山奈酚腹腔注射后脑出血大鼠脑组织神经炎症反应及PI3K/AKT信号通路相关蛋白表达观察

Observation of neuroinflammatory response and PI3K/AKT signaling pathway-related protein expression in brain tissues of ICH rats after intraperitoneal injection of kaempferol

目的观察山奈酚腹腔注射后脑出血大鼠脑组织神经炎症反应及PI3K/AKT信号通路相关蛋白表达,以探讨山奈酚对大鼠脑出血后神经炎症反应的抑制作用及机制。方法72只SD大鼠随机被均分为4组,分别为山奈酚组、山奈酚+PI3K抑制剂LY294002组(抑制剂组)、脑出血组、假手术组。山奈酚组大鼠先建立脑出血模型,成功后腹腔注射山奈酚20 mg/(kg·d),1次/天,连续3 d;抑制剂组大鼠侧脑室先注入PI3K抑制剂LY294002(10µL,10 mmol/L),然后建立脑出血模型,其余步骤同山奈酚组;脑出血组建立脑出血模型后,腹腔注射等体积生理盐水,1次/天,连续3 d;假手术组不建立脑出血模型,仅腹腔注射等体积生理盐水,用法同脑出血组。处理结束后,观察各组脑组织神经炎症反应[神经功能缺损程度(mNSS评分)、脑组织含水量占比、脑血肿周围组织形态结构、脑组织小胶质细胞数量和形态、小胶质细胞标志物Iba-1蛋白及脑组织IL-1β、IL-6、TNF-α表达水平)]情况及脑组织磷脂酰肌醇3激酶(PI3K)/蛋白激酶B(AKT)信号通路相关蛋白(P-PI3K/PI3K、P-AKT/AKT、P-NF-κB p65/NF-κB p65)表达情况。结果与假手术组比较,脑出血组神经功能评分及脑组织含水量占比均升高(P均<0.05),血肿周围组织排列紊乱、细胞间隙增大、可见大量炎性细胞及红细胞浸润、免疫荧光下可见处于激活状态的小胶质细胞,脑组织Iba-1蛋白相对表达量高(P<0.05),脑组织IL-1β、IL-6、TNF-α等炎性因子表达水平升高(P均<0.05);与脑出血组比较,山奈酚组大鼠神经功能评分及脑组织含水占比均下降(P均<0.05),脑组织病理形态上均得到改善、小胶质细胞趋于静息态改变,脑组织Iba-1蛋白相对表达量低(P<0.05),脑组织IL-1β、IL-6、TNF-α表达水平下降(P均<0.05);与山奈酚组比较,抑制剂组神经功能评分及脑组织含水量占比均升高(P均<0.05),血肿周围组织排列较紊乱、可见较多的炎性细胞及红细胞浸润、被激活的小胶质细胞增多,脑组织Iba-1蛋白相对表达量高(P<0.05),脑组织IL-1β、IL-6、TNF-α水平升高(P均<0.05)。与假手术组比较,脑出血组脑组织P-PI3K/PI3K、P-AKT/AKT下降,P-NF-κB p65/NF-κB p65上升(P均<0.05);与脑出血组比较,山奈酚组脑组织P-PI3K/PI3K、P-AKT/AKT上升,P-NF-κB p65/NF-κB p65下降(P均<0.05);与山奈酚组比较,抑制剂组脑组织P-PI3K/PI3K、P-AKT/AKT下降,P-NF-κB p65/NF-κB p65上升(P均<0.05)。结论山奈酚腹腔注射后脑出血大鼠脑组织神经炎症反应减轻,PI3K/AKT信号通路激活。山奈酚能有效抑制大鼠脑出血后神经炎症反应,可能是通过靶向调控PI3K/AKT信号通路起作用的。

Objective To observe the neuroinflammatory response and the expression of PI3K/AKT signaling pathway-related proteins in brain tissues of rats with intracerebral hemorrhage(ICH)after intraperitoneal injection of kaempferol in order to explore the inhibitory effect and mechanism of kaempferol on the neuroinflammatory response after ICH in rats.Methods Seventy-two SD rats were randomly divided into four groups:the kaempferol group,the kaempferol+PI3K inhibitor LY294002 group(inhibitor group),the ICH group,and the sham operation group,respectively.The model of ICH was established in the kaempferol group,and then kaempferol 20 mg/(kg.d)was injected intraperitoneally once a day for 3 consecutive days.In the inhibitor group,PI3K inhibitor LY294002(10µL,10 mmol/L)was injected into the lateral ventricle before ICH was induced,and the other steps were the same as those in the kaempferol group.After the ICH model was established,the rats in the ICH group were intraperitoneally injected with the same volume of normal saline once a day for 3 consecutive days.The ICH model was not established in the sham operation group,and the equal volume of normal saline was injected intraperitoneally with the same method as the ICH group.At the end of the treatment,the neuroinflammatory response of brain tissues in each group was observed,including the degree of neurological deficit(mNSS score),the proportion of water content in the brain tissues,the morphological structure of tissue around cerebral hematoma,the number and morphology of microglia in brain tissue,the expression levels of microglia marker Iba-1 and interleukin-1β(IL-1β),interleukin-6(IL-6)and tumor necrosis factor-α(TNF-α)in the brain tissues,and the expression of phosphatidylinositol 3 kinase(PI3K)/protein kinase B(AKT)signaling pathway-related proteins(P-PI3K/PI3K,PAKT/AKT,P-NF-κB p65/NF-κB p65)in the brain tissues.Results Compared with the sham operation group,the neurological function score and the proportion of water content in brain tissue increased(all P<0.05),and the tissue arrangement around the hematoma was disordered,the intercellular space was enlarged,a large number of inflammatory cells and red blood cells were infiltrated,and activated microglia were observed under immunofluorescence,the relative expression of Iba-1 protein in brain tissue increased,and the expression levels of inflammatory factors such as IL-1β,IL-6 and TNF-αin the brain tissue increased in the ICH group(all P<0.05).Compared with the ICH group,the nerve function score and brain tissue water proportion decreased(all P<0.05),and brain tissue pathological morphology was improved,the microglia tended to resting state changes,Iba-1 protein expression was lower,and the expression levels of IL-1β,IL-6 and TNFαin the brain tissues decreased in the kaempferol group(all P<0.05).Compared with the kaempferol group,the neurological function score and the proportion of water content in the brain tissues significantly increased,the tissues around the hematoma were more disorderly arranged,with more inflammatory cells and red blood cells infiltration and increased activated microglia,the relative expression of Iba-1 protein in brain tissue increased,and the levels of IL-1β,IL-6 and TNF-αin the brain tissues increased in the inhibitor group(all P<0.05).Compared with the sham operation group,the P-PI3K/PI3K and P-AKT/AKT decreased,and the P-NF-κB p65/NF-κB p65 increased in the ICH group(all P<0.05).Compared with the ICH group,the ratios of P-PI3K/PI3K and P-AKT/AKT increased,and the ratio of P-NF-κb p65/NF-κB p65 decreased in the kaempferol group(all P<0.05).Compared with the kaempferol group,the ratio of P-PI3K/PI3K and P-AKT/AKT decreased,and the ratio of the P-NF-κb p65/NF-κB p65 increased in the inhibitor group(all P<0.05).Conclusions Intraperitoneal injection of kaempferol can reduce the neuroinflammatory response and activate PI3K/AKT signaling pathway in the brain tissues of rats with ICH.Kaempferol can effectively inhibit the neuroinflammatory response after ICH in rats possibly by regulating the PI3K/AKT signaling pathway.