To explore the mechanism of Dahuang Lingxian Formula in relieving inflammatory response of bile duct cells based on IL-6/JAK/STAT3 signaling pathway

Objective:To explore the mechanism of action of Dahuang Lingxian Formula in alleviating the inflammatory response of bile duct cells in LPS-induced intrahepatic bile duct inflammation model rats based on IL-6/JAK/STAT3 signaling pathway.Methods:Fifty SD rats were randomly divided into five groups,blank group,model group,choling tablets(0.5 g/kg),and low and high concentration groups(2.4 g/kg and 4.8 g/kg)of Dahuang Lingxian Formula,ten rats in each group.Except for the blank group,the rats in each group were injected with 1.25 mg/kg LPS at the common bile duct at one time to construct an animal model of intrahepatic bile duct infection.After gavage on day 8,liver tissues were taken from rats at the hepatic hilum,and the histopathological changes of the hepatic hilum and biliary tree were observed by HE staining.The expression levels of serum glutamic alanine transaminase(ALT),glutamic oxalacetic transaminase(AST),malondialdehyde(MDA)and superoxide dismutase(SOD)were measured by biochemical method.The expression levels of interleukin 6(IL-6),Janus protein tyrosine kinase 2(JAK2),signal transducer and activator of transcription 3(STAT3)in rat serum were measured by enzyme-linked immunosorbent assay(ELISA).Protein immunoblotting(WB)and real-time fluorescence quantitative PCR(RT-qPCR)were used to detect the expression levels of IL-6,JAK2,STAT3 protein and mRNA in biliary tree tissues.Results:①Compared with the blank group,the structures such as interlobular bile ducts in the hepatic sinusoids and portal duct area of the model rats were destroyed,and inflammatory cells infiltrated around them.The expression of ALT,AST,MDA,IL-6,JAK2 and STAT3 in the serum increased significantly,the expression level of SOD decreased,and the expression levels of IL-6,JAK2 and STAT3 proteins and mRNA increased.②Compared with the model group,the degree of liver pathological damage in rats in the Chiling Ning tablet group and the low and high concentration groups of Dahuang Lingxian Formula were improved,which could significantly reduce the expression levels of ALT,AST,MDA,IL-6,JAK2,STAT3 and up-regulate SOD in serum,and down-regulate the expression of IL-6,JAK2,STAT3 protein and mRNA,with the best effect in the high concentration group of Dahuang Lingxian Formula.③Compared with the choling tablet group,the rats in the low and high concentration groups of Dahuang Lingxian Formula tended to normalize the degree of liver pathological damage,without obvious inflammatory cell infiltration,and the expression levels of ALT,AST,MDA,IL-6,JAK2,STAT3 and the expression levels of IL-6,JAK2,STAT3 protein and mRNA in serum were reduced,and the expression levels of SOD were increased,with the best effect of Dahuang Lingxian Formula The treatment effect was best in the high concentration group.Conclusion:The mechanism may be related to the down-regulation of IL-6/JAK/STAT3 signaling pathway activation,and the best therapeutic effect was achieved by the high concentration group of Dahuang Lingxian Formula.

Mechanism of Yanghe Pingchaun granules on airway remodeling in asthmatic rats based on IL-6/JAK2/STAT3 signaling axis

Objective: To investigate the effects of Yanghe Pingchuan Granules on airway remodeling in asthmatic rats, and to explore the mechanism of Interleukin-6/Janus kinase 2/ Signal transducing activator of transcription 3(IL-6/JAK2/STAT3) signal axis. Methods: We separated 42 healthy male SD rats into two groups, a control group (7) and a model group (35).The model group was sensitized with a combination of ovalbumin (OVA) and aluminum hydroxide for 2 weeks, while the control group was given an equal amount of physiological saline.After 2 weeks, the modeling group was randomly divided into Model group, Yanghe Pingchuan Granules high, medium and low dose groups and Dexamethasone group, each group consisted of 7 animals. After 4 weeks, OVA atomization and gavage were used for stimulation and treatment. Yanghe Pingchuan Granules high, middle and low groups were given 15.48, 7.74, 3.87 g∙kg-1 Yanghe Pingchuan Granules daily, dexamethasone group was given 0.0625 mg∙kg-1 dexamethasone daily, and the other groups were given the same amount of normal saline. HE, PAS and Masson staining were used to observe the lung histopathological changes in rats. The levels of interleukin-6, IL-23 and IL-17A were detected by ELISA. The expression levels of JAK-2, P-JAK2, STAT3 and P-STAT3 in lung tissues were detected by Western blot. Real-time quantitative polymerase chain reaction (qRT-PCR) was used to detect the mRNA expression levels of IL-6, JAK2 and STAT3 in rat lung tissue. Results: The lung tissue structure of the model group was severely damaged compared to the control group, accompanied by a great many of inflammatory cell infiltration, goblet cell hyperplasia, subepithelial collagen fiber deposition and airway epithelial thickening were more obvious. The expressions of IL-6, IL- 23 and IL-17A in serum were significantly increased (P<0.01), the protein expression levels of JAK-2, P-JAK2, STAT3 and P-STAT3 and the mRNA expression levels of IL-6, JAK2 and STAT3 in lung tissue were significantly increased (P<0.01);Compared with the model group, inflammatory cell infiltration, goblet cell proliferation, subepithelial collagen fiber deposition and airway epithelial thickening were significantly reduced in each administration group, and the expressions of IL-6, IL-23 and IL-17A in serum were significantly decreased (P< 0.01). The protein expression levels of JAK-2, P-JAK2, STAT3 and P-STAT3 and mRNA expression levels of IL-6, JAK2 and STAT3 in lung tissue were significantly decreased (P<0.01). Conclusion: Yanghe Pingchuan Granules can significantly alleviate airway remodeling in asthmatic rats, and its mechanism may be through inhibiting the IL-6/JAK2/STAT3 signal axis.

