摘要
旨在通过脂多糖(LPS)建立RAW264.7细胞与BALB/c小鼠炎症模型进行体内外试验,探讨熊果酸对LPS诱导炎症的保护作用。体外采用MTT法测定熊果酸和LPS对RAW264.7细胞活力的影响,中性红测试熊果酸对LPS刺激RAW264.7细胞吞噬能力的影响,Griess法测定NO释放水平;荧光定量PCR(RT-qPCR)测定JAK激酶1(JAK1)/信号转导子和转录激活子3(STAT3)信号通路和炎症因子mRNA表达水平。BALB/c小鼠灌胃熊果酸分为25 mg/kg的低剂量组(UA-L)、50 mg/kg的中剂量组(UA-M)和100 mg/kg的高剂量组(UA-H),每只小鼠200μL/d,灌胃7 d,腹腔注射LPS诱导炎症反应后采集脾脏,计算脾脏指数,使用流式细胞术测定脾脏T细胞分化和Th17/Treg平衡,RT-qPCR方法验证α肿瘤坏死因子(TNF-α)、白细胞介素(IL)-1、JAK1和STAT3的mRNA表达水平。结果:熊果酸在12.5μg/mL范围内对细胞活力无影响(P>0.05),LPS浓度为1μg/mL时,细胞存活率为54.55%;LPS刺激后RAW264.7细胞吞噬能力显著下降(P<0.05),NO释放水平显著上升(P<0.05);熊果酸干预后,细胞吞噬能力呈剂量依赖式上升,NO释放水平下降。RT-qPCR结果表明,RAW264.7细胞经LPS刺激后,一氧化氮合酶(iNOS)、IL-1、IL-6、TNF-α、JAK1和STAT3的mRNA表达水平均显著上升(P<0.05);熊果酸干预后,上述因子mRNA表达水平均显著下降(P<0.05)。体内试验结果表明,腹腔注射LPS后,小鼠脾脏指数显著高于空白对照组(P<0.05),CD4^(+)/CD8^(+)和Th17/Treg细胞出现失衡;不同剂量熊果酸干预后脾脏指数均显著降低(P<0.05),对小鼠CD4^(+)和CD8^(+)细胞分化具有明显促进作用,同时改善Th17/Treg失衡;腹腔注射LPS的小鼠脾脏IL-1、TNF-α、JAK1和STAT3的mRNA表达水平均显著上升(P<0.05),提前饲喂熊果酸能改善这一情况。综上,熊果酸通过调节炎症因子、改善T细胞分化失衡,从而降低由LPS诱导的RAW264.7细胞和小鼠的炎症反应。
This study was to establish an inflammatory model of RAW264.7 cells and BALB/c mice by LPS,and to detect the protective effect of ursolic acid on LPS-induced inflammation in mice by in vivo and in vitro experiments.The effects of ursolic acid and LPS on the viability of RAW264.7 cells were measured by MTT assay in vitro.Then,the effect of ursolic acid on the phagocytosis of RAW264.7 cells stimulated by LPS was detected by neutral red test,and RT-qPCR was used to measure the mRNA expression of the JAK1/STAT3 signaling pathway and inflammatory factors.Next,BALB/c mice were divided into the low dose group(UA-L,25 mg/kg),the middle dose group(UA-M,50 mg/kg)and the high dose group(UA-H,100 mg/kg)by gavage of ursolic acid.Each mouse was treated with 200μL/d by gavage for 7 days.Flow cytometry was used to measure the differentiation of splenic T cells and Th17/Treg balance.Finally,RT-qPCR was used to verify the mRNA expression levels of TNF-α,IL-1,JAK1 and STAT3.The results were as follows:Ursolic acid had no effect on cell viability in the range of 12.5μg/mL(P>0.05).When the concentration of LPS was 1μg/mL,the cell survival rate was 54.55%.After LPS stimulation,the phagocytic ability of the RAW264.7 cells decreased significantly(P<0.05),and the release of NO increased signifi-cantly(P<0.05).After ursolic acid intervention,the phagocytic ability of the RAW264.7 cells increased in a dose-dependent manner,and the release of NO decreased.The results of RT-qPCR showed that the mRNA expression levels of iNOS,IL-1,IL-6,TNF-α,JAK1 and STAT3 in the RAW264.7 cells were significantly increased after LPS stimulation(P<0.05),and the mRNA expression levels of the above were significantly decreased after ursolic acid intervention(P<0.05).The results of in vivo experiment showed that,after intraperitoneal in-jection of LPS,the spleen index of the mice was significantly higher than that of the blank control group(P<0.05),and the imbalance of CD4^(+)/CD8^(+)and Th17/Treg cells was observed.After intervention with ursolic acid at different doses,the spleen index was significantly de-creased(P<0.05),which promoted the differentiation of CD4^(+)and CD8^(+)cells and improved the imbalance of Th17/Treg in the mice.The mRNA expressions of IL-1,TNF-α,JAK1 and STAT3 in the spleen of the mice with intraperitoneal injection of LPS were increased(P<0.05),and early feeding of ursolic acid could improve the situation.In conclusion,ursolic acid reduced the LPS-stimulated inflammatory response in RAW264.7 cells and in mice by regulating inflammatory factors,and restoring the imbalance of T cell differentiation.
作者
刘恋
刘佳丽
黄永熙
余捷
汤锋
白若男
刘满
朱君
郭利伟
刘国平
杨小林
LIU Lian;LIU Jiali;HUANG Yongxi;YU Jie;TANG Feng;BAI Ruonan;LIU Man;ZHU Jun;GUO Liwei;LIU Guoping;YANG Xiaolin(College of Animal Scienceand Technology,Yangtze University,Jingzhou 434000,China)
出处
《畜牧与兽医》
CAS
北大核心
2024年第10期39-48,共10页
Animal Husbandry & Veterinary Medicine
基金
国家自然科学青年基金资助项目(31602099)
湖北省重点研发计划项目(2023BBB045)。
