参苓白术散通过IκBα/NF-κB通路调控Lewis肺癌小鼠肿瘤自噬的研究

Study of IκBα/NF-κB Pathway about Regulation of Autophagy in Lewis Lung Cancer Mice Through Reinforcing Shenlingbaizhu Powder

目的观察培土生金法代表方参苓白术散对Lewis肺癌小鼠模型肿瘤组织IκBα/NF-κB信号通路以及肿瘤自噬的干预作用,探讨参苓白术散联合顺铂对肺癌的部分治疗机制。方法选取SPF级雄性C57BL/6J小鼠40只,采用腋窝皮下接种肺癌Lewis细胞悬液法建立Lewis肺癌小鼠模型,建模成功后随机分为4组:模型组、顺铂组、中药组和中药+顺铂组,每组10只。模型组给予等量蒸馏水灌胃;顺铂组每周给与腹腔注射顺铂(5 mg·kg^(-1))一次,共2次;中药组每日给与参苓白术散(9.36g·kg^(-1))灌胃一次,连续14天;中药+顺铂组每日给与参苓白术散(9.36g·kg^(-1))灌胃一次,连续14天,同时每周给与腹腔注射顺铂(5mg·kg^(-1))一次,共2次。给药14天后,测定各组小鼠肿瘤体积,并计算相对肿瘤体积和相对肿瘤增殖率;采用ELISA法测定肿瘤组织中肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)和白细胞介素-1(interleukin-1β,IL-1β)的含量;采用透射电镜观察肿瘤细胞超微形态的变化;采用免疫组化法测定核因子κB抑制蛋白α(NF-kappa-B inhibitor alpha,IκBα)和核因子-κB(nuclear factor-κ-gene binding,NF-κB)P65的蛋白表达水平;采用Western blot法测定自噬相关基因3(autophagy related gene 3,Atg3)、自噬相关基因5(autophagy related gene5,Atg5)、自噬特异性基因(Beclin-1)和微管相关蛋白1轻链3(Microtubule-associated protein 1 light chain 3,LC3)的蛋白表达水平。结果与模型组比较,顺铂组、中药组、中药+顺铂组小鼠肿瘤体积和相对肿瘤体积显著减小(P<0.01),肿瘤组织TNF-α、IL-1β含量显著降低(P<0.01),IκBα蛋白表达水平显著升高(P<0.01)、NF-κB P65蛋白入核率显著降低(P<0.01),Atg3、Atg5、Beclin-1和LC3II/LC3I的蛋白表达水平显著升高(P<0.01),和顺铂组比较,中药组和中药+顺铂组肿瘤组织TNF-α、IL-1β含量显著降低(P<0.01),中药+顺铂组肿瘤组织LC3II/LC3I的蛋白表达水平显著升高(P<0.01)。结论参苓白术散能够上调IκBα蛋白表达,抑制NF-κB的激活,降低肿瘤组织中促炎因子TNF-α、IL-1β的含量,调控肿瘤组织炎症反应,改变肿瘤微环境,并上调肺癌组织Atg3、Atg5、Beclin-1和LC3II/LC3I的蛋白表达水平,激活肿瘤发生自噬,抑制肿瘤的生长。

Objective To observe the intervention effect of Shenlingbaizhu powder on autophagy of Lewis lung cancer mice through IκBα/NF-κB pathway, and to explore the mechanism of Shenlingbaizhu power combined with cisplatin on autophagy of lung cancer. Methods 40 C57-B6 male mice of SPF grade were selected. Lewis lung cancer cell suspension was inoculated subcutaneously in the right armpit of mice to establish mice models of Lewis lung cancer.Then these successful models were divided into four groups randomly named model group, cisplatin group,Shenlingbaizhu powder group and Shenlingbaizhu powder with cisplatin group, each group had 10 mice in it. The cisplatin group mice were given cisplatin(5 mg·kg^(-1)) once a week for two weeks by intraperitoneal injection. The Shenlingbaizhu powder group mice were given Shenlingbaizhu powder(9.36 g·kg^(-1)) once a day and lasted 14 days by intragastric administration. The model group mice were given same amount of normal saline by intragastric administration. The Shenlingbaizhu powder with cisplatin group mice were given Shenlingbaizhu powder(9.36 g·kg^(-1))once a day and lasted 14 days by intragastric administration and cisplatin(5 mg·kg^(-1)) once a week for two weeks by intraperitoneal injection. After 14 days, the tumor volume was measured, and the relative tumor volume and relative tumor increment rate were calculated. ELISA was used to detect the content of TNF-α and IL-1β in tumor tissue.Transmission electron microscope was used to observe the ultrastructural changes of tumor cells. Immunohistochemistry was used to determine protein expression level related protein IκBα and protein NF-κB P65. Immunoblotting was used to determine protein expression level related protein Atg3, Atg5, Beclin-1 and LC3. Results Compared with model group, the tumor volume and relative tumor volume was reduced(P<0.01), and the content of TNF-α and IL-1β in tumor tissue decreased(P<0.01). The protein expression level related IκBα was increased(P<0.01), and the rate of nuclear import related NF-κB P65 was decreased(P<0.01). The protein expression level related Atg3, Atg5, Beclin-1and LC3II/LC3I was increased(P<0.01) in cisplatin group, Shenlingbaizhu powder group and Shenlingbaizhu powder with cisplatin group. Compared with cisplatin group, the contents of TNF-α and IL-1β in tumor tissue were significantly decreased(P<0.01) in Shenlingbaizhu powder group and Shenlingbaizhu powder with cisplatin group, and the protein expression level of LC3II/LC3I in Shenlingbaizhu powder group and Shenlingbaizhu powder with cisplatin group was significantly increased(P<0.01). Conclusion Shenlingbaizhu powder combined with cisplatin can up-regulate the expression of IκBα protein and inhibit the activation of NF-κB. It can reduce the content of pro-inflammatory factors TNF-α and IL-1β in tumor tissues and regulate the inflammatory response of tumor tissues to change the tumor microenvironment. Then it can up-regulate the protein expression levels of Atg3, Atg5, Beclin-1 and LC3II/LC3I in lung cancer tissues to activate tumor cell autophagy and inhibit the growth of lung cancer cells.