Exploring the mechanism of electroacupuncture at different acupoints on acute colitis rats based on JAK2/STAT3/SOCS1 signaling pathway

Objective:To investigate the mechanism of JAK2/STAT3/SOCS1 signaling pathway in electroacupuncture of different acupoints on acute colitis rats.Methods:36 SPF SD rats were randomly divided into 6 groups,with 6 rats in each group.The rat model of acute colitis was prepared by enema with glacial acetic acid solution.After the model was established,electroacupuncture was given to each acupoint group,with density wave,frequency 2Hz-50 Hz,intensity 2 mA,muscle tremor as the degree 20 min/time,1 time/day,for 3 consecutive days.Observe the general condition of rats;the pathological changes of colonic mucosa in rats were observed by HE method.The contents of serum interleukin-4(IL-4)and interleukin-8(IL-8)were detected by ELISA.Western blot and RT-PCR were used to detect the expression of JAK2,STAT3,SOCS1 protein and mRNA in rat colon tissue.Results:In contrast to the normal group,the overall condition of the model group was worse,the colonic mucosa was severely damaged,even necrotic,and the ulcer surface was obvious.The content of IL-4 in serum was obviously reduced,and the content of IL-8 was obviously go up(P<0.01).The protein content of JAK2,STAT3 and the expression of JAK2,STAT3 mRNA in colon tissue of rats were obviously go up,while the protein content of SOCS1 and the expression of SOCS1 mRNA were obviously reduced(P<0.01).In contrast to the model group,the general condition of rats in each acupoint group was significantly improved,the damage and necrosis of colonic mucosa and ulcer surface were obviously alleviated,the content of IL-4 in serum was obviously go up,and the content of IL-8 was significantly decreased(P<0.01).The protein content of JAK2,STAT3 and the expression of JAK2,STAT3 mRNA in colon tissue of rats were obviously reduced,while the protein content of SOCS1 and the expression of SOCS1 mRNA were obviously go up(P<0.05,P<0.01).Comparison of different acupoint groups,the colonic mucosal injury in the Zusanli group was significantly reduced,the content of serum IL-4 was significantly increased,and the content of IL-8 was significantly decreased(P<0.05,P<0.01).The protein content and mRNA expression of JAK2 and STAT3 in colon tissue were significantly down-regulated,while the protein content and mRNA expression of SOCS1 were significantly go up(P<0.05,P<0.01).Conclusion:Electroacupuncture at each acupoint can improve the damage of colonic mucosa and reduce the inflammatory response.The therapeutic effect of Zusanli(ST36)is better than that of Tianshu(ST25),Dachangshu(BL25)and Shangjuxu(ST37).The mechanism may be related to the regulation of JAK2/STAT3/SOCS1 signaling pathway related proteins and inflammatory cytokines IL-4 and IL-8.

Oleanolic acid inhibits colon cancer cell stemness and reverses chemoresistance by suppressing JAK2/STAT3 signaling pathway

Background:Oleanolic acid(OA),a pentacyclic triterpenoid exhibiting specific anti-cancer properties and highly effective antioxidant activity,was isolated from traditional Chinese medicinal herbs.Conversely,the OA that impacts colon cancer(CC)cells and its underlying mechanisms remain poorly understood.Methods:The cytotoxic effect of OA alone or OA-5-Fluorouracil(5-FU)combination on normal and CC cells was analyzed by methyl thiazolyl diphenyl-tetrazolium bromide(MTT).Then,the impact of OA on CC cell lines(LoVo and HT-29)proliferation and stemness were measured using colon formation and tumorsphere formation assays.Octamer-binding transcription factor 4(Oct4),Prominin-1(CD133),Nanog,and transcription factor SOX-2(SOX2)are cell stemness-related indicators whose expression was assessed usingfluorescence qPCR assay,Western blotting,and immunohistochemistry.The effect of OA on the proliferative potency of CC cells was evaluated using an in vivo model.Results:The stem-like characteristics and clone production of colon cancer cells were markedly reduced by OA alone or in combination with OA-5-FU.Moreover,OA increases the susceptibility of CC cells to 5-FU by blocking the cell stemness-related markers(CD133,Nanog,SOX2,and Oct4)expression levels both in vitro and in vivo,as well as by inactivating the activator of transcription 3(STAT3 signaling)and Janus kinase 2/signal transducer(JAK2).Conclusion:Thesefindings imply that oleanolic acid,both in vitro and in vivo,suppresses the JAK2/STAT3 pathway,which in turn reverses chemoresistance and decreases colon cancer cell stemness.Therefore,by reducing the recommended amount of 5-FU,this strategy may improve chemotherapeutic effectiveness and minimize undesired side effects.

