附子理中丸通过调节MAPK信号通路改善顺铂诱导CIPN模型小鼠损伤的作用机制

Mechanism of Fuzi Lizhongwan Improving Injury in Cisplatin-induced CIPN Mice by Regulating MAPK Signaling Pathway

目的:基于丝裂原活化蛋白激酶(MAPK)信号通路,探讨附子理中丸改善顺铂诱导化疗所致周围神经病变(CIPN)模型小鼠损伤的作用机制。方法:KM雌性小鼠40只,随机分为正常组,模型组,附子理中丸组(3.5 g·kg^(-1)),阿司匹林组(0.026 g·kg^(-1))。每日腹腔注射顺铂(3 mg·kg^(-1)),连续5 d,制备CIPN模型;造模同时灌胃给予相应药物,连续12 d。观察其一般情况和行为学表现;末次给药后取材,苏木素-伊红(HE)染色观察足掌部皮肤组织病理变化。采用生化学检测血清中超氧化物歧化酶(SOD),过氧化氢(H_(2)O_(2)),丙二醛(MDA),一氧化氮(NO)的水平;采用酶联免疫吸附测定法(ELISA)检测肾脏组织中白细胞介素-6(IL-6),白细胞介素-1β(IL-1β),肿瘤坏死因子-α(TNF-α),谷胱甘肽过氧化物酶-3(GPX-3)的含量。蛋白免疫印迹法(Western blot)检测肾脏组织中细胞外调节蛋白激酶1/2(ERK1/2)及其磷酸化(p-ERK1/2),p38 MAPK及其磷酸化p38 MAPK(p-p38 MAPK)的表达水平。结果:与正常组比较,模型组小鼠足掌部皮肤病理损伤明显,表皮角化过度呈网篮状结构,棘层萎缩,细胞数量减少,细胞内水肿;与模型组比较,附子理中丸组足掌部皮肤组织的病理损伤明显降低。与正常组比较,模型组小鼠体质量、机械痛阈值、热痛阈值显著下降(P<0.01),SOD活性明显降低(P<0.05),H_(2)O_(2),MDA,NO含量显著升高(P<0.01),IL-6,IL-1β,TNF-α表达显著升高(P<0.01);与模型组比较,附子理中丸组小鼠体质量、机械痛阈值、热痛阈值显著升高(P<0.01),SOD活性明显升高,H_(2)O_(2),MDA和NO含量明显降低(P<0.05),IL-6,IL-1β,TNF-α表达显著降低(P<0.01);与正常组比较,模型组ERK1/2,p-ERK1/2,p38 MAPK,p-p38 MAPK蛋白表达显著上升(P<0.01);与模型组比较,附子理中丸组ERK1/2,p-ERK1/2,p38 MAPK,p-p38 MAPK蛋白表达显著下降(P<0.01)。结论:附子理中丸能够改善顺铂诱导CIPN模型小鼠的神经功能损伤,提高小鼠的疼痛阈值,其作用机制可能与调节MAPK信号通路,抑制炎性反应及机体氧化应激水平相关。

Objective: To explore the mechanism of Fuzi Lizhongwan alleviating the damage of chemotherapy-induced peripheral neuropathy(CIPN) mice caused by cisplatin based on mitogen-activated protein kinase(MAPK)signaling pathway. Method:A total of 40 female KM mice were randomized into blank group(distilled water,ig),model group(distilled water,ig),Fuzi Lizhongwan group(3.5 g·kg^(-1),ig),and aspirin group(0.026 g·kg^(-1),ig). Cisplatin(3 mg·kg^(-1),ip,5 days)was used to induce CIPN in mice.Administration began while modeling and lasted 12 days. The general conditions and behaviors of mice were observed. After the last administration,samples were collected. Pathological changes of the soles were observed based on hematoxylin-eosin(HE)staining. Biochemical assay was employed to determine the levels of serum superoxide dismutase(SOD),hydrogen peroxide(H_(2)O_(2)),malondialdehyde(MDA),and nitric oxide(NO),enzyme-linked immunosorbent assay(ELISA)the content of interleukin-6(IL-6),interleukin-1β(IL-1β),tumor necrosis factor-α(TNF-α),and glutathione peroxidase-3(GPX-3)in kidney tissue,and Western blotting the expression of extracellular signal-regulated kinase1/2(ERK1/2),phosphorylated-ERK1/2(p-ERK1/2),p38 MAPK,and phosphorylated-p38 MAPK(p-p38 MAPK)in kidney tissue. Result: Compared with the blank group,model group demonstrated obvious pathological damage on the soles,hyperkeratosis of the epidermis with a basketweave pattern,atrophy of stratum spinosum,reduction of cells,and intracellular edema. Compared with the model group,Fuzi Lizhongwan significantly alleviated the pathological damage of the skin tissue of the soles. The model group showed lower body weight,mechanical pain threshold,thermal pain threshold(P<0.01),and SOD activity(P<0.05),higher content of H_(2)O_(2),MDA,and NO(P<0.01),and higher expression of IL-6,IL-1β,and TNF-α(P<0.01)than the blank group. Fuzi Lizhongwan group demonstrated higher body weight,mechanical pain threshold,thermal pain threshold(P<0.01),and SOD activity(P<0.05),lower content of H_(2)O_(2),MDA,and NO(P<0.05),and lower expression of IL-6,IL-1β,and TNF-α(P<0.01)than the model group. The expression of ERK1/2,p-ERK1/2,p38 MAPK,and p-p38 MAPK increased significantly(P<0.01)in the model group compared with that in the blank group,while the expression decreased significantly(P<0.01)in the Fuzi Lizhongwan group compared with that in the model group. Conclusion:Fuzi Lizhongwan can relieve the neurological injury of cisplatin-induced CIPN mice and increase the pain threshold of mice,possibly by regulating the MAPK signaling pathway and inhibiting inflammatory response and oxidative stress.