四君子汤改善吲哚美辛所致大鼠小肠黏膜损伤及其可能的作用机制

Improvement of Sijunzi decoction on rat intestinal mucosal injuries induced by indomethacin and its possible mechanism

目的探讨四君子汤改善小肠黏膜损伤的作用机制。方法制备四君子汤水提物(SJZDA),通过醇沉和Severge法去蛋白得到四君子汤多糖(SJZDP);高效液相色谱法(HPLC)检测SJZDP样品图谱,苯酚-硫酸法测定SJZDP糖含量。SD雄性大鼠分组为正常对照、模型对照、模型+SJZDA 5和15 g·kg^(-1)及模型+SJZDP 0.358和1.074 g·kg^(-1)组。大鼠造模前1 h,模型+SJZDA和模型+SJZDP组分别ig给予相应剂量SJZDA和SJZDP,正常对照组和模型对照组给予等体积纯水;1 h后除正常对照组外,各组大鼠均皮下注射吲哚美辛6 mg·kg^(-1)。SJZDA和SJZDP及吲哚美辛均每天给药1次,共6 d。末次给药后24 h麻醉大鼠,取腹主动脉血制备血浆;摘除小肠观察小肠黏膜大体损伤并评分;在黏膜损伤明显处取2~3 cm小肠常规石蜡包埋切片,HE染色观察组织病理变化;余小肠冰上刮取黏膜组织,制备组织匀浆。柱前衍生HPLC法检测小肠黏膜组织中多胺含量,邻甲酚酞络合酮比色法检测小肠黏膜组织Ca^(2+)含量,Amplite荧光法检测血浆D-乳酸含量,免疫组织化学法检测小肠黏膜组织闭锁小带蛋白、闭合蛋白、封闭蛋白3、E钙黏蛋白、α连环蛋白和β连环蛋白表达。结果 HPLC图谱显示SJZDP为杂多糖,苯酚-硫酸法测得其多糖含量为81.6%。与正常对照组比较,模型对照组大鼠小肠黏膜损伤大体和组织病理评分均明显增加(P<0.01),提示小肠黏膜损伤模型制备成功。与模型对照组比较,模型+SJZDA 15 g·kg^(-1)、模型+SJZDP 0.358和1.074 g·kg^(-1)组大鼠小肠黏膜损伤大体和组织病理评分降低(P<0.05,P<0.01),小肠黏膜组织多胺含量提高(P<0.05,P<0.01);模型+SJZDA 15 g·kg^(-1)和模型+SJZDP 1.074 g·kg^(-1)组黏膜组织Ca^(2+)含量升高(P<0.05),血浆中D-乳酸浓度降低(P<0.05,P<0.01)。免疫组织化学法检测结果表明,与模型对照组比较,模型+SJZDA 15 g·kg^(-1)和模型+SJZDP 1.074 g·kg^(-1)组黏膜组织中闭锁小带蛋白、封闭蛋白、E钙黏蛋白、α连环蛋白和β连环蛋白表达水平升高(P<0.05,P<0.01),模型+SJZDP 0.358和1.074 g·kg^(-1)组黏膜组织中闭合蛋白表达水平升高(P<0.01)。结论四君子汤对吲哚美辛所致大鼠小肠黏膜损伤具有改善作用,该作用可能与其影响小肠黏膜组组多胺及其调控的黏膜屏障有关。

OBJECTIVE To explore the mechanism by which Sijunzi decoction improves smal intestinal mucosal injuries.METHODS Sijunzi decoction was soaked,decocted and freeze-dried to obtain Sijunzi decoction aqueous extract(SJZDA).Sijunzi decoction polysaccharides(SJZDP) were obtained through ethanol precipitation and severge deproteinization.HPLC was used to detected the polysaccharide content of SJZDP.The rats were randomly divided into the normal control group,model control groups,model+SJZDA 5 and 15 g·kg^(-1) groups and model+SJZDP 0.358 and 1.074 g·kg^(-1) group.The rats in drug treatment groups were ig given SJZDA or SJZDP.Those in normal and model control groups were perfused with an equal volume of water at the same time.An hour later,the rats were injected subcutaneously with indomethacin 6 mg·kg^(-1),once daily,for 6 d,except those of the normal control group.On the 7 th day,the rats were sacrificed by anesthesia,and the gross appearance of intestinal mucosal injuries was scored.HE staining was used for pathological scores of intestinal mucosal injuries.The content of polyamines in the intestinal mucosa was detected with pre-column derivatization HPLC.O-cresolphthalein complexone was used to measure the Ca^(2+)content in the intestinal mucosa.The D-lactate(effective index of intestinal permeability) in plasma was detected by Amplite fluorescence method.Immunohistochemical staining was adopted to evaluate the expressions of related proteins(ZO-1,occludin,claudin-3,E-cadherin,α-catenin,and β-catenin) in the small intestinal mucosa.RESULTS HPLC analysis showed that SJZDP was a type of heteropolysaccharides and the content of polysaccharides was81.6%.Compared with the normal control group,the gross score and pathological score of the model group were significantly increased(P<0.01),indicating that the model was established.Compared with the model group,SJZDA 15 g·kg^(-1),SJZDP 0.358 and 1.074 g·kg^(-1) decreased the gross and pathological scores of its small intestinal mucosal injuries(P<0.05,P<0.01).SJZDA 15 g·kg^(-1),SJZDP 0.358 and1.074 g·kg^(-1) elevated the polyamines(spermine) of the small intestinal mucosa in the model rats(P<0.05,P<0.01),but SJZDA 15 g·kg^(-1) and SJZDP 1.074 g·kg^(-1) increased the total Ca^(2+)content in the intestinal mucosa(P<0.05) and D-lactate in plasma(P<0.05,P<0.01).The results of immunohistochemical staining showed that the expressions of ZO-1,claudin-3,E-cadherin,α-catenin and β-catenin were increased in model+SJZDA 15 g·kg^(-1) and model+SJZDP 1.074 g·kg^(-1) groups(P<0.05,P<0.01),and the expression of occludin was increased in modle+SJZDP 0.358 and 1.074 g·kg^(-1) goups compared with the model control group(P<0.01).CONCLUSION The improvement of Sijunzi decoction on indomethacin-induced small intestinal mucosal injuries may be related to polyamines and its regulation of the mucosal barrier.