知母、干姜对PI3K/AKT通路异常激活介导的非小细胞肺癌吉非替尼耐药的影响及机制研究

Effect and Mechanism Study of Anemarrhenae Rhizoma and Zingiberis Rhizoma Recens on Gefitinib Resistance in Non-small Cell Lung Cancer Mediated by Abnormal Activation of PI3K/AKT Pathway

目的以知母、干姜分别作为清热和温阳治则的代表中药,联合表皮生长因子受体酪氨酸激酶抑制剂(EGFR-TKIs)代表药物吉非替尼,探索寒、热不同药性中药对PI3K/AKT通路异常激活介导的非小细胞肺癌(NSCLC)耐药细胞生长及代谢的影响。方法采用三维(3D)法培养PC-9-PIK3CA-Mutation(PC-9-PIK3CA-M,EGFR 19外显子突变合并PIK3CA突变)细胞和H1650(EGFR突变合并PTEN缺失)细胞,作为PI3K/AKT通路异常活化导致的EGFR-TKIs(吉非替尼)耐药模型。NSCLC-3D细胞分组:对照组、知母组、干姜组、吉非替尼组、吉非替尼+知母组、吉非替尼+干姜组。知母、干姜冻干粉浓度均为3200μg·mL^(-1),吉非替尼浓度为20μmol·L^(-1),干预24 h。采用ATP法检测细胞活性;Annexin V-FITC/PI双染法检测细胞凋亡情况;活性氧(ROS)荧光探针法检测细胞ROS水平。结果(1)在PC-9-PIK3CA-M-3D和H1650-3D细胞中,与对照组比较,10~80μmol·L^(-1)吉非替尼能显著抑制细胞活力(P<0.01);与吉非替尼组比较,吉非替尼+知母能显著抑制细胞活力(P<0.01),吉非替尼+干姜能显著促进细胞活力(P<0.05,P<0.01)。知母能协同促进吉非替尼的抗肿瘤作用,而干姜则拮抗吉非替尼的抗肿瘤作用。(2)在PC-9-PIK3CA-M-3D细胞中,与对照组比较,吉非替尼对细胞凋亡无显著影响(P>0.05),吉非替尼+知母能显著促进细胞凋亡(P<0.01),而吉非替尼+干姜能显著抑制细胞凋亡(P<0.05);与吉非替尼组比较,吉非替尼+知母能显著促进细胞凋亡(P<0.01)。在H1650-3D细胞中,与对照组比较,吉非替尼和吉非替尼+干姜对细胞凋亡无显著影响(P>0.05),吉非替尼+知母可显著促进细胞凋亡(P<0.01);与吉非替尼组比较,吉非替尼+知母可显著促进细胞凋亡(P<0.01)。(3)在PC-9-PIK3CA-M-3D细胞中,与对照组比较,吉非替尼组细胞线粒体ROS水平明显降低(P<0.05);在H1650-3D细胞中,与对照组比较,吉非替尼组细胞线粒体ROS水平无明显变化(P>0.05)。在PC-9-PIK3CA-M-3D和H1650-3D细胞中,与对照组比较,吉非替尼+知母组细胞粒体ROS水平明显升高(P<0.05,P<0.01),吉非替尼+干姜组细胞线粒体ROS水平明显降低(P<0.05,P<0.01)。与吉非替尼组比较,吉非替尼+知母组细胞线粒体ROS水平显著升高(P<0.01)。结论清热药知母可能通过上调线粒体ROS水平,进而促进氧化应激诱导的细胞死亡,逆转PI3K/AKT通路异常活化介导的吉非替尼耐药,而温阳药干姜可能通过下调线粒体ROS水平而部分拮抗吉非替尼的抗肿瘤作用。