基于IL-6/JAK2/STAT3信号轴研究阳和平喘颗粒调控哮喘大鼠气道重塑作用机制

目的:研究阳和平喘颗粒对哮喘大鼠气道重塑及白细胞介素-6(IL-6)/Janus蛋白酪氨酸激酶2(JAK2)/信号转导和转录活化因子3(STAT3)信号轴在其中的作用机制。方法:选取健康雄性SD大鼠42只,随机数字法分为正常对照组7只与造模组35只,造模组采用卵清蛋白(OVA)联合氢氧化铝腹腔注射2周的方式进行致敏,正常对照组采用等量的生理盐水;2周后将造模组随机分为模型组、阳和平喘高、中、低剂量组和地塞米松组,每组7只;后4周采用OVA雾化+灌胃的方式进行激发和治疗,阳和平喘高中低组每日分别予以15.48、7.74、3.87 g/kg阳和平喘颗粒灌胃,地塞米松组予以0.0625 mg/kg地塞米松进行灌胃,其余组灌胃等量生理盐水。HE、PAS、Masson染色观察大鼠肺组织病理学变化;ELISA检测大鼠血清中IL-6、IL-23、IL-17A水平;Western blot检测肺组织中JAK-2、P-JAK2、STAT3、P-STAT3蛋白表达量;qRT-PCR检测大鼠肺组织中IL-6、JAK2、STAT3的mRNA水平。结果:与正常对照组比较,模型组大鼠肺组织有大量炎性细胞浸润,杯状细胞增生、上皮下胶原纤维沉积、气道上皮增厚较为明显;血清中IL-6、IL-23、IL-17A水平显著升高(P<0.01),肺组织JAK-2、P-JAK2、STAT3、P-STAT3的蛋白表达量和IL-6、JAK2、STAT3的mRNA表达水平显著升高(P<0.01);与模型组比较,各给药组炎性细胞浸润、杯状细胞增生、上皮下胶原纤维沉积、气道上皮增厚程度明显减轻,血清中IL-6、IL-23、IL-17A水平显著降低(P<0.01),肺组织JAK-2、P-JAK2、STAT3、P-STAT3的蛋白表达量和IL-6、JAK2、STAT3的mRNA水平显著降低(P<0.01)。结论:阳和平喘颗粒可明显缓解哮喘大鼠气道重塑,其机制可能是通过抑制IL-6/JAK2/STAT3信号轴发挥作用。

IL-6通过激活JAK2/STAT3信号通路增强小鼠肺泡巨噬细胞的吞噬功能

目的 探讨白细胞介素6(IL-6)对MH-S小鼠肺泡巨噬细胞吞噬功能的影响及其相关机制。方法 脂多糖(LPS)经气道滴入激发构建小鼠急性肺损伤(ALI)模型,ELISA检测支气管肺泡灌洗液(BALF)中IL-6的含量。体外培养MH-S细胞,在信号转导子与转录激活子3 (STAT3)抑制剂Stattic(5μmol/L)存在与否的情况下,再加入IL-6(10 ng/mL~500 ng/mL)刺激6 h,加入荧光微球孵育2 h后,采用流式细胞术检测MH-S细胞吞噬荧光微球情况;Western blot法检测磷酸化的Janus激酶2(p-JAK2)、磷酸化的STAT3(p-STAT3)、肌动蛋白相关蛋白2(Arp2)、纤维型肌动蛋白(F-actin)的表达水平。结果 鼻腔滴入LPS后,小鼠BALF中IL-6含量显著升高。随着IL-6刺激剂量的增加,MH-S细胞吞噬荧光微球的作用增强,Arp2、 F-actin蛋白的表达水平升高。100 ng/mL IL-6刺激MH-S细胞后,p-JAK2和p-STAT3蛋白的表达水平升高。阻断MH-S细胞STAT3信号后,IL-6促进细胞吞噬的效应完全消失,IL-6诱导的Arp2、 F-actin蛋白表达增加被抑制。结论 IL-6通过激活JAK2/STAT3信号通路促进MH-S细胞Arp2、 F-actin蛋白的表达增强吞噬功能。

基于IL-6介导的JAK1/STAT3信号通路探讨竹叶石膏汤合清气化痰丸对COPD大鼠的改善作用

目的探讨竹叶石膏汤合清气化痰丸对慢性阻塞性肺疾病(COPD)大鼠IL-6介导JAK1/STAT3信号通路的调控作用。方法将48只大鼠随机分为正常组,模型组,中药低、中、高剂量组(5、10、15 g/kg竹叶石膏汤合清气化痰丸)及中药低、中、高剂量(5、10、15 g/kg竹叶石膏汤合清气化痰丸)+伊他替尼(30 mg/kg)组,每组8只。采用气道滴注脂多糖(LPS)联合烟熏方法建立COPD模型,造模28 d后给药干预。给药2周后,采用肺功能检测仪测定肺功能指标[吸气峰流速(PIF)、呼气峰流速(PEF)、分钟通气量(MV)],HE染色观察肺组织病理变化,RT-qPCR、免疫印迹分析和免疫组化法检测肺组织IL-6、JAK1、STAT3、SOCS3 mRNA和蛋白表达。结果与模型组比较,中药各剂量组PIF、PEF、MV增加(P<0.05),肺组织炎症细胞浸润和充血水肿减轻,肺大泡减少,肺泡腔的破坏、扩张和融合得到缓解,IL-6、JAK1、STAT3 mRNA和蛋白表达降低(P<0.05),SOCS3 mRNA和蛋白表达升高(P<0.05);给予伊他替尼干预后,中药各剂量组的作用均被逆转(P<0.05)。结论竹叶石膏汤合清气化痰丸可下调IL-6介导的JAK/STAT信号通路过度表达和持续活化,并抑制IL-6表达,减轻气道炎症,抑制COPD症状。

