丹参酮ⅡA通过抑制JAK2/STAT3通路减轻慢性萎缩性胃炎大鼠胃黏膜损伤的机制研究

Study on the mechanism of Tanshinone ⅡA alleviates gastric mucosal injury in rats with chronic atrophic gastritis by inhibiting JAK2/STAT3 pathway

关键词:  慢性萎缩性胃炎  丹参酮ⅡA  胃黏膜  炎症  凋亡  JAK2/STAT3通路

Key words:  chronic atrophic gastritis  Tanshinone ⅡA  gastric mucosa  inflammation  apoptosis  JAK2/STAT3 pathway

摘要:

[目的] 研究丹参酮ⅡA（Tan ⅡA）对慢性萎缩性胃炎（CAG）大鼠胃黏膜的影响，并基于Janus激酶2（JAK2）/信号转导与转录激活子3（STAT3）通路探讨其机制。[方法] 将80只Wistar大鼠随机分为5组（n=16）：正常（Normal）组、模型（Model）组、Tan ⅡA（5 mg/kg）组、Tan ⅡA（5 mg/kg）+AG490（JAK2抑制剂，5 mg/kg）组和Tan IIA（5 mg/kg）+C-A1（JAK2激动剂，50 mg/kg）组。除Normal组外，其他4组均采用N-甲基-N'-硝基-N-亚硝基胍溶液（MNNG，0.04 g/mL）自由饮联合雷尼替丁灌胃、饥饱失常饮食的综合法构建CAG大鼠模型。各组分别每日1次连续给药治疗12周后，中性红清除法检测胃黏膜血流量，酶联免疫吸附法（ELISA）法检测血清胃泌素（GAS）、血浆胃动素（MTL）及胃黏膜炎症因子水平，苏木精-伊红（HE）染色法观察胃黏膜组织病理学改变并行萎缩评分，TUNEL染色法观察胃黏膜细胞凋亡状况并计算凋亡指数，逆转录聚合酶链反应（RT-PCR）法、蛋白免疫印迹（Western Blot）法检测胃黏膜JAK2/STAT3通路相关mRNA和蛋白表达。[结果] 与Model组相比，Tan ⅡA组大鼠胃黏膜血流量、血清GAS水平、血浆MTL水平均明显升高（P<0.05）；胃黏膜中肿瘤坏死因子-α（TNF-α）、白细胞介素-1β（IL-1β）、IL-6等炎症因子水平明显降低（P<0.05）；胃黏膜病理学改变明显改善，萎缩评分明显降低（P<0.05）；胃黏膜凋亡细胞数量明显减少，凋亡指数明显降低（P<0.05）；胃黏膜JAK2、STAT3 mRNA表达量明显降低（P<0.05）；B淋巴细胞瘤2（Bcl-2）蛋白表达量明显升高，Bcl-2相关X蛋白（Bax）、激活型半胱氨酸蛋白酶3（C-Cas-3）、C-Cas-9蛋白表达量及p-JAK2/JAK2、p-STAT3/STAT3、胞核核因子-κB（NF-κB）p65/胞浆NF-κB p65表达比值明显降低（P<0.05）。AG490能够明显增强Tan ⅡA对CAG大鼠各检测指标的调控作用，C-A1则能够明显逆转Tan ⅡA对CAG大鼠各检测指标的调控作用（P<0.05）。[结论] Tan ⅡA可能通过抑制JAK2/STAT3通路活化及NF-κB核转位，减轻炎症反应和细胞凋亡，从而减轻CAG大鼠胃黏膜结构和功能损伤。

Abstract:

[Objective] To investigate the effect of Tanshinone ⅡA（Tan ⅡA） on gastric mucosa in rats with chronic atrophic gastritis（CAG），and explore its mechanism based on the janus kinase 2/signal transduction and transcriptional activator 3（JAK2/STAT3） pathway. [Methods] The 80 Wistar rats were randomly divided into 5 groups（n=16）：Normal group，Model group，Tan ⅡA（5 mg/kg） group，Tan ⅡA（5 mg/kg） + AG490（JAK2 inhibitor，5 mg/kg） group and Tan ⅡA（5 mg/kg） + C-A1（JAK2 agonist，50 mg/kg） group. Except for normal group，the rats in other 4 groups were treated with free drinking of N-methyl-N’-nitro-N-nitroguanidine solution（MNNG， 0.04 g/mL） combined with ranitidine gavage and starvation and satiety disorder diet. After 12 weeks of continuous administration once a day in each group，the gastric mucosal blood flow was detected by neutral red clearance. The gastrin（GAS） in serum，motilin（MTL） in plasma and the inflammatory factors in gastric mucosa were detected by ELISA. The gastric mucosal histopathological changes was observed through HE staining，and the atrophy score was performed. The apoptosis of gastric mucosa cells was observed through TUNEL staining，and the apoptosis index was calculated. The mRNA and protein expression of JAK2/STAT3 pathway in gastric mucosa were detected by RT-PCR or Western blotting. [Results] Compared with the Model group，the gastric mucosal blood flow，the level of GAS in serum and the level of MTL in plasma of Tan ⅡA group were significantly increased（P<0.05）. The level of tumor necrosis factor-α（TNF-α），interleukin-1β（IL-1β），IL-6 in gastric mucosa were significantly decreased（P<0.05）. The pathological changes of gastric mucosa were significantly improved，and the atrophy score was significantly decreased（P<0.05）. The number of apoptotic cells was decreased significantly，and the apoptotic index was significantly decreased（P<0.05）. The mRNA expressions of JAK2，STAT3 in gastric mucosa were significantly decreased（P<0.05）. The expression of B lymphoblastoma 2（Bcl-2） protein was significantly increased（P<0.05）. The protein expression level of Bcl-2 associated X protein（Bax），cleaved Caspase-3（C-Cas-3），cleaved Caspase-9（C-Cas-9） and the ratio of P-JAK2/JAK2，P-STAT3/STAT3，nuclear NF-κB p65/cytoplasmic NF-κB p65 were significantly decreased（P<0.05）. AG490 could significantly enhance the regulatory effect of Tan ⅡA on various detection indexes in CAG rats，and C-A1 could significantly reverse the regulatory effect of Tan ⅡA on CAG rats（P<0.05）. [Conclusion] Tan ⅡA can reduce the structural and functional damage of gastric mucosa in CAG rats by inhibiting JAK2/STAT3 pathway activation and NF-κB nuclear translocation，and reducing inflammatory response and apoptosis.