莪连颗粒调控JAK2/STAT3通路治疗慢性萎缩性胃炎的机制

Mechanism of Elian Granules modulating JAK2/STAT3 pathway for treatment of chronic atrophic gastritis

目的通过转录组测序探讨莪连颗粒对慢性萎缩性胃炎(CAG)大鼠Janus激酶2(JAK2)/信号转导和转录激活因子3(STAT3)通路的影响,揭示莪连颗粒治疗CAG的机制。方法18只6周龄雄性SD大鼠适应性喂养1周后,随机分为正常组、模型组和莪连颗粒组。除正常组外,模型组、莪连颗粒组采用N-甲基-N'-硝基-N-亚硝基胍(MNNG)综合法构建CAG大鼠模型,莪连颗粒组予莪连颗粒水溶液灌胃(剂量3.24 g/kg),干预周期均为40周。苏木精-伊红(HE)染色法观察大鼠胃窦组织病理;转录组学法进行建库测序及生物信息学分析;蛋白免疫印迹(Western blot)法检测各组大鼠胃窦组织JAK2、磷酸化Janus激酶2(p-JAK2)、STAT3、磷酸化信号转导和转录激活因子3(p-STAT3)、细胞因子信号抑制因子3(SOCS3)、B淋巴细胞瘤-2基因蛋白(Bcl-2)、Bcl-2相关X蛋白(Bax)的表达。结果正常组大鼠胃黏膜上皮结构完整,腺体形态规则,排列整齐规律;模型组大鼠胃黏膜腺体萎缩,排列紊乱;莪连颗粒组大鼠胃黏膜腺体形态尚完整,排列尚规律。与正常组比较,模型组差异基因3595个,其中上调1203个,下调2392个;与模型组比较,莪连颗粒组差异基因561个,其中上调174个,下调387个。京都基因和基因组百科全书数据库(KEGG)富集分析结果显示,莪连颗粒干预CAG作用机制可能与趋化因子信号传导途径、磷脂酰肌醇3-激酶(PI3K)-蛋白激酶B(Akt)信号通路、癌症通路等密切相关。Western blot提示,与正常组比较,模型组大鼠胃窦组织p‑JAK2、p-STAT3、Bcl-2蛋白表达显著升高(P<0.01,P<0.001),SOCS3、Bax蛋白表达显著降低(P<0.01);与模型组比较,莪连颗粒组大鼠胃窦组织p-JAK2、p-STAT3、Bcl-2蛋白表达显著降低(P<0.01,P<0.001),SOCS3、Bax蛋白表达显著升高(P<0.05)。结论本研究通过MNNG综合法成功复制CAG大鼠模型,借助转录组测序发现并经验证表明莪连颗粒可能通过抑制JAK2/STAT3通路治疗CAG。

Objective To explore the effect of Elian Granules on the Janus kinase 2/signal transducer and activator of transcription 3(JAK2/STAT3)pathway in rats with chronic atrophic gastritis(CAG)using transcriptomic sequencing,and to elucidate the mechanisms behind its therapeutic action.Methods Eighteen 6-week-old male Sprague-Dawley(SD)rats were acclimatized for one week and then divided into three groups:a normal group,a model group,and an Elian Granules group.Apart from the normal control group,the model group and the Elian Granules group were induced with chronic atrophic gastritis using the N-methyl-N'-nitro-N-nitrosoguanidine(MNNG)synthesis method.The Elian Granules group was administered an oral gavage of Elian Granules solution at a dosage of 3.24 g/kg.The intervention lasted for 40 weeks.The pathological morphology of rat gastric tissues was detected using Hematoxylin-Eosin(HE)staining;transcriptomics was performed for library sequencing and raw signal analysis,and JAK2,phosphorylated Janus kinase 2(p-JAK2),STAT3,phosphorylated signal transducer and activator of transcription 3(p-STAT3),suppressor of cytokine signaling 3(SOCS3),B-cell lymphoma-2(Bcl-2),and Bcl-2-associated X protein(Bax)protein expression were detected using Western blot in each group of rat gastric sinus tissues.Results The epithelial structure of the gastric mucosa was intact in the control group of rats,and the glands had normal morphology and were arranged in a neat and regular manner.In the model group,the gastric mucosal glands of rats were atrophied,with disorderly arrangement.In contrast,in the Elian Granules group,the glandular morphology was maintained relatively intact with regular arrangement.Compared to the normal group,the model group expressed 3,595 differentially regulated genes(DEGs),including 1,203 upregulated and 2,392 downregulated genes.Compared to the model group,the Elian Granules group exhibited 561 DEGs,including 174 upregulated and 387 downregulated genes.Enrichment analysis through Kyoto Encyclopedia of Genes and Genomes(KEGG)suggested that the mechanisms of CAG intervention by Elian Granules might be closely associated with signaling pathways such as chemokine signaling pathway,PI3K-Akt signaling pathway,and oncogenic pathways.Western blot results indicated that compared to the control group,the model group had significantly higher expression of p-JAK2,p-STAT3,and Bcl-2 proteins(P<0.01,P<0.001),and significantly lower SOCS3 and Bax proteins(P<0.01).The Elian Granules group showed significant reductions in p-JAK2,p-STAT3,and Bcl-2 protein expressions compared to the model group(P<0.01,P<0.001),with a significant increase in SOCS3 and Bax protein expressions(P<0.05).Conclusion In this study,we successfully replicated the CAG rat model using the MNNG synthesis method,and with the help of transcriptomic sequencing and validation,we found and empirically demonstrated that Elian granules may be able to treat CAG by inhibiting the JAK2/STAT3 pathway.