川芎嗪对坏死性小肠结肠炎新生大鼠保护作用的研究

Protective effect of tetramethylpyrazine on newborn rats with necrotizing enterocolitis

目的探讨川芎嗪对缺氧冷应激联合配方奶侵袭性喂养所致坏死性小肠结肠炎(NEC)新生大鼠的保护作用。方法将50只出生48 h内的健康新生Sprague-Dawley大鼠按随机数字表法分为空白组、模型组、低剂量组、中剂量组和高剂量组,每组10只。除空白组外,其他各组新生大鼠均采用缺氧冷应激联合配方奶侵袭性喂养法建立NEC模型。同时低、中、高剂量组分别给予2.09、5.18、10.36 mg/kg川芎嗪,连续给药3 d;模型组给予等体积的氯化钠注射液;空白组则不予任何处理,均由哺乳期母鼠代乳。末次人工喂养12 h后,采用颈椎脱臼法处死各组大鼠,取小肠回盲部组织,检测回盲部肠组织匀浆中的丙二醛、过氧化氢酶(CAT)、谷胱甘肽过氧