清化凉血护络汤通过PI3K/Akt通路预防大鼠急性放射性肠炎的机制研究

Mechanisms of Qinghua Liangxue Huluo Decoction in preventing acute radiation enteritis in rats through the PI3K/Akt pathway

目的探讨清化凉血护络汤对大鼠急性放射性肠炎中氧化应激和炎症反应的调节作用及其对PI3K/Akt通路的影响。方法随机区组法将36只SD大鼠分为对照组、模型组、低剂量组(6.17 g/kg)、高剂量组(24.68 g/kg),每组各9只。17.5 GyX射线照射腹部建立大鼠急性放射性肠炎模型,照射前后各连续灌胃给药7 d。照射后7 d观察各组大鼠肛周和粪便情况,取直肠组织研磨,采用酶联免疫吸附法(ELISA)测定直肠组织中超氧化物歧化酶(SOD)活性、过氧化氢酶(CAT)表达及脂质过氧化过程中丙二醛(MDA)含量。qRT-PCR检测各组直肠组织中肿瘤坏死因子ɑ(TNF-ɑ)、白介素6(IL-6)和白介素1β(IL-1β)mRNA的表达变化。Western blot检测各组直肠组织中PI3K/Akt信号通路相关蛋白的表达变化。将IEC-6细胞分为对照组、辐射组、空白组和给药组,除对照组外,其余各组细胞接受10 Gy单次照射。ELISA法测定各组细胞上清中SOD、CAT、MDA、TNF-ɑ、IL-6和IL-1β的含量。Western blot检测各组细胞中PI3K/Akt信号通路相关蛋白的表达变化。结果与模型组相比,低剂量组和高剂量组大鼠肛周和粪便情况趋向正常。与模型组比较,清化凉血护络汤低剂量组和高剂量组中SOD活性上升(t=4.86、8.50,P<0.05),CAT表达增加(t=8.72、14.28,P<0.05),MDA含量降低(t=6.94、10.66,P<0.05);低剂量组和高剂量组给药后能够明显抑制直肠组织中TNF-ɑ、IL-6和IL-1βmRNA的表达(t=5.60、2.95、4.31、9.16、4.66、13.35,P<0.05),且两组直肠组织p-PI3K/PI3K和p-Akt/Akt比值低于模型组(t=22.35、13.56、18.23、13.85,P<0.05)。与辐射组相比,10%含药血清给药后,细胞上清中SOD和CAT表达上升(t=6.85、10.44,P<0.05),MDA表达下降(t=10.44,P<0.05),而且能够明显抑制细胞上清中TNF-ɑ、IL-6和IL-1β的含量(t=12.07、6.87、14.80,P<0.05)。10%含药血清给药后,细胞中p-PI3K/PI3K和p-Akt/Akt比值低于辐射组(t=10.95、5.59,P<0.05)。结论清化凉血护络汤能减轻大鼠急性放射性肠炎氧化应激损伤和炎症因子的表达,其机制可能与负向调控PI3K/Akt信号通路有关。

Objective To explore the regulatory effects of Qinghua Liangxue Huluo Decoction on oxidative stress and inflammation in acute radiation enteritis in rats,as well as its impact on the PI3K/Akt pathway.Methods A total of 36 SD rats were randomly divided into four groups using block randomization,namely the control,model,low-dose group(6.17 g/kg),and high-dose(24.68 g/kg)groups,with nine rats in each group.These rats were exposed to X-ray irradiation at a dose of 17.5 Gy to induce acute radiation enteritis,followed by continuous intragastric administration for seven days pre-and post-irradiation.Seven days post-irradiation,the perianal and fecal conditions of rats in each group were observed,and rectal tissues were collected and ground.Enzyme-linked immunosorbent assay(ELISA)was employed to assess the activity of superoxide dismutase(SOD),catalase(CAT)expression,and malondialdehyde(MDA)levels indicative of lipid peroxidation.Quantitative real-time polymerase chain reaction(qRT-PCR)was utilized to analyze the mRNA expression of tumor necrosis factor-ɑ(TNF-ɑ),interleukin-6(IL-6),and interleukin-1β(IL-1β)in the rectal tissues of each group.Additionally,Western blot was conducted to examine the expression of proteins associated with the PI3K/Akt signaling pathway in rectal tissues.The IEC-6 cells were categorized into the control,radiation,blank,and drug administration groups,with all these groups except for the control group subjected to 10 Gy single irradiation.ELISA was then employed to determine the concentrations of SOD,CAT,MDA,TNF-ɑ,IL-6,and IL-1βin cell supernatants,while Western blot was utilized to assess the expression of PI3K/Akt signaling pathway-related proteins in each group.Results Compared to the model group,rats in the low-dose and high-dose groups exhibited a trend toward normal perianal and fecal conditions,increased SOD activity(t=4.86,8.50,P<0.05),elevated CAT expression(t=8.72,14.28,P<0.05),and decreased MDA level(t=6.94,10.66,P<0.05).Furthermore,the mRNA expression of TNF-ɑ,IL-6,and IL-1βin rectal tissues was significantly inhibited in both low-dose and high-dose groups(t=5.60,2.95,4.31,9.16,4.66,13.35,P<0.05),along with lower p-PI3K/PI3K and p-Akt/Akt ratios in rectal tissues compared to the model group(t=22.35,13.56,18.23,13.85,P<0.05).Compared to the radiation group,the drug administration groups(10%drug-containing serum)exhibited increased SOD and CAT expressions(t=6.85,10.44,P<0.05),as well as decreased MDA expression(t=10.44,P<0.05),in the supernatant.Furthermore,compared to the radiation group,this group displayed significantly inhibited TNF-ɑ,IL-6,and IL-1βconcentrations in the cell supernatant(t=12.07,6.87,14.80,P<0.05),while lowering p-PI3K/PI3K and p-Akt/Akt ratios in cells(t=10.95,5.59,P<0.05).Conclusions Qinghua Liangxue Huluo Decoction demonstrates the potential for mitigating oxidative stress-induced injury and suppressing the expressions of inflammatory factors in rats with acute radiation enteritis.The mechanism behind the potential is likely associated with the negative regulation of the PI3K/Akt signaling pathway.