木瓜总三萜对幽门螺杆菌诱导小鼠胃炎的保护作用研究

Protective effect of total triterpenoids from Chaenomeles speciosa against Helicobacter pylori-induced gastritis in mice

观察木瓜总三萜对幽门螺杆菌(Helicobacter pylori,Hp)诱导小鼠胃炎保护作用,探寻其可能作用机制。将慢性萎缩性胃炎(chronic atrophic gastritis,CAG)模型制作成功小鼠随机分为模型组、木瓜总三萜不同剂量(50、100 mg·kg^(-1))组和三联疗法组,另取未感染Hp C57BL/6J小鼠作为正常组;治疗组小鼠分别给予相应的药物,每日1次,连续4周。然后取血和胃组织进行血液中活性氧自由基(reactive oxygen species,ROS)、单核细胞趋化蛋白1(monocyte chemotactic protein 1,MCP-1)、角质细胞趋化因子(keratinocyte chemokines,KC)、TNF-α、IL-1β、IL-6、IL-18、IL-4、IL-10含量,胃组织中LDH、MPO、SOD、GSH-Px、CAT和MDA活性和含量及血液、胃组织和溶酶体中β-葡萄糖醛酸酶、β-半乳糖苷酶、组织蛋白酶B和D活性的检测,HE染色法观察胃组织形态学改变;实时定量PCR法测定胃组织中Toll样受体4(Toll like receptor 4,TLR4)、髓样分化因子(myeloid differentiation factor 88,MyD88)、Bcl-2、Bcl-xl、Bax、Bad mRNA表达,Western blot法测定胃组织中TLR4、MyD88、p-IKKβ、p-IκBα、NOD样受体家族3(NOD-like receptor family 3,NLRP3)、凋亡相关斑点样蛋白(apoptosis-associated speck-like protein,ASC)、pro-caspase-1、caspase-1、硫氧还蛋白互作蛋白(thioredoxin-interacting protein,TXNIP)、pro-IL-1β、pro-IL-18、Bcl-2、Bcl-xl、Bax、Bad、cytochrome C、凋亡酶激活因子(apoptotic protease activating factor-1,Apaf-1)、pro-caspase-9、pro-caspase-3、cleaved-caspase-9、cleaved-caspase-3、聚腺苷二磷酸核糖聚合酶-1[poly(ADP-ribose)polymerase 1,PARP-1]、cleaved-PARP-1及胞浆和胞核中NF-κB p65蛋白表达。结果显示,木瓜总三萜可显著抑制Hp增殖,减轻胃黏膜损伤,改善淋巴细胞浸润和腺体萎缩;降低血液、胃组织中β-葡萄糖醛酸酶、β-半乳糖苷酶、组织蛋白酶B和D活性及溶酶体细胞器胞质中β-葡萄糖醛酸酶、组织蛋白酶D的活性,升高溶酶体细胞器溶酶体中β-葡萄糖醛酸酶、组织蛋白酶D活性;降低血液中ROS、MCP-1、KC、TNF-α、IL-1β、IL-6、IL-18和胃组织中MDA含量及MPO、LDH活性,升高血液中IL-4、IL-10含量和胃组织中SOD、CAT和GSH-Px活性;下调胃组织中TLR4、MyD88、Bax、Bad mRNA和蛋白表达,下调胃组织中p-IKKβ、p-IκBα、NLRP3、ASC、pro-caspase-1、caspase-1、TXNIP、pro-IL-1β、pro-IL-18、cytochrome C、Apaf-1、cleaved-caspase-9、cleaved-caspase-3、cleaved-PARP-1和胞核中NF-κB p65蛋白表达,降低p-IKKβ/IKKβ和p-IκBα/IκBα比值;上调胃组织中Bcl-2、Bcl-xl mRNA和蛋白表达,升高Bcl-2/Bax和Bcl-xl/Bad比值,上调胃组织中pro-caspase-9、pro-caspase-3和胞浆中NF-κB p65蛋白表达。以上研究表明木瓜总三萜对Hp诱导小鼠CAG有显著保护作用,其机制可能与增强内源性抗氧化系统功能、抑制Hp诱导CAG小鼠氧化应激、炎症反应、纠正溶酶体功能障碍及TLR4/NF-κB/NLRP3炎症小体信号通路炎性激活,进而抑制线粒体介导的细胞凋亡有关。

