通肠清胰汤调控TLR4/NF-κB通路减轻重症急性胰腺炎大鼠肺肠损伤的实验研究

Experimental Study of Tongchangqingyi Decoction Regulating TLR4/NF-κB Pathway to Reduce Lung and Intestinal Injury in Rats with Severe Acute Pancreatitis

目的探讨通肠清胰汤对重症急性胰腺炎(severe acute pancreatitis,SAP)大鼠肺、肠损伤的保护作用及其可能作用机制。方法将24只SPF级雄性SD大鼠随机分为假手术组、模型组、通肠清胰汤组、乌司他丁组,每组6只。假手术组麻醉暴露腹腔后,仅轻轻翻动十二指肠和胰腺数次后缝合腹部,其余组均经胆胰管穿刺逆行注射牛磺胆酸钠制备SAP大鼠模型,大鼠造模成功后4 h开始给药。通肠清胰汤组予通肠清胰汤煎剂灌胃,模型组和假手术组大鼠予等体积生理盐水灌胃,乌司他丁组予乌司他丁注射液尾静脉注射,均每隔8 h给药1次,共给药3次。造模后24 h将大鼠麻醉、取材,生化法检测血清淀粉酶(amylase,AMS)、脂肪酶(lipase,LPS)含量;酶联免疫吸附分析(enzyme-linked immunosorbent assay,ELISA)检测血清C反应蛋白(C-reactive protein,CRP)、肿瘤坏死因子α(tumor necrosis factor alpha,TNF-α)、白细胞介素6(interleukin 6,IL-6)、白细胞介素10(interleukin 10,IL-10)水平;苏木精-伊红(hematoxylin-eosin,HE)染色法观察大鼠胰腺组织、结肠组织、肺组织病理变化;蛋白免疫印迹法(Western blot,WB)检测大鼠结肠组织咬合蛋白(Occludin)、闭合蛋白(Claudin)、闭锁小带蛋白1(zonula occludens 1,ZO-1)含量,大鼠结肠组织和肺组织Toll样受体4(Toll-like receptor 4,TLR4)、核转录因子κB(nuclear factor kappa B,NF-κB)p65蛋白表达量。结果HE染色显示假手术组胰腺、结肠、肺组织无明显病理损伤变化,模型组胰腺腺泡间隙明显扩张、间质水肿、小叶间隙扩大、大量炎性细胞浸润、组织片状坏死,模型组结肠细胞肿胀、炎性细胞浸润、黏膜腺体减少、肠绒毛剥脱,模型组肺组织细胞水肿明显、炎性细胞浸润、间质出血,与模型组比较,通肠清胰汤组和乌司他丁组胰腺腺泡结构破坏、间质水肿、出血坏死均减轻,结肠炎性细胞浸润减少,肺组织细胞水肿和炎性浸润亦减轻。与假手术组比较,模型组大鼠血清AMS、LPS、CRP、TNF-α、IL-6含量均显著升高(P均<0.01),IL-10含量显著降低(P<0.01),结肠组织Occludin、Claudin、ZO-1蛋白表达量显著降低(P均<0.01),结肠组织和肺组织TLR4、NF-κB p65蛋白表达量显著升高(P均<0.01),与模型组比较,通肠清胰汤组、乌司他丁组大鼠血清AMS、LPS、CRP、TNF-α、IL-6含量显著降低(P均<0.01)、IL-10含量显著升高(P<0.01),结肠组织Occludin、Claudin、ZO-1蛋白表达量显著升高(P均<0.01),结肠组织和肺组织TLR4、NF-κB p65蛋白表达量显著降低(P均<0.01)。与乌司他丁组比较,通肠清胰汤组大鼠血清AMS、LPS、CRP含量显著升高(P均<0.05)、IL-10含量显著降低(P<0.01),结肠组织Occludin、ZO-1蛋白表达量显著降低(P均<0.05),结肠组织和肺组织TLR4蛋白表达量显著升高(P均<0.05),而血清TNF-α及IL-6含量、结肠组织Claudin及NF-κB p65表达量、肺组织NF-κB p65表达量差异均无统计学意义(P均>0.05)。结论通肠清胰汤可减轻SAP大鼠的肺肠损伤,可减轻炎性因子释放、保护肠黏膜屏障,其机制可能与调控TLR4/NF-κB通路相关。

