摘要
目的：在临床应用及计算机分子对接模拟基础上推断FYG具有免疫调节功效,首先利用动物模型确认了FYG的免疫调节作用,继而在细胞水平进行了部分作用机制的探讨；回归临床评估扶正抑瘤颗粒(Fuzheng Yiliu Granules, FYG)影响消化道肿瘤患者围化疗期免疫调节及生活质量的临床疗效。
方法：
1.建立H22小鼠肝癌细胞的皮下移植瘤模型,然后随机分配到对照组(Vehicle,灌胃生理盐水),5-FU组(10mg/kg),5-FU+FYG组(10mg/kg+18g/kg)和FYG组(18g/kg),给药5天后测量肿瘤重量和体积,检测CD3、CD4、CD8、Treg、NK细胞的百分含量、血清中白介素2(interleukin-2, IL-2)和肿瘤坏死因子(tumor necrosis factor α, TNF-α)的浓度,肿瘤组织的凋亡情况及相关凋亡因子蛋白和基因的表达(Bax、Bcl-2、p53、C-myc)及5-FU靶酶胸苷酸合成酶(Thymidylate Synthase, TS)的表达水平。培养人肝癌细胞HepG2, FYG生药单独给药及联合5-FU给药后,噻唑蓝(Methylthiazolyldiphenyl-tetrazolium bromide, MTT)检测细胞活力。SD大鼠灌胃FYG7天(高剂量组：25.2g/kg;中剂量组：12.6g/kg;氐剂量组：6.3g/kg),取含药血清后检测工L-2和TNF-α的浓度,含药血清干预HepG2细胞后检测细胞活力、细胞增殖及凋亡情况。
2.按标准入选胃癌、大肠癌患者患者85例,按最小化随机分为治疗组50例、对照组35例；治疗组给予益气养阴中药联合FOLFOX4化疗方案,对照组给予安慰剂联合FOLFOX化疗方案。定期评估体力状况、生存质量、安全性。于基线期、化疗后第4周、第8周、研究结束时检测外周血T淋巴细胞及亚群(CD3、CD4、CD8、调节T细胞(Regulatory T cells, Treg)及CD4/CD8),自然杀伤细胞(Natural killing cells, NK),免疫球蛋白(IgA、IgG、IgM)及补体(C3、C4)浓度。
结果：
1.FYG单独使用表现出一定的抗肿瘤功效和诱导凋亡作用(与对照组相比P＜0.05),肿瘤组织中Bax基因表达明显上调(P＜0.05),FYG+5-FU组小鼠肿瘤组织中野生型p53基因表达显著上调(P＜O.05)。FYG联合5-FU给药后显著抑制了小鼠皮下移植瘤的生长且诱导肿瘤组织发生凋亡(与对照组相比P＜0.0l,与5-FU或FYG组相比P＜0.05),但未体现协同作用。FYG联合5-FU后上调了胸腺指数(与对照组相比P＜0.05)。,FYG上调了被5-FU抑制的WBC及LY细胞数量(FYG+5-FU vs5-FU, P<0.01; FYG vs vehicle, P<0.01)。FYG组与对照组相比显著增加外周血中CD3(R0.01),CD4(P＜0.01)及NK(P＜0.01)细胞的水平,抑制了Treg细胞的表达(P＜0.01)。FYG联合5-FU后增加了CD3和CD4的百分含量(与对照组相比P＜0.01),增加了CD3和NK细胞的百分比(与5-FU组相比＜0.05),降低了Treg的百分含量(与对照组相比P＜0.01,与5-FU组相比P＜0.01)。FYG组和FYG+5-FU组与对照组相比显著升高了血清中TNF-α的浓度(P＜0.01)上调肿瘤组织中Bax基因表达(与对照组相比P<0.05, P<0.01); FYG+5-FU组小鼠肿瘤组织中野生型p53基因表达显著上调(与对照组相比P＜0.05,P＜0.01)。
2.FY复方中多糖含量约占其干重的63%,FY单独或联合5-FU使用体外均未表现明显的抗肿瘤功效。FYG含药血清与对照组血清相比含有更高浓度的IL-2和TNF-α(P＜0.01,P＜0.05),剂量依赖性的抑制肿瘤细胞活力(P＜0.01),诱导肿瘤细胞凋亡(P＜0.01)、将肿瘤细胞阻滞在S期(P＜0.01),抑制肿瘤细胞增殖(P＜0.05)。
3.临床共入组病例85例,3例出组,可评价疗效病例82例,其中治疗组48例、对照组34例;治疗前二组基线资料分布均衡(P＞O.05)。治疗组相较对照组治疗后体力状况改善率提高了29.8%(P=0.025)；总生活质量评分提高(P=0.040)；二组间症状领域中疲劳、纳差项目评分下降有显著差异(P＜0.05)；治疗期间未见明显益气养阴中药相关的不良反应。治疗组治疗后CD3、CD4、CD4/CD8、IgG、IgA、IgM、C3、NK细胞均有明显提高(P＜0.05),CD8、C4无明显提高(P>0.05)：Treg明显降低(P＜0.05)。与对照组相比,CD3、CD4、CD4/CD8、IgG、IgA、C3、NK细胞差异均有统计学意义(P＜O.05)。
结论：
1.FYG显著改善小鼠肝癌模型接受化疗药物后的免疫功能状态,促进肿瘤组织的凋
2.FYG需经体内代谢后才具有抑制肿瘤细胞增殖及促进凋亡的作用。
3.FYG改善化疗期胃癌、大肠癌患者临床症状,使生理状况好转,减轻疲倦、纳差症状。提高患者的免疫功能,改善其免疫抑制状态,具有双向调节作用。
Objective:The objective of the study was to examine the health-related quality of life and immune function during treatment in patients with gastric or rectal cancer who receive FOLFOX4and Fuzheng Yiliu Granules(FYG) and to examine the antitumor activity and biological mechanisms of FYG in vitro and in vivo.
