甘肃道地药材当归红芪超滤物通过调控肝癌细胞自噬对化疗的减毒增敏效应研究

Liver cancer is one of the most common malignant carcinomas. Chemotherapy for liver cancer achieved some results, but hepatoma carcinoma cells (HCC) are not sensitive to chemotherapeutic drugs, and the drugs has greater toxic side effects, which is the bottleneck in the clinical application of chemotherapy. And but, the attenuation and sensitization effects of Chemotherapeutics is one of the particularly important effects in Chinese medicine for chemotherapy. Many studies found that the active ingredient of Angelica sinensis and Radix Hedysari have certain effects of anti-tumor, improve the immune functions and protect the hematopoietic system. In addition, some studies found that HCC had appeared the decreased of autophagy in the pre-cancerous. After chemotherapy, autophagy occurs in HCC, and activation of autophagy may lead to autophagic death for HCC, which could inhibits HCC growth, and then some Chinese medicines could inhibit tumor growth by regulating autophagy, As a result, a reasonable hypothesis that Angelica and Radix Hedysari may be reduction poison and enhance chemosensitivity of HCC by autophagy. Therefore, in this study, we use conventional chemotherapy of Cisplatin and intervene with the ultra-filtration extract mixture from Angelica Sinensis and Radix Hedysari on hepatoma carcinoma cells H22 and tumor-burdened mouse. Use of molecular biological methods, from autophagy perspective, based on autophagy signaling pathway of Class III PI3K, observe Beclin-1 mRNA, PI3KⅢmRNA, LC3-ⅡmRNA, Bcl-2mRNA and their protein expression and tumor-burdened mouse cell immunity and humoral immune, In order to elucidate the mechanism of attenuation and sensitization effects of Chemotherapeutics through autophagy expression regulated by the ultra-filtration extract mixture of Angelica Sinensis and Radix Hedysari, which provides certain experiment basis for assistant antitumor clinical application of Gansu genuine medicinal Angelica Sinensis and Radix Hedysari.

肝癌是我国最常见的恶性肿瘤之一，尽管对肝癌的化疗取得了一定的进展，但癌细胞对化疗药物不敏感且毒副作用大是其在临床应用的瓶颈。当归红芪有效成分具有抗肿瘤、提高免疫和保护造血系统的作用。而肝癌细胞在癌变前期就已出现自噬活性下降，化疗后，肿瘤细胞中出现自噬现象，而激活自噬可导致肿瘤细胞发生自噬性死亡，从而达到抑制肿瘤增殖的目的。据此推测：当归红芪有效成分可能通过调控自噬达到对化疗药物产生减毒增敏的效果。为此，本研究拟借助肝癌细胞株H22及荷瘤小鼠，采用顺铂化疗，辅以超滤技术纯化的当归红芪干预，利用分子生物学方法，从自噬角度出发，基于Class III PI3K信号通路，观察对Beclin-1、PI3KⅢ、LC3-Ⅱ、Bcl-2 mRNA及其蛋白表达以及荷瘤小鼠细胞免疫、体液免疫的影响，阐明当归红芪通过调控肝癌细胞自噬对化疗药物减毒增敏的作用机制，为甘肃道地药材当归红芪抗肿瘤的临床应用提供实验依据

虽然临床对肝癌化疗取得较好的进展，但化疗药物毒副作用仍是其应用的瓶颈，当归红芪有效成分具有抗肿瘤、提高免疫和保护造血作用，为此，我们借助肝癌H22细胞株和荷瘤小鼠模型，用顺铂化疗，辅以当归红芪超滤物干预，从自噬角度观察对化疗药物减毒增敏作用。.目的：观察当归红芪超滤物通过调控肝癌细胞自噬，研究其对顺铂治疗肿瘤的减毒增敏作用及机制。.方法：1、超滤技术制备当归红芪超滤膜提取物（RAS-RH）；CCK-8法检测增殖抑制率，并计算IC20；克隆集落实验检测克隆形成；显微镜和电镜观察自噬性囊泡和自噬小体；流式细胞术检测细胞周期、凋亡率；real-time PCR和Western blotting法检测Beclin-1、PI3KⅢ、LC3-Ⅱ、Bcl-2表达；2、建立人肝癌H22细胞荷瘤小鼠模型；观察实体瘤体积、自噬小体、抑瘤率、TGD、TGT3、增敏系数，测定脾脏、胸腺指数；检测白细胞、红细胞含量，T淋巴细胞亚群，C3、IFN-γ、IL-2 含量；免疫组织化学法和Western blotting法检测Beclin-1、PI3KⅢ、LC3-Ⅱ、Bcl-2的表达；.结果：100kDa的RAS-RH能抑制肝癌H22细胞的增殖，呈现一定的时间剂量依赖性，其IC20为117.6±2.15 mg/L；与对照组、药物组和化疗组比较，RAS-RH与顺铂联合可以增加自噬性囊泡和自噬小体（p＜0.05），可能通过上调Beclin-1、PI3Ⅲ、LC3-Ⅱ的mRNA和蛋白的表达（p＜0.01），下调Bcl-2 的表达（p＜0.01），促进H22细胞的自噬，引起H22细胞G2/M期阻滞，凋亡率增加，具有化疗增敏作用；动物学实验显示，通过上调Beclin-1、PI3KⅢ、LC3-Ⅱ（p＜0.01），下调Bcl-2 的表达（p＜0.01），能改善荷瘤小鼠的一般情况，增加白细胞、红细胞、T淋巴细胞亚群数含量，提高C3、IFN-γ、IL-2水平，降低脾脏、胸腺指数，抑制TGT3、TGD，提高增敏系数，达到具有减毒増敏作用。.结论：当归红芪超滤物能增加肝癌对顺铂的化疗敏感性，其作用机制可能在于上调自噬因子Beclin-1、PI3KⅢ、LC3-Ⅱ的mRNA和蛋白的表达，下调Bcl-2 的表达，从而通过调控肝癌H22细胞自噬，促进凋亡，强化机体免疫，达到对顺铂减毒増敏的作用。