摘要
目的探究巴马汀通过调控Janus激酶2(Janus kinase 2,JAK2)/信号转导和转录激活因子3(signal transducer and activator of transcription 3,STAT3)通路抗脓毒症的作用机制。方法将90只BALB/c小鼠随机分为空白组、模型组、巴马汀组(5.0 mg/kg)、Coumermycin A1组(JAK2激活剂,4.0 mg/kg)、AG490组(JAK2抑制剂,8.0 mg/kg),以及巴马汀+Coumermycin A1组,每组15只。用红外额温度计检测体温,苏木精伊红染色观察肺、肝、肾组织病理,酶联免疫吸附测定法(enzyme-linked immunosorbent assay,ELISA)检测血清肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)、白细胞介素-6(interleukin-6,IL-6)和C-反应蛋白水平,Western blot检测血液中JAK2、STAT3、高迁移率族蛋白-1、TOLL样受体4(TOLL-like receptor 4,TLR4)蛋白表达。将小鼠RAW264.7巨噬细胞分为对照组、脂多糖(lipopolysaccharide,LPS)组及巴马汀(16、64、128 mg/L)组,用四甲基偶氮唑蓝(methyl thiazolyl tetrazolium,MTT)法、比色法分别检测细胞增殖和乳酸脱氢酶释放,免疫细胞化学染色检测TLR4及JAK2阳性表达。另取RAW264.7细胞分为正常对照组、LPS组、巴马汀组(128 mg/L)、Ad-JAK2组、Ad-NC组及巴马汀+Ad-JAK2组,MTT法检测细胞增殖,免疫细胞化学染色法检测TLR4及JAK2阳性表达,ELISA法检测TNF-α、IL-6水平,反转录酶-聚合酶链反应法检测JAK2、STAT3基因表达。结果与空白组比较,模型组小鼠腹泻、死亡及肝、肾、肺组织炎性浸润严重,体温升高,血清炎症因子及血液中单核细胞JAK2/STAT3通路及相关蛋白表达升高(P<0.05);巴马汀及AG490可抑制JAK2/STAT3通路及相关蛋白表达,缓解小鼠死亡及肝、肾、肺组织炎性浸润,抑制小鼠体温升高及血清炎性因子表达(P<0.05),相反Coumermycin A1可逆转巴马汀上述作用(P<0.05);体外实验发现,巴马汀可抑制LPS诱导的RAW264.7细胞增殖、JAK2及STAT3表达且呈剂量依赖性,但不影响乳酸脱氢酶释放(P<0.05);上调JAK2表达可逆转巴马汀抑制RAW264.7细胞增殖、TNF-α及IL-6炎性因子分泌、JAK2/STAT3通路及TLR4表达的作用(P<0.05)。结论巴马汀抗脓毒症炎性反应的作用机制与抑制JAK2/STAT3通路有关。
Objective To explore the mechanism of palmatine exerting anti-septic effects by regulating the Janus kinase 2(JAK2)/signal transducer and activator of transcription 3(STAT3)pathway.Methods Ninety BALB/c mice were randomly divided into blank group,model group,palmatine group(5.0 mg/kg),Coumermycin A1 group(JAK2 activator,4.0 mg/kg),AG490 group(JAK2 inhibitor,8.0 mg/kg),and palmatine+Coumermycin A1 group,with 15 mice in each group.Body temperature was measured using an infrared forehead thermometer.Hematoxylin eosin staining was used to observe the histopathology of lung,liver and kidney tissues.The levels of serum tumor necrosis factor-α(TNF-α),interleukin-6(IL-6)and C-reactive protein were detected by enzyme-linked immunosorbent assay(ELISA).The protein expression levels of JAK2,STAT3,high mobility group protein-1 and TOLL-like receptor 4(TLR4)in the blood were detected with Western blot.Mouse RAW264.7 macrophages were divided into control group,LPS group,and palmatine(16,64,128 mg/L)group.The cell proliferation and lactate dehydrogenase release were detected with methyl thiazolyl tetrazolium(MTT)assay and colorimetric method,respectively.The immunopositivity of TLR4 and JAK2 was detected with immunocytochemical staining.In addition,RAW264.7 cells were divided into normal control group,LPS group,palmatine group(128 mg/L),Ad-JAK2 group,and Ad-NC group,palmatine+Ad-JAK2 group.MTT assay was used to detect cell proliferation.Immunocytochemical staining was used to detect the immunopositivity of TLR4 and JAK2.ELISA was used to detect the levels of TNF-αand IL-6.Reverse transcription-polymerase chain reaction was used to detect the expression levels of JAK2 and STAT3 genes.Results Compared with the blank group,mice in the model group had severe diarrhea,death,and inflammatory infiltration in liver,kidney,and lung tissues,as well as increased body temperature,serum inflammatory factors,and expression of monocyte JAK2/STAT3 pathway and related proteins in the blood(P<0.05).Palmatine and AG490 could inhibit the expression of JAK2/STAT3 pathway and related proteins,alleviating the death and Inflammatory infiltration of liver,kidney,and lung tissues of mice and inhibiting the increase of body temperature and the expression of serum inflammatory factors in mice(P<0.05).However,Coumermycin A1 could reverse the above-mentioned effects of palmatine(P<0.05).In vitro experiments,it was found that palmatine could inhibit LPS-induced proliferation and expression of JAK2 and STAT3 in RAW264.7 cells in a dose-dependent manner(P<0.05),but did not affect the release of lactate dehydrogenase.After up-regulating the expression of JAK2,the inhibitory effects of palmatine were reversed on the proliferation of RAW264.7 cells,the secretion of TNF-αand IL-6 inflammatory factors,and the expression of JAK2/STAT3 pathway and TLR4(P<0.05).Conclusion The mechanism of palmatine's anti-inflammatory response in sepsis may be related to the inhibition of JAK2/STAT3 pathway.
作者
熊晓灿
余平
XIONG Xiaocan;YU Ping(Department of Emergency,Wuhan Third Hospital,Wuhan 430071,Hubei,China)
出处
《贵州医科大学学报》
2025年第8期1196-1206,共11页
Journal of Guizhou Medical University
基金
湖北省自然科学基金计划(2023AFB843)。