Methods:
1. Hepatoma22(H22) tumor-bearing mice were treated with FYG (18g/kg, ig),5-FU (10mg/kg, ip), or5-FU plus FYG for5days. The relative tumor proliferation rates and weight were measured. White blood cell (WBC) and lymphocyte (LY) were counted. Percentages of immune cells in the peripheral blood and cytokines (IL-2) and TNF-a in the serum were measured. The apoptosis of tumor tissue was determined. The apoptosis of tissue was examined by TUNNEL. Protein expression of apoptosis factors (Bax,Bcl-2) and TS (5-FU target enzyme) were evaluated by immunhistochemistry. Transcription of apoptosis factors (Bax,Bcl-2, p53,C-myc) and TS was evaluated by RT-PCR.
2. FYG along and FYG plus5-FU were administrated to HepG2cell line with different dosage for24h,48h and72h respectively and then the cell viability was detected by MTT. FYG-containing serum was prepared from SD rats treated for7days (high dose:25.2g/kg; intermidiate dose:12.6g/kg; low dose:6.3g/kg). Cell cycle, cell viability and apoptosis were evaluated after HepG2cells were cultured in FYG-containing serum for48h. The levels of IL-2and TNF-a in FYG-containing serum were also determined.
3. Patients with gastric or rectal cancer who receive FOLFOX4were randomized into2groups. Patients in the TCM group received additional FYG. Patients in the control group received placebo. Health-related quality of life was assessed at baseline (pre-treatment) and at5-week intervals during treatment, using the European Organization for Research and Treatment of Cancer QLQ-C30questionnaires. Health-related quality of life data for five courses of treatment were then analyzed longitudinally. At baseline, at4-week,8-week after chemotherapy and the end of the study to examine the percentage of T lymphocyte subsets in peripheral blood (CD3, CD4, CD8,Treg), NK and concentrations of immunoglobulin (IgA, IgG, IgM), and complement (C3, C4).
Results:
1. FYG had no apparent toxicity. FYG alone had antitumor effect and could induce apoptosis (P<0.05, vs vehicle group). Combination of5-FU and FYG produced a more potent antitumor effect and caused more marked apoptosis in tumor tissue (compared with vehicle, P<0.01; compared with5-FU or FYG, P<0.05), although no synergistic effect was observed. Mice treated with5-FU plus FYG had higher thymus index (P<0.05) compared with the vehicle group. The numbers of both WBC and LY were decreased by5-FU (compared with vehicle, P<0.01), which was significantly reversed after FYG was administered (5-FU+FYG vs5-FU, P<0.01and P<0.05, FYG vs vehicle, P<0.01). FYG significantly increased the percentages of CD3+(P<0.01), CD4+(P<0.01), natural killer (NK; P<0.01) and ratio of CD4+/CD8+(P<0.05) compared with the vehicle group. Combination of5-FU and FYG increased the percentages of CD3+, CD4+, and ratio of CD4+/CD8+compared with the vehicle group (P<.01) and the percentages of CD3+, NK, and ratio of CD4+/CD8+compared with the5-FU group(P<0.05). Mice receiving FYG alone or FYG plus5-FU had higher serum levels of TNF-a (P<0.01) compared with the vehicle. Compared with vehicle group, expression of Bax in FYG group,5-FU group and5-FU+FYG group was significant up-regulated(P<0.05or P<0.01). The expression of p53in5-FU group and5-FU+FYG group and the Bax gene in FYG group and5-FU+FYG group were higher than model group(P<0.05or P<0.01).
2. The polysaccharide content in FYG was about63%, the FYG alone or in combination with5-FU in vitro did not show any anti-tumor effect. FYG serum significantly decreased HepG2cell viability, inhibited cell proliferation (P<0.05) and induced apoptosis (P<0.01). In addition, the levels of IL-2and TNF-a (P<0.01) of high dose serum were higher than the blank serum.
3. Eighty-five patients (50in the TCM group and35in the control group respectively) completed the baseline and post-treatment health-related quality of life assessments. The post-treatment assessments changed significantly from the baseline values in both groups. The post-treatment assessments favored the TCM group for the total scales (P=0.040) and fatigue and appetite scales (P<0.05). After treatment, CD3, CD4, CD4/CD8, IgG, IgA, IgM, C3, NK cells showed significant increase (P<0.05), CD8and C4had no significant changes (P>0.05); Treg was significantly decrease (P<0.05). Compared with the control group, CD3, CD4, CD4/CD8, IgG, IgA, and C3, NK cells showed significant differences (P<0.05).
Conclusions:
1. FYG can regulate the immune function while alleviating its side effects and induce the apoptosis of tumor in Hepatoma22tumor-bearing mice during adjuvant chemotherapy with5-FU
2. FYG extract did not inhibit the tumor cell proliferation and promote apoptosis until it was metabolized in vivo.
3. Overall health-related quality of life was improved and immune system was modulated by FYG during adjuvant chemotherapy with oral uracil/tegafur plus leucovorin in patients with gastric or rectal cancer.
引文
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