IL-6/JAK2/STAT3通路中葛花解酲汤对脾虚湿热型溃疡性结肠炎“炎-癌转化”的预防作用

目的:探讨葛花解酲汤对脾虚湿热型溃疡性结肠炎“炎-癌转化”(UC-UCAC)小鼠结肠组织IL-6/JAK2/STAT3信号通路的影响。方法:80只SPF级C57BL/6雄性小鼠随机选出10只作为空白组,其余70只为造模组。造模组在建立脾虚湿热模型后随机分为模型组(第1、2、3周期)、葛花解酲汤高、中、低剂量组、美沙拉嗪组,10只/组,以氧化偶氮甲烷(AOM)/葡聚糖硫酸钠(DSS)继续建立UC-UCAC转化模型。各组给予相应药物治疗4周。观察小鼠一般状态;统计小鼠疾病活动指数(DAI)评分;HE染色观察小鼠结肠黏膜组织病理;Western blot、IHC和RT-q PCR检测小鼠结肠组织EGFR、IL-6、JAK2、STAT3、 p-STAT3蛋白和基因表达。结果:与空白组相比,模型组(第3周期)小鼠一般状态较差,结肠黏膜组织出现癌变,DAI评分、各目标蛋白及基因表达显著升高(P<0.01);与模型组(第3周期)相比,各治疗组小鼠一般状态有所恢复,结肠组织病理不同程度改善,除葛花解酲汤低剂量组外,其他各治疗组各目标蛋白及基因表达显著下降(P<0.01)。结论:葛花解酲汤可能通过抑制IL-6/JAK2/STAT3信号通路激活破坏肿瘤炎症微环境,修复受损结肠黏膜组织,延缓UC-UCAC进程,预防UCAC。

基于IL-6/JAK2/STAT3信号通路探讨电针对脑卒中肢体痉挛大鼠的影响

目的观察电针对脑卒中肢体痉挛(PSS)大鼠中枢炎症反应及神经递质释放的影响,基于IL-6/JAK2/STAT3信号通路探讨其治疗PSS的作用机制。方法30只雄性SD大鼠随机分为假手术组、模型组和电针组,每组10只。采用线栓+内囊注射N-甲基-D-天冬氨酸受体法制备PSS大鼠模型,电针组电针患侧“曲池”“阳陵泉”,30 min/d,连续7 d。假手术组、模型组仅固定不干预。治疗前后进行Zea Longa神经功能评分和改良Ashworth肌张力评分,HE染色观察缺血侧大脑皮质病理变化,ELISA检测缺血侧皮质白细胞介素-6(IL-6)、肿瘤坏死因子-α(TNF-α)、γ-氨基丁酸(GABA)含量,生化试剂盒检测缺血侧皮质谷氨酸(Glu)含量,Western blot检测缺血侧皮质酪氨酸激酶2(JAK2)、p-JAK2、信号转导和转录激活因子3(STAT3)、p-STAT3蛋白表达,RT-PCR检测缺血侧皮质JAK2、STAT3 mRNA表达。结果与假手术组比较,模型组大鼠神经功能评分、肌张力评分明显升高(P<0.01),大脑皮质神经元紊乱、细胞核固缩,IL-6、TNF-α、Glu含量明显增加,GABA含量明显减少(P<0.01),p-JAK2、p-STAT3蛋白及JAK2、STAT3 mRNA表达均明显升高(P<0.01,P<0.05);与模型组比较,电针组大鼠神经功能评分、肌张力评分明显降低(P<0.05),大脑皮质神经元损伤程度减轻,细胞轮廓清晰,IL-6、TNF-α、Glu含量明显减少,GABA含量明显增加(P<0.05,P<0.01),p-JAK2、p-STAT3蛋白及JAK2、STAT3 mRNA表达均明显降低(P<0.01)。结论电针可能通过抑制IL-6/JAK2/STAT3信号通路减轻PSS模型大鼠中枢炎症反应,改善肢体痉挛状态。

基于IL-6/JAK2/STAT3通路探讨斑蝥酸钠对人胃癌细胞增殖、凋亡及炎症免疫的影响

目的探讨去甲斑蝥酸钠(SNCTD)基于白细胞介素-6(IL-6)/酪氨酸蛋白激酶2(JAK2)/信号转导及转录激活因子3(STAT3)通路对胃癌细胞增殖、凋亡和炎症免疫的影响。方法以人胃癌HGC-27细胞为研究对象,加入不同浓度SNCTD(0.25、0.5、1、2、4、-8、16μmol·L^(-1))分别处理24、48h后,用CCK-8法检测各浓度组细胞生长抑制率。不同浓-度SNCTD(1、2、4μmol·L^(-1))作用细胞24h后,ELISA法检测细胞上清液中IL-6水平;流式细胞术检测各浓度组细胞凋亡率;Western blot法和RT-qPCR法分别对IL-6/JAK2/STAT3通路基因(IL-6、GP130、JAK2、STAT3)、细胞增殖和凋亡相关基因(c-Myc、Bcl-2、Bax)进行mRNA和蛋白水平上的表达情况检测。结果与对照组相比,SNCTD组细胞生长抑制率、凋亡率均明显升高(P<0.05);细胞上清液中IL-6水平下降(P<0.01);细胞c-Myc、Bcl-2的mRNA和蛋白表达量均明显下降(P<0.05),而Bax的mRNA和蛋白表达量则明显上升(P<0.05);细胞IL-6、GP130、p-JAK2/JAK2、p-STAT3/STAT3蛋白表达量及JAK2、STAT3 mRNA表达明显下降(P<0.05)。结论SNCTD能有效抑制胃癌细胞增殖并诱导凋亡,其机制可能与SNCTD降低胃癌细胞微环境中炎症因子IL-6的表达有关,通过抑制IL-6/JAK2/STAT3通路的激活,达到调控特定基因的转录与翻译,包括下调c-Myc和Bcl-2、上调Bax的表达。