This study investigated the protective effect of total triterpenoids from Chaenomeles speciosa against Helicobacter pylori(Hp)-induced gastritis in mice and explored its possible mechanism.The chronic atrophic gastritis(CAG)model mice were randomly divided into four groups of model,total triterpenoids from C.speciosa(50 and 100 mg·kg^(-1))and triple therapy,with C57 BL/6 J mice without Hp infection taken as the normal group.Mice in the treatment groups were given corresponding drugs once a day for 4 weeks.Then the following indexes were detected:the contents of reactive oxygen species(ROS),monocyte chemotactic protein 1(MCP-1),keratinocyte chemokines(KC),TNF-α,IL-1β,IL-6,IL-18,IL-4 and IL-10 in blood and gastric tissue,the activities and contents of LDH,MPO,SOD,GSH-Px,CAT and MDA in gastric tissue and the activities of β-glucuronidase,β-galactosidase,cathepsins B and D in blood,gastric tissue and lysosome.Besides,the mRNA expression levels of Toll-like receptor 4(TLR4),myeloid differentiation factor 88(MyD88),Bcl-2,Bcl-xl,Bax and Bad in gastric tissue were determined by quantitative real-time PCR.Western blot was employed to detect the protein expression levels of TLR4,MyD88,p-IKKβ,p-IκBα,NOD-like receptor 3(NLRP3),apoptosis-associated speck-like protein(ASC),pro-caspase-1,caspase-1,thioredoxin-interacting protein(TXNIP),pro-IL-1β,pro-IL-18,Bcl-2,Bcl-xl,Bax,Bad,cytochrome C,apoptotic protease-activating factor-1(Apaf-1),pro-caspase-9,pro-caspase-3,cleaved-caspase-9,cleaved-caspase-3,poly(ADP-ribose)polymerase 1(PARP-1),cleaved-PARP-1 and cytosol and nucleus NF-κB p65 in gastric tissue.The results indicated that the total triterpenoids from C.speciosa significantly suppressed Hp proliferation,alleviated the damage to gastric mucosa and improved lymphocyte infiltration and gland atrophy.They were also effective in reducing the activities of β-glucuronidase,β-galactosidase,cathepsins B and D in blood and gastric tissue,elevating the activities of β-glucuronidase and cathepsin D in lysosomal organelles,decreasing the contents of ROS,MCP-1,KC,TNF-α,IL-1β,IL-6,IL-18 in blood,MDA content and MPO and LDH activities in gastric tissue and increasing the contents of IL-4 and IL-10 in blood and activities of SOD,CAT and GSH-Px in gastric tissue.Other phenomena were also observed after the treatment with total triterpenoids from C.speciosa,including the down-regulation of the mRNA and protein expression levels of TLR4,MyD88,Bax and Bad,the protein expression levels of p-IKKβ,p-IκBα,NLRP3,ASC,pro-caspase-1,caspase-1,TXNIP,pro-IL-1β,pro-IL-18,cytochrome C,Apaf-1,cleaved-caspase-9,cleaved-caspase-3,cleaved-PARP-1 and nuclear NF-κB p65,reduction of p-IKKβ/IKKβ and p-IκBα/IκBα ratios and up-regulation of the mRNA and protein expression levels of Bcl-2 and Bcl-xl,up-regulation of pro-caspase-9,pro-caspace-3,cytosol NF-κB p65 protein expression levels and Bcl-2/Bax and Bcl-xl/Bad ratios in gastric tissue.These aforementioned results suggest that the total triterpenoids from C.speciosa have significant protective effects against CAG induced by Hp,and its mechanism may be related to enhancing the function of endogenous antioxidant system,suppressing the oxidative stress and inflammatory reaction induced by Hp,correcting lysosomal dysfunction and inflammatory activation of TLR4/NF-κB/NLRP3 inflammasome signaling pathway and thus inhibiting mitochondria-mediated apoptosis.