Objective To investigate the effects of Dangshen Yigong Decoction on the learning memory ability of D-galactose-induced Alzheimer’s disease(AD)mice and its mechanism of action.Methods(1)The potential targets of Dangshen Yigong San for the treatment of AD were obtained by network pharmacological analysis,and the potential targets were subjected to protein-protein interaction(PPI)network analysis,then the key targets were analyzed by the CentiScaPe plug-in of Cytoscape software,and KEGG pathway enrichment analysis was carried out.(2)Male KM mice were randomly divided into blank group,model group,Donepezil(Aricept)group(1.5 mg·kg^(-1))and Dangshen Yigong San high-(7.5 g·kg^(-1))and low-(3.75 g·kg^(-1))dose groups,with 8 mice in each group.Except for the blank group,mice in each group were subcutaneously injected with 200 mg·kg^(-1)D-galactose at the back of the neck to replicate the AD mouse model once a day for 30 days.Meanwhile,mice in the drugadministered group were gavaged with the corresponding dose of the drug(10 mL·kg^(-1))once a day for 30 days.The Morris water maze experiment was performed on the day 26 to the day 30 of the drug administration,to test the learning and memory ability of the mice.HE staining was used to observe the histopathological changes in mouse hippocampus and to score the pathology of hippocampal damaged neuronal cells;immunofluorescence was used to detect the expressions of glial fibrillary acidic protein(GFAP),ionic calcium-binding adapter molecule 1(IBA1),and Synaptophysin in mouse hippocampus;and levels of glutamate,tumor necrosis factorα(TNF-α),interleukin10(IL-10),transforming growth factorα(TGF-α)and TGF-βin the hippocampus and cortex of mice were determined by ELISA;the protein expression levels of tumor necrosis factor receptor 1(TNFR1)and tumor necrosis factor receptor 2(TNFR2)were detected in the hippocampus and cortex of mice by Western Blot.Results(1)A total of 187 potential targets and 38 key targets were obtained for the treatment of AD by Dangshen Yigong Decoction,involving 107 signaling pathways related to AD disease,including the neural TNF signaling pathway,PI3K-Akt signaling pathway,MAPK signaling pathway,IL-17 signaling pathway and so on.(2)Compared with the blank group,mice in the model group showed a significantly longer latency to escape on day 5(P<0.01),a significantly lower number of crossings of the plateau quadrant and residence time(P<0.05);hippocampal neuronal cells were disarranged,some cells were missing,intercellular gaps were enlarged,and nuclei were seen to be consolidated,denatured,or dead,with a significantly higher pathological score(P<0.01);the areas of GFAP and IBA1-positive expression area were significantly increased(P<0.01),and Synaptophysin-positive expression area was significantly decreased(P<0.01);the contents of glutamate,TNF-α,TGF-αand TGF-βin brain tissue were all significantly increased(P<0.01),and the content of IL-10 was significantly decreased(P<0.01);the TNFR1 protein expression in brain tissue was significantly up-regulated(P<0.05)and TNFR2 protein expression was significantly down-regulated(P<0.01).Compared with the model group,the frequency and residence time of crossing the platform quadrant in the high-and low-dose groups of Dangshen Yigong San were significantly increased(P<0.05,P<0.01);the hippocampal nerve cells were arranged relatively neatly and tightly,with clear structure and occasional degeneration or death of individual nerve cells,and the pathological score was significantly decreased(P<0.05,P<0.01).The positive expression area of GFAP and IBA1 in hippocampus were significantly decreased,while the positive expression area of Synaptophysin was significantly increased,and the contents of glutamate,TNF-αand TGF-αin brain tissue were significantly decreased(P<0.01)and the IL-10 content was significantly increased.On the fifth day,the escape latency of mice in the highdose of Dangshen Yigong San group was significantly shortened,the protein expression of TNFR1 in brain tissue was significantly down-regulated,the TNFR2 protein expression was significantly up-regulated,and the content of TGF-βin brain tissue was significantly decreased in low-dose Dangshen Yigong San group(P<0.01).Conclusion Dangshen Yigong San was able to improve learning memory ability and ameliorate hippocampal histopathological damage in D-galactose-induced AD mice,and its mechanism of action may be related to the modulation of TNF-αTNFRs signalling in the brain to attenuate neuroinflammatory responses.