IL-6/JAK2/STAT3信号通路在电针调控胰岛素抵抗模型大鼠骨代谢中的作用

目的研究电针对胰岛素抵抗(insulin resistance,IR)大鼠骨代谢及炎症状态的影响。方法8只大鼠按照随机数字表单列为空白组,另外32只大鼠给予高脂饲料喂养8周以建立IR大鼠模型,选取造模成功的24只大鼠,随机分为模型组、假电针组、电针组,每组8只,空白组、假电针组不做干预,电针组选取“中脘”“关元”、“足三里”、“丰隆”4穴进行电针治疗,假电针组选取上述4穴旁边的皮下后不进行电针治疗,各组均治疗8周。在治疗前后,测量各组大鼠的体质量(body mass,BM)、空腹血糖(fasting blood-glucose,FBG)及餐后血糖水平(postprandial blood-glucoser,PBG)、葡萄糖输注速率(glucose infusion rate,GIR);各组大鼠血清中Ⅰ型胶原氨基端前肽(N-terminal propeptide of typeⅠcollagen,P1NP)、β-Ⅰ型胶原C末端肽(C-terminal telopeptides of typeⅠcollagen,β-CTX)含量用酶联免疫吸附测定(enzyme-linked immunosorbent assay,ELISA)检测;大鼠骨组织的骨密度(bone mineral density,BMD)、骨体积分数(BV/TV)、骨小梁数量(trabecularNumber,Tb.N)和骨小梁分离度(trabecularSeparation/Spacing,Tb.Sp)采用微计算机断层扫描技术(micro CT)检测;骨组织中白细胞介素-6(interleukin-6,IL-6)、Janus蛋白酪氨酸激酶2(Janus Protein Tyrosine Kinase2,JAK2)、信号转导与转录活化因子3(Signal Transducers and Activators of Transcription 3,STAT3)mRNA及蛋白的表达分别采用实时荧光定量聚合酶链式反应(Real-time PCR)与蛋白免疫印迹法(Western blot)检测。结果治疗前与空白组相比,模型组、假电针组、电针组大鼠BMD、PBG值明显升高(P<0.01或P<0.05),GIR明显降低(P<0.01);治疗结束后,与空白组相比,电针组大鼠BM、FBG、PBG、β-CTX、IL-6、JAK2、STAT3 mRNA及蛋白浓度明显升高(P<0.01),GIR、P1NP、BMD、BV/TV、Tb.N和Tb.Sp明显降低(P<0.01);与模型组比较,电针组大鼠的BM、PBG、β-CTX、IL-6、JAK2、STAT3 mRNA及蛋白浓度均明显降低,BMD、BV/TV、Tb.N、Tb.Sp、GIR均明显升高(P<0.05或P<0.01);假电针组大鼠BM、PBG、JAK2 mRNA、STAT3蛋白表达明显降低(P<0.05),而P1NP、β-CTX、BMD、BV/TV、Tb.N、Tb.Sp、IL-6、STAT3 mRNA、IL-6、JAK2蛋白表达均无统计学差异(P>0.05)。结论电针能够通过调控IL-6/JAK2/STAT3信号通路降低机体炎症反应改善胰岛素抵抗,并能一定程度的降低机体破骨细胞活性,减少骨吸收。

Yangyin Huowei mixture alleviates chronic atrophic gastritis by inhibiting the IL-10/JAK1/STAT3 pathway

BACKGROUND The Chinese medicine Yangyin Huowei mixture(YYHWM)exhibits good clinical efficacy in the treatment of chronic atrophic gastritis(CAG),but the mechanisms underlying its activity remain unclear.AIM To investigate the therapeutic effects of YYHWM and its underlying mechanisms in a CAG rat model.METHODS Sprague-Dawley rats were allocated into control,model,vitacoenzyme,and low,medium,and high-dose YYHWM groups.CAG was induced in rats using Nmethyl-N′-nitro-N-nitrosoguanidine,ranitidine hydrochloride,hunger and satiety perturbation,and ethanol gavage.Following an 8-wk intervention period,stomach samples were taken,stained,and examined for histopathological changes.ELISA was utilized to quantify serum levels of PG-I,PG-II,G-17,IL-1β,IL-6,and TNF-α.Western blot analysis was performed to evaluate protein expression of IL-10,JAK1,and STAT3.RESULTS The model group showed gastric mucosal layer disruption and inflammatory cell infiltration.Compared with the blank control group,serum levels of PGI,PGII,and G-17 in the model group were significantly reduced(82.41±3.53 vs 38.52±1.71,23.06±0.96 vs 11.06±0.70,and 493.09±12.17 vs 225.52±17.44,P<0.01 for all),whereas those of IL-1β,IL-6,and TNF-αwere significantly increased(30.15±3.07 vs 80.98±4.47,69.05±12.72 vs 110.85±6.68,and 209.24±11.62 vs 313.37±36.77,P<0.01 for all),and the protein levels of IL-10,JAK1,and STAT3 were higher in gastric mucosal tissues(0.47±0.10 vs 1.11±0.09,0.49±0.05 vs 0.99±0.07,and 0.24±0.05 vs 1.04±0.14,P<0.01 for all).Compared with the model group,high-dose YYHWM treatment significantly improved the gastric mucosal tissue damage,increased the levels of PGI,PGII,and G-17(38.52±1.71 vs 50.41±3.53,11.06±0.70 vs 15.33±1.24,and 225.52±17.44 vs 329.22±29.11,P<0.01 for all),decreased the levels of IL-1β,IL-6,and TNF-α(80.98±4.47 vs 61.56±4.02,110.85±6.68 vs 89.20±8.48,and 313.37±36.77 vs 267.30±9.31,P<0.01 for all),and evidently decreased the protein levels of IL-10 and STAT3 in gastric mucosal tissues(1.11±0.09 vs 0.19±0.07 and 1.04±0.14 vs 0.55±0.09,P<0.01 for both).CONCLUSION YYHWM reduces the release of inflammatory factors by inhibiting the IL-10/JAK1/STAT3 pathway,alleviating gastric mucosal damage,and enhancing gastric secretory function,thereby ameliorating CAG development and cancer transformation.