Objective To investigate the protective effects of Tongchangqingyi Decoction on lung and intestinal injury in rats with severe acute pancreatitis(SAP)and its possible mechanisms.Methods 24 SPF male SD rats were randomly divided into sham operation group,model group,Tongchangqingyi Decoction group and ulinastatin group,with 6 rats in each group.After the rats of sham operation group were anesthetized and exposed to the abdominal cavity,the duodenum and pancreas were gently turned several times and the abdomen was sutured,SAP rat models were prepared by retrograde injection of sodium taurine cholate through bile pancreatic duct puncture in other groups.The rats were given the drug at 4 hours after the modeling was successful.Rats of Tongchangqingyi Decoction group were given Tongchangqingyi Decoction by intragastric administration,rats in model group and sham operation group were given equal volume normal saline instillation,ulinastatin group rats were given uli-nastatin injection by tail vein,once every 8 hours,for a total of 3 times.24 hours after the modeling,the rats were anesthe-tized and sampled,the contents of amylase(AMS)and lipase(LPS)in serum were determined by biochemical method,se-rum levels of C-reactive protein(CRP),tumor necrosis factor alpha(TNF-α),interleukin 6(IL-6)and interleukin 10(IL-10)were detected by enzyme-linked immunosorbent assay(ELISA),the pathological changes of pancreatic,colon and lung tissues of rats were observed by hematoxylin-eosin(HE),and the contents of occludin,claudin and zonula occludens-1(ZO-1),protein expression of Toll-like receptor 4(TLR4)and nuclear transcription factor kappa B(NF-κB)in colon tis-sue and lung tissue of rats were detected by Western blot.Results HE staining showed no significant pathological changes in pancreas,colon and lung tissues in the sham operation group.In the model group,pancreatic acinous space was signifi-cantly expanded,interstitial edema,lobular space was enlarged,a large number of inflammatory cells infiltrated and tissue sheet necrosis was observed,colon cells were swollen,inflammatory cells were infiltrated,mucosal glands were reduced and intestinal villi was exfoliated.Lung tissue cell edema,inflammatory cell infiltration and interstitial bleeding were obvious in the model group.Compared with the model group,The destruction of pancreatic acinar structure,interstitial edema,hem-orrhage and necrosis were alleviated in Tongchangqingyi Decoction group and ulinastatin group,the inflammatory cell infil-tration of colon was reduced,the pulmonary tissue cell edema and inflammatory infiltration were also alleviated.Compared with sham operation group,the contents of serum AMS,LPS,CRP,TNF-αand IL-6 in model group were significantly increased(all P<0.01),the content of IL-10 was significantly decreased(P<0.01),the protein expressions of Occludin,Claudin and ZO-1 in colon tissue were significantly decreased(all P<0.01);compared with model group,the contents of AMS,LPS,CRP,TNF-αand IL-6 in serum of Tongchangqingyi Decoction group and ulinastatin group were significantly decreased(all P<0.01),and the content of IL-10 was significantly increased(P<0.01),the expression of Occludin,Claudin and ZO-1 in colon tissue were significantly increased(all P<0.01),while the protein expressions of TLR4 and NF-κB p65 in colon tissue were significantly decreased(all P<0.01).Compared with ulinastatin group,the contents of serum AMS,LPS and CRP in Tongchangqingyi Decoction group were significantly increased(all P<0.05),the content of IL-10 was significantly decreased(P<0.01),and the protein expression of Occludin and ZO-1 in colon tissue was sig-nificantly decreased(all P<0.05),the protein expression levels of TLR4 in colon and lung were significantly increased(all P<0.05),but the contents of TNF-αand IL-6 in serum,the expression levels of Claudin and NF-κB p65 in colon and NF-κB p65 in lung were not statistically significant(all P>0.05).Conclusion Tongchangqingyi Decoction can alleviate lung-intestinal injury,reduce the release of inflammatory factors and protect intestinal mucosal barrier in rats with severe acute pancreatitis,and its mechanism may be related to the regulation of TLR4/NF-κB pathway.