bax蛋白的表达,抑制Bcl-2蛋白的表达,明显上调Bax/Bcl-2蛋白的比值,其中低中高剂量组中,随着药物浓度的增加,这种作用不断增强。小鼠肿瘤组织内活化的caspase-3蛋白水平表达趋势也与上面免疫组化的结果类似,与模型组相比,低中高剂量组和阳性组小鼠肿瘤组织内活化的caspase-3蛋白水平明显增加,其中低中高剂量组中,活化的casapse-3蛋白水平的增加同剂量呈现一定的依赖关系。
结论
1.参桃软肝胶囊含药血清可能抑制HepG2人肝癌细胞生长,并诱导其凋亡。
2.参桃软肝胶囊可能抑制H22肝癌小鼠肿瘤细胞生长,并诱导凋亡的作用。
3.参桃软肝胶囊可能具有改善机体的免疫功能状态的作用。
4.参桃软肝胶囊可能具有抑制肿瘤血管生成的作用。
5.参桃软肝胶囊可能通过调节Bax/Bcl-2比值,活化casapse-3蛋白来实现抗肿瘤的作用。
Objective
This trial aims to study the therapeutic mechanism of Sen Tao Ruan Gan Capsules (STRGC) for hepatoma from three perspectives, including in vivo, in vitro and Immunohistochemistry.
Methods
1. In vitro study
We prepared low, medium and high dosage of drug serums and tumor cells in increased logarithmic phase. Six groups were established in the trial, including normal control group, normal serum group, three drug serum groups (low, medium and high dosages) and positive control group. With MTT T assay, inhibition ratios of drug serums was determined in the cells of hepatoma HepG2, respectively;with Flow cytometry. effect of drug serum on early apoptosis in human hepatoma HepG2cells; with RT-PCR, effect of drug serum on levels of bax and bcl-2mRNA in human hepatoma HepG2cells; with wb, effect of drug serum on levels of bax, bcl-2and activated caspase-3proteins in cells.
2. In vivo study
The tumor-bearing mouse models were randomized into five groups,8in each, with the aim to explore anti-hepatoma mechanism of STRGC. The negative control group was assigned to saline; low, medium and high dosage groups to STRGC Suspension0.35g/kg,0.7g/kg and1.4g/kg, respectively; positive control group to Cisplatin Injection2.Omg/kg; twice daily, three weeks in total. The samples were harvested24hours after the withdrawal. We observed the drug effect on tumor inhibition and apoptosis in tumor cells in mice and determine the contents of TNF-a, IL-1β and IL-6in serums and tumor tissues of mice as well as the killing quality of splenic lymphocytes on H22cells, the proportion of CD4+and CD8+in peripheral blood and splenic lymphocytes and drug effect on mice's immunity. In tumor tissues, with immunohistochemistry, the levels of CD34and VEGF and activated caspase-3were determined; with RT-PCR, the levels of bcl-2and bax-RNA; wi th western blot, the levels of bcl-2, activated caspase-3proteins.
Results
1. In vitro study
1.1After the administration of STRGC-containing serum, morphology change was noted in the human hepatoma HepG2cells as well as a decline in cell number.
1.2The role of drug serum in hepatoma cell proliferation:the normal mouse serum plays'no role in the hepatoma cell proliferation (P>0.05). Low, medium and high dosage of drug serum and Cisplatin showed significant inhibitory action (P<0.05). The inhibitory action grew with the increased concentration of STRGC in serum in three drug serum groups (low, medium and high dosages) and had dose-dependent quality.
1.3Flow cytometry revealed that:normally cultured human hepatcma hepG2cells have5%-10%natural apoptosis; normal mouse serum has no remarkable effect on apoptosis of hepG2cells; low, medium and high dosage of drug serum and Cisplatin had significant role in inducing apoptosis of hepG2cells.
1.4RT-PCR showed that:normal mouse serum had no significant effect on the levels of Bax and Bcl-2mRNA in human hepatoma HepG2cells (P>0.05). Low, medium and high dosage drug serums and positive chemo drug played a significant role in inducing the transcription of Bax gene (P<0.01), inhibiting the transcription of Bcl-2gene (P<0.01) and elevating the value of Bax/Bcl-2. The increase in the Bax mRNA level and decrease in Bcl-2mRNA level in low, medium and high dosage drug serum groups were to some extent dependent upon the STRGC concentration.