淫羊藿素通过调控IL-6/JAK2/STAT3途径抑制卵巢癌的发生与恶性生长

目的:探讨淫羊藿素通过调控IL-6/JAK2/STAT3信号通路对卵巢癌A2780和SKOV3细胞增殖、凋亡和侵袭及对小鼠卵巢癌移植瘤模型的影响。方法:将人卵巢癌A2780和SKOV3细胞置于细胞培养箱中培养,设置空白对照组和淫羊藿素低、中、高剂量组(5.00、10.00、20.00μmol/L),药物干预48 h。采用克隆形成实验检测细胞增殖,流式细胞术检测细胞凋亡,Transwell实验检测细胞侵袭。Western blotting检测Ki67、PCNA、cleaved Caspase-3、cleaved Caspase-9、E-cadherin、N-cadherin、VEGF、IL-6、p-JAK2和p-STAT3蛋白相对表达量。建立裸鼠卵巢癌移植瘤模型,检测移植瘤质量和体积,采用Western blotting检测Ki67、cleaved Caspase-3、E-cadherin、N-cadherin、VEGF、IL-6、p-JAK2和p-STAT3蛋白的表达量。结果:与空白对照组比较,淫羊藿素中剂量和高剂量治疗后,卵巢癌A2780和SKOV3细胞增殖、侵袭降低,凋亡增加,体内外Ki67、PCNA、cleaved Caspase-3、cleaved Caspase-9、N-cadherin、VEGF、IL-6、p-JAK2和p-STAT3蛋白的表达量下调,E-cadherin蛋白的表达量上调。结论:淫羊藿素可抑制卵巢癌A2780和SKOV3细胞的增殖和侵袭,促进细胞凋亡,其机制与淫羊藿素影响IL-6、JAK2和STAT3的表达有关。

西黄胶囊调控IL-6/JAK2/STAT3信号通路促进非哺乳期乳腺炎大鼠模型创面愈合的作用机制研究

目的观察西黄胶囊对非哺乳期乳腺炎大鼠模型创面愈合及白细胞介素-6(interleukin-6,IL-6)/非受体型酪氨酸蛋白激酶2(Janus kinase 2,JAK2)/信号转导及转录激活蛋白3(signal transducer and activator of transcription 3,STAT3)信号通路的影响。方法复制非哺乳期乳腺炎大鼠模型,随机分为模型对照组、阳性药物组、中药治疗组、联合用药组,每组10只,另选取10只同批次SPF级健康SD大鼠作为假手术组。干预后观察并评估各组大鼠的创面愈合情况及乳腺炎症指数,HE染色法观察各组大鼠乳腺组织的病理改变;免疫组织化学法检测各组大鼠乳腺组织B淋巴细胞瘤-2基因关联X蛋白(B-cell lymphoma-2-associated X,BAX)、B淋巴细胞瘤-2基因(B-cell lymphoma-2,Bcl-2)的平均光密度值;Western blot法及RT-PCR法检测各组大鼠乳腺组织IL-6、JAK2、STAT3、人胱天蛋白酶9(Caspase-9)蛋白及mRNA的表达水平。结果与假手术组大鼠比较,模型对照组大鼠的创面愈合率明显降低(P<0.05),而乳腺炎症指数,乳腺组织BAX、Bcl-2平均光密度值,IL-6、JAK2、STAT3、Caspase-9蛋白及mRNA的表达水平显著升高(P<0.05)。HE染色可见模型对照组大鼠乳腺腺叶存在明显的肉芽肿变性,周围充斥大量炎症细胞,阳性药物组、中药治疗组、联合用药组大鼠的病理情况存在不同程度的缓解,炎性细胞浸润及肉芽肿组织明显减少,“空泡样”坏死存在不同程度的愈合。与模型对照组比较,阳性药物组、中药治疗组、联合用药组的创面愈合率、Bcl-2光密度值均明显升高(P<0.05);其中联合用药组的创面愈合率、Bcl-2光密度值明显高于阳性药物组及中药治疗组(P<0.05);阳性药物组的创面愈合率高于中药治疗组(P<0.05),Bcl-2光密度值与中药治疗组比较差异无统计学意义(P>0.05)。与模型对照组比较,阳性药物组、中药治疗组、联合用药组的BAX光密度值、乳腺炎症指数均明显下降(P<0.05);其中联合用药组的BAX光密度值、乳腺炎症指数明显低于阳性药物组及中药治疗组(P<0.05);阳性药物组的乳腺炎症指数低于中药治疗组(P<0.05),BAX光密度值与中药治疗组比较差异无统计学意义(P>0.05)。与模型对照组比较,阳性药物组、中药治疗组、联合用药组的IL-6、JAK2、STAT3、Caspase-9蛋白及mRNA的表达水平均明显降低(P<0.05);其中联合用药组上述蛋白及mRNA表达水平明显低于阳性药物组及中药治疗组(P<0.05),而阳性药物组的上述蛋白及mRNA表达水平均明显高于中药治疗组(P<0.05)。结论西黄胶囊能有效促进非哺乳期乳腺炎大鼠模型的乳腺组织创面愈合,抑制乳腺组织细胞凋亡及炎症反应,其作用机制可能与西黄胶囊抑制IL-6/JAK2/STAT3信号通路相关。

