基于转录组测序技术研究香砂六君子汤治疗慢性萎缩性胃炎的分子机制

Molecular mechanism of Xiangsha Liujunzi Decoction in treating chronic atrophic gastritis based on transcriptome sequencing technology

基于转录组学技术探讨香砂六君子汤治疗慢性萎缩性胃炎(CAG)的分子机制并进行实验验证。通过1-甲基-3-硝基-1-亚硝基胍(MNNG)复合多因素法建立CAG大鼠模型,香砂六君子汤干预90 d。测定各组大鼠体质量和3 h进食量,采用HE染色观察各组大鼠胃组织病理改变,采用转录组学测序技术(RNA-Seq)测定各组大鼠胃组织中基因表达,对差异基因进行KEGG富集分析并对富集到的关键信号通路进行验证。结果显示,与空白组相比,模型组大鼠体质量、3 h进食量显著降低,胃组织表现典型的萎缩之征;与模型组相比,给药组大鼠体质量、3 h进食量显著增加,胃组织病理损伤均得到明显改善。RNA-Seq测序共获得香砂六君子汤改善CAG的相关基因944个,KEGG富集分析发现这些基因主要富集于多种涉及炎症、增殖、凋亡的信号转导途径以及环磷酸腺苷(cAMP)和环磷酸鸟苷(cGMP)/蛋白激酶G(PKG)通路。与空白组比较,模型组胃组织中腺苷A1受体(Adora1)、钙激活钾通道蛋白β2(Kcnmb2)mRNA表达显著升高,B型内皮素受体(Ednrb)、人Rho关联含卷曲螺旋蛋白激酶2(Rock2)、cGMP依赖性蛋白激酶Ⅱ(Prkg2)、环腺苷酸应答元件结合蛋白H抗体(Creb3l3)mRNA表达显著降低;与模型组比较,给药组胃组织中Adora1、Kcnmb2 mRNA表达显著降低,Ednrb、Rock2、Prkg2、Creb3l3 mRNA表达显著升高。与空白组比较,模型组胃组织和血浆中cAMP含量以及胃组织中Ras同源基因家族成员A(RhoA)、ROCK2、磷酸化肌球蛋白磷酸酶靶标亚基1(p-MYPT1)、磷酸化肌球蛋白轻链20(p-MLC20)蛋白表达均显著降低,胃组织和血浆中cGMP含量以及胃组织中PKG蛋白表达显著升高;与模型组比较,给药组胃组织和血浆中cGMP含量以及胃组织中PKG蛋白表达显著降低,胃组织中RhoA、ROCK2、p-MYPT1、p-MLC20蛋白表达显著升高。同时RT-qPCR结果表明cGMP/PKG通路关键基因的表达与RNA-Seq结果基本一致。综上所述,香砂六君子汤对CAG大鼠具有显著的治疗作用,其具体机制可能与调控cGMP/PKG通路及下游相关信号分子的异常表达相关。

Based on transcriptomics technology,this study investigated the molecular mechanisms of Xiangsha Liujunzi Decoction in treating chronic atrophic gastritis(CAG),which were confirmed through experimental validation.The CAG rat model was built by the MNNG composite multi-factor method,followed by a 90-day administration of Xiangsha Liujunzi Decoction.The study measured the rat body mass and 3-hour food intake in each group and observed the pathological changes in gastric tissue using HE staining.It used RNA sequencing(RNA-Seq)to determine the gene expression in the gastric tissue of rats in each group and then analyzed differential genes through KEGG enrichment analysis to validate enriched key signaling pathways.The results indicated that the body mass and 3-hour food intake in the model group were significantly lower than those in the blank group,alongside typical signs of gastric tissue atrophy.In contrast,the treatment group exhibited a significant increase in body mass and 3-hour food intake and significant improvements in the damage of gastric tissue pathology.A total of 944 genes associated with the improvement of CAG by Xiangsha Liujunzi Decoction were obtained via RNA-Seq.KEGG enrichment analysis revealed that these genes were mainly enriched in multiple signal transduction pathways involving signaling pathways such as inflammation,proliferation,apoptosis,as well as cyclic adenosine monophosphate(cAMP)and cyclic guanosine monophosphate(cGMP)/protein kinases(PKG)pathways.Compared to the blank group,the RNA-Seq results showed significant increases in adenosine A1 receptor recombina(Adora1)and BK channel subunit beta 3(Kcnmb2)mRNA expression in gastric tissue in the model group,while the expression of endothelin receptor type B gene(Ednrb),recombinant Rho associated coiled coil containing protein kinase 2(Rock2),cGMP-dependent protein kinase 2(Prkg2),and cAMP responsive element binding protein 3 like 3 gene(Creb3l3)mRNA was significantly decreased.Compared to the model group,the expression of Adora1 and Kcnmb2 mRNA in gastric tissue in the treatment group decreased significantly,while the expression of Ednrb,Rock2,Prkg2,and Creb3l3 mRNA increased significantly.Molecular biology experiments revealed significant decreases in cAMP content in gastric tissue and plasma,as well as decreased expression levels of Ras homolog gene family member A(RhoA),ROCK2,phospho-myosin phosphatase target subunit 1(p-MYPT1),and phospho-Myosin light chain(Ser20)(p-MLC20)proteins in gastric tissue in the model group,accompanied by significantly increased cGMP content in gastric tissue and plasma and significantly increased expression of PKG protein in gastric tissue.When the treatment group was compared with the model group,the results showed significant decreases in cGMP content in gastric tissue and plasma and PKG protein expression in gastric tissue,alongside increased expression le-vels of RhoA,ROCK2,p-MYPT1,and p-MLC20 in gastric tissue.At the same time,the results of RT-qPCR confirmed that the expression of key genes in the cGMP/PKG pathway was consistent with the RNA-Seq findings.In summary,Xiangsha Liujunzi Decoction demonstrates significant therapeutic efficacy in CAG rats,and the specific mechanism may be through the abnormal expression of signaling molecules that regulate the cGMP/PKG pathway and downstream processes.