黄芪-莪术基于C5a/NETs途径抑制Lewis肺癌小鼠肿瘤转移的机制

Astragali Radix-Curcumae Rhizoma Inhibits Metastasis of Lewis Lung Cancer in Mice via C5a/NETs Pathway

目的:基于补体C5a/C5a受体(C5aR)对中性粒细胞胞外诱捕网(NETs)形成的调控机制,探讨黄芪-莪术(HQ-EZ)改善高凝状态、抑制肿瘤生长转移的机制。方法:40只雄性C57BL/6小鼠随机分为4组,其中设空白组,另外3组建立Lewis肺癌小鼠模型后分为模型组、黄芪-莪术组、PMX53组,每组各10只。建模后第3天开始给药,中药组按8.2 g·kg^(-1)剂量连续14 d给予中药浓煎液灌胃,PMX53组分别于第3、6、9、12、15天按1 mg·kg^(-1)剂量给予腹腔注射PMX53;每2 d测量小鼠体质量和肿瘤长短径,末次给药次日各组小鼠行活体肺部microCT检测转移瘤情况,麻醉后眼球取血,处死剖取肺脏、瘤块,观察肺脏大体情况,称取瘤重计算抑瘤率,酶联免疫吸附试验(ELISA)检测血清和肿瘤组织中补体C5a、中性粒细胞弹性蛋白酶(NE)、瓜氨酸化组蛋白-H3(Cit-H3)、髓过氧化物酶(MPO)、基质金属蛋白酶-9(MMP-9)、NETs、血管性血友病因子(vWF)、组织因子(TF)、P-选择素含量,原位末端转移酶标记法(TUNEL)检测肿瘤组织细胞凋亡情况,苏木素-伊红(HE)染色观察肺内转移情况,免疫荧光(IF)观察肿瘤组织内NETs等的表达情况,蛋白免疫印迹法(Western blot)检测肿瘤组织C5aR、MPO、Cit-H3蛋白表达情况。结果:与空白组比较,模型组肺内可见肿块,肺内X射线透射度低区域增多,模型组肺脏大体可见结节灶及多处出血灶,整体肺脏颜色较深;小鼠血清补体C5a、NETs及相关蛋白、vWF、TF、P-选择素水平显著增高(P<0.01)。与模型组比较,HQ-EZ组与PMX53组肺内转移灶数量较少,X射线透射度低区域相对较少,肺脏大体未见明显结节,整体肺脏颜色相对较浅;与模型组比较,两用药干预组肿瘤生长曲线较缓,瘤重显著降低(P<0.01),肿瘤细胞凋亡率显著升高(P<0.01),血清和肿瘤组织中补体C5a、NETs及相关蛋白、vWF、TF、P-选择素水平明显降低(P<0.05),补体C5aR、MPO、Cit-H3的蛋白表达水平明显降低(P<0.05)。结论:黄芪-莪术通过抑制补体C5a/C5aR通路抑制了NETs的表达,进一步改善机体的高凝状态,起到抑制肿瘤生长转移的作用。

Objective:To explore the mechanisms of Astragali Radix-Curcumae Rhizoma(HQ-EZ)in alleviating hypercoagulability and inhibiting tumor growth and metastasis by modulating the formation of neutrophil extracellular traps(NETs)via the complement component 5a(C5a)/C5a receptor(C5aR)pathway.Method:Forty male C57BL/6 mice were randomized into four groups:Blank,model,HQ-EZ(8.2 g·kg^(-1)),and PMX53(1 mg·kg^(-1)).The mouse model of Lewis lung cancer was established in other three groups except the blank group.Mice were administrated with corresponding drugs from day 3 after modeling.Specifically,the HQ-EZ decoction was administrated for 14 consecutive days,while intraperitoneal injection of PMX53 was implemented on days 3,6,9,12,and 15.Mouse body weight and tumor diameter were measured every two days.On the next day of the last administration,lung microCT was performed to observe the tumor metastasis in vivo.Blood samples were collected from the eyeball after anesthetization,and tumor and lungs were collected after the mice were sacrificed.Tumor weight was measured to calculate the tumor growth inhibitory rate.Enzyme-linked immunosorbent assay was employed to measure the levels of C5a,neutrophil elastase(NE),citrullinated histone-H3(Cit-H3),myeloperoxidase(MPO),matrix metallopeptidase-9(MMP-9),NETs,von Willebrand Factor(vWF),tissue factor(TF),and P-selectin in the serum and tumor tissue.Terminaldeoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling was conducted to assess apoptosis in the tumor tissue.Hematoxylin-eosin staining was conducted to observe lung metastasis,and immunofluorescence(IF)was employed to observe the expression of NETs in the tumor tissue.Western blot was employed to determine the protein levels of C5aR,MPO,and Cit-H3 in the tumor tissue.Result:Compared with the blank group,the model group had nodules in the lung,increased areas with low X-ray transmittance,appearance of nodular foci and multiple hemorrhagic foci in the lungs,and darkening lung color.Furthermore,the modeling elevated the serum levels of C5a,NETs and related proteins,vWF,TF,and P-selectin(P<0.01).Compared with the model group,HQ-EZ and PMX53 reduced the lung metastases,areas with low X-ray transmittance,and nodules in the lungs and lightened the lung color.Compared with the model group,the two drug intervention groups showed flat tumor growth curves,decreased tumor weight(P<0.01),increased apoptosis of tumor cells(P<0.01),lowered levels of C5a,NETs and related proteins,vWF,TF,and P-selectin both in the serum and tumor tissue(P<0.05),and down-regulated protein levels of C5aR,MPO,and Cit-H3(P<0.05).Conclusion:HQ-EZ inhibited the expression of NETs by suppressing the C5a/C5aR pathway,thereby alleviating hypercoagulability and inhibiting tumor growth and metastasis.