Effects of plumbagin on migration and invasion of human hepatoma cell line via JAK2/STAT3 signaling pathway

Objective:To study the effect of plumbagin(PL)on the migration and invasion of human hepatocellular carcinoma(HCC)cells and its possible mechanism.Methods:The cell counting kit(CCK-8)was used to detect the effects of different concentrations of plumbagin on the proliferation of human hepatocellular carcinoma Huh-7 and LM3 cells.The effect of plumbagin on the migration ability of Huh-7 and LM3 cells was detected by scratch test and Transwell migration test,and the effect of on the invasion ability of Huh-7 and LM3 cells was detected by Transwell invasion test.Western Blot was used to detect the expression of E-cadherin,N-cadherin,matrix metalloproteinase-2 and related proteins in JAK2/STAT3 signaling pathway in Huh-7 and LM3 cells.Results:Plumbagin could inhibit the proliferation of Huh-7 and LM3 cells in a time-and concentration-dependent manner.Plumbagin inhibited the migration and invasion of Huh-7 and LM3 cells in a concentration dependent manner,and it can down-regulate the expression of N-cadherin and MMP-2 protein,up-regulate the expression of E-cadherin protein,and inhibit the activation of JAK2/STAT3 signaling pathway.Conclusion:Plumbagin can inhibit the migration and invasion of human hepatocellular carcinoma Huh-7 and LM3 cells,and the molecular mechanism of this process may be related to the inhibition of JAK2/STAT3 signaling pathway activation.

基于IL-6/JAK/STAT3信号通路探讨大黄灵仙方缓解胆管细胞炎性反应的作用机制

目的:基于IL-6/JAK/STAT3信号通路探讨大黄灵仙方缓解脂多糖(LPS)诱导肝内胆管炎症模型大鼠的胆管细胞炎性反应的作用机制。方法:将50只SD大鼠随机分为5组,空白组、模型组、胆宁片(0.5 g/kg)、大黄灵仙方低、高浓度组(2.4 g/kg、4.8 g/kg),每组10只。除空白组以外,其余各组大鼠于胆总管处一次性注射1.25 mg/kg LPS以构建肝内胆管感染动物模型。第8天灌胃结束后取材,取大鼠肝门处肝脏组织,采用HE染色观察肝门和胆管树组织病理变化。生化法测定血清中谷丙转氨酶(ALT)、谷草转氨酶(AST)、丙二醛(MDA)、超氧化物歧化酶(SOD)的表达水平。酶联免疫吸附法(ELISA)检测大鼠血清中白介素6(IL-6)、Janus蛋白酪氨酸激酶2(JAK2)、信号转导和转录活化因子3(STAT3)的表达水平。蛋白免疫印迹法(WB)、实时荧光定量PCR(RT-qPCR)检测胆管树组织中的IL-6、JAK2、STAT3蛋白及mRNA的表达水平。结果:(1)与空白组对比,模型组大鼠的肝窦和门管区小叶间胆管等结构均被破坏,周围存在炎性细胞浸润,血清中ALT、AST、MDA、IL-6、JAK2、STAT3表达量明显上升,SOD的表达水平下降,IL-6、JAK2、STAT3蛋白及mRNA表达水平升高。(2)与模型组比较,胆宁片组、大黄灵仙方低、高浓度组的大鼠肝脏病理损伤程度均有改善,能显著降低血清中ALT、AST、MDA、IL-6、JAK2、STAT3和上调SOD的表达水平,下调IL-6、JAK2、STAT3蛋白及mRNA的表达,以大黄灵仙方高浓度组效果最佳。(3)与胆宁片组相比,大黄灵仙方低、高浓度组的大鼠肝脏病理损伤程度趋于正常,无明显炎性细胞浸润,血清中ALT、AST、MDA、IL-6、JAK2、STAT3表达量以及IL-6、JAK2、STAT3蛋白及mRNA表达水平均降低,SOD的表达水平上升,以大黄灵仙方高浓度组治疗效果最佳。结论:大黄灵仙方可有效减缓LPS诱导的大鼠胆管炎症反应,促使胆道恢复至非炎症状态,其机制可能与下调IL-6/JAK/STAT3信号通路的活化有关,以大黄灵仙方高浓度组治疗效果最佳。

肉桂醛调节IL-6/JAK2/STAT3信号通路对变应性鼻炎大鼠鼻黏膜细胞凋亡的影响

目的探讨肉桂醛对变应性鼻炎(allergic rhinitis,AR)大鼠鼻粘膜细胞凋亡的影响及机制。方法构建大鼠AR模型,并随机分为AR组、肉桂醛低、中、高剂量组和氯雷他定组,另选10只大鼠为假手术组(sham组)。检测大鼠血清IL-6水平、鼻粘膜组织细胞凋亡和IL-6、JAK2、STAT3 mRNA表达以及JAK2、p-JAK2、STAT3、p-STAT3、Bcl-2及Bax蛋白表达。结果与sham组比,AR组大鼠血清IL-6水平、鼻粘膜组织IL-6 mRNA及p-JAK2/JAK2、p-STAT3/STAT3和Bcl-2蛋白表达均升高(P<0.05),细胞凋亡指数(AI)和Bax蛋白表达降低(P<0.05);与AR组比,肉桂醛低、中、高剂量组和氯雷他定组大鼠血清IL-6水平、鼻粘膜组织IL-6 mRNA及p-JAK2/JAK2、p-STAT3/STAT3和Bcl-2蛋白表达均降低(P<0.05),AI和Bax蛋白表达升高(P<0.05)。结论肉桂醛可促进AR大鼠鼻粘膜细胞凋亡,其机制可能与抑制IL-6/JAK2/STAT3通路有关。