1.5western blot suggested that:normal mouse serum had no significant effect on the levels of Bax, Bcl-2and caspase-3proteins in human hepatoma HepG2cells (P>0.05). Low, medium and high dosage drug serums and positive chemo drug played a significant role in inducing the expression of Bax protein, inhibiting the expression of Bcl-2protein, elevating the ratio of Bax/Bcl-2and activating caspase-3. This action grew with the increased concentration of STRGC in low, medium and high dosage groups.
2. In vivo study
2.1The maximum tumor size and weight were noted in model group while the minimum in positive control group with the low, medium and high dosage group in between. The anti-tumor effect grew with the increased concentration of drug and compared to the model group, a significant reduction was noted in tumor weight in all other groups, which is significantly different (P<0.01).
2.2The mouse tumor tissue in model group has2%-5%natural apoptosis. The low, medium and high dosage of STRGC and peritoneal injection of positive chemo drug played a significant role in inducing the apoptosis of tumor cells in vivo. The apoptosis ratio was significantly elevated and the highest of which was noted in positive control group (50%-70%), followed successively by high dosage group (40%-60%), medium dosage group (30%-50%), and the low dosage group (20%-30%).
2.3The content of cytokine in serum of tumor-bearing mouse, which was obviously lower than that in the normal control group (P<0.01), was significantly elevated with the increased concentration of STRGC, which is significantly different (P<0.01), compared to the model group. No improvement was made in the cytokine content in serum of mouse managed with positive chemo drug (P>0.05). The cytokine content in the tumor tissue of mouse held the trend that was similar to blood serum. The STRGC was able to significantly elevate the content of cytokine in local tumor tissue, thus improving the immunity, but not the positive chemo drug.
2.4Compared with the normal mouse, a reduction was noted in CD4+cells and an increase in CD8+cells in the peripheral blood and splenic T lymphocytes, resulting in a reduction in the value of CD4+/CD8+.The low, medium and high dosage of STRGC was capable of significantly improving the cellular immunity in mice (P<0.01), but not the positive chemo drug (P>0.05).
2.5Compared with the normal mouse, the ability of splenic lymphocytes to kill the H22cells in tumor-bearing mouse is significantly weakened. The low, medium and high dosage of STRGC can significantly enhance the killing ability of splenic lymphocytes (P<0.01) which is stronger than that in normal mouse, but not the positive chemo drug (P>0.05).
2.6Compared with the model group, a significant reduction in the MVD counts was noted in the positive control group, low, medium and high dosage group, which is statistically significant (P<0.01). There's no definite difference in VEGF expression level between groups.
2.7STRGC in low, medium and high dosage groups and positive chemo drug played a significant role in inducing the transcription of Bax gene (P<0.01), inhibiting the transcription of Bcl-2gene (P<0.01) and elevating the value of Bax/Bcl-2. The increase in the Bax mRNA level and decrease in Bcl-2mRNA level in low, medium and high dosage drug serum groups were to some extent dependent upon the STRGC concentration.
2.8The minimum proportion of activated caspase-3cell was noted in model group while the maximum in positive group, with low, medium and high dosage group in between, which was to some extent dependent upon the dosage.
2.9Low, medium and high dosage of STRGC and positive chemo drug played a role in inducing the expression of Bax protein, inhibiting the expression of Bcl-2protein and elevating the value of Bax/Bcl-2. In low, medium and high dosage groups, these effects grew with the increased concentration of STRGC. The tread of expression of activated caspase-3protein in tumor tissues of mice was similar to that in the immunohistochemistry study and compared with the model group, a significant increase in the level of activated caspase-3protein was noted in the tumor tissues of mice in the low, medium and high dosage groups (depending on the dosage) and positive group.
Conclusions
1. STRGC-containing serum could play a role in inhibition and apoptosis of the human hepatoma HepG2cells.
2. STRGC could play a role in tumor growth inhibition and apoptosis of tumor cells in H22hepatoma-bearing mouse.
3. STRGC could contribute to improving the immunity.
4. STRGC could play a role in inhibition of tumor angiogenesis.
5. STRGC could play a role in activating caspase-3protein for suppress tumor by modulating the ratio of Bax/Bcl-2.
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