基于JAK2/STAT3信号通路探讨黄芪-山茱萸对高糖诱导足细胞损伤的保护作用

目的:探究黄芪-山茱萸对高糖诱导肾足细胞损伤的保护作用及机制。方法:CCK8法检测黄芪-山茱萸(0、175、350、700、1400、2800 mg/L)对足细胞(MPC-5)活性的影响。30 mmol/L葡萄糖诱导MPC-5细胞损伤,350、700、1400 mg/L黄芪-山茱萸进行干预;ELISA测定MPC-5细胞IL-6、IL-1β水平,流式细胞术测定细胞凋亡,免疫荧光染色测定MPC-5肾病蛋白(nephrin)、足细胞素(podocin)表达,qRT-PCR测定MPC-5细胞nephrin、podocin、结蛋白(desmin)、Wilm瘤基因1(WT-1)基因表达,Western blot测定细胞Janus激酶2/信号转导和转录激活因子3(JAK2/STAT3)信号通路磷酸化表达。结果:2800 mg/L黄芪-山茱萸可显著抑制MPC-5细胞活性(P<0.01)。MPC-5细胞经高糖诱导后,IL-6、IL-1β水平升高,凋亡增加,nephrin、podocin、WT-1表达降低,desmin表达升高,JAK2/STAT3信号通路磷酸化表达增加(P<0.01)。350、700、1400 mg/L黄芪-山茱萸可抑制高糖诱导MPC-5细胞IL-6、IL-1β水平升高和凋亡,增强nephrin、podocin、WT-1表达,降低desmin表达,抑制JAK2/STAT3信号通路磷酸化表达(P<0.05)。结论:黄芪-山茱萸可减轻高糖诱导足细胞炎症和凋亡损伤,其机制与抑制JAK2/STAT3通路磷酸化有关。

白细胞介素-1β通过IL-6/JAK2/STAT3轴促进脑胶质瘤细胞的侵袭

目的探讨白细胞介素-1β(IL-1β)在脑胶质瘤细胞侵袭中的作用及其机制。方法使用ELISA检测IL-1β和IL-6在人星形胶质细胞(NHA)、U87、U251、A172胶质瘤细胞培养液上清中的浓度,根据结果挑选实验细胞,随后检测IL-1β刺激下的IL-6生成。在不同浓度的IL-1β刺激、改变IL-6含量、下调JAK2和STAT3基因表达水平等不同条件下,使用Transwell小室实验检测胶质瘤细胞侵袭能力的变化以及Western blot检测JAK2、STAT3磷酸化蛋白水平的改变。结果与NHA相比,U87、U251的培养液中IL-1β水平明显升高,U87、U251、A172的培养液中IL-6水平均明显升高。在U87、U251细胞中,IL-1β不仅能促进细胞的侵袭,而且能促进其IL-6的产生。在IL-1β的刺激下,IL-6蛋白能进一步促进细胞的侵袭,而IL-6中和抗体则明显抑制IL-1β的促侵袭作用。研究表明IL-1β刺激可促进U87、U251细胞JAK2和STAT3的磷酸化蛋白表达。在IL-1β的刺激下,IL-6蛋白能进一步促进它们的表达,而IL-6抗体则明显抑制其表达。同时,下调JAK2和STAT3也能显著抑制IL-1β刺激下的U87、U251细胞的侵袭能力。结论IL-1β通过IL-6/JAK2/STAT3轴促进脑胶质瘤细胞的侵袭。

Effects of IL-6/JAK2/STAT3 on the Biological Behavior of Oral Squamous Cell Carcinoma

Objective:To investigate the effect of interleukin 6/Janus kinase 2/signal transducer and activator of transcription 3(IL-6/JAK2/STAT3)on the biological behavior of oral squamous cell carcinoma(OSCC).Methods:OSCC cells were transfected with the designed lentiviral vector plasmid pGMLV-SB3(experimental group)and the corresponding negative control plasmid pGMLV-SB3-shNC(control group);48 hours after transfection,a liposome transfection kit(Sigma,USA)was used for lentivirus packaging;after virus packaging,a medium containing pGMLV-SB3 lentiviral vector was added and cultured for 24 h;the cells were harvested,and RNA was extracted;Transwell chamber assay(Sigma,USA)was used to detect cell migration and invasion ability;dot-enzyme-linked immunosorbent assay(ELISA)kit was used to detect the level of interleukin 6(IL-6)in the culture supernatant,while serum IL-6 level was measured by ELISA.Results:The expressions of IL-6,JAK2,and STAT3 in the experimental group were significantly raised,as compared to the control group(P<0.05);the apoptosis rate of OSCC cells in the experimental group,which was detected by flow cytometry 48 h after transfection,was significantly higher than that of cells in the control group(P<0.05);and there was a significant improvement in the experimental group’s cell migration and invasion ability,as compared to that of the control group(P<0.05).Conclusion:The IL-6/JAK2/STAT3 signaling pathway plays an important role in the migration and invasion of OSCC cells.Inhibiting the expression of IL-6 can inhibit the growth and proliferation of OSCC cells as well as reduce their ability to invade and migrate.These results provide a new target for the treatment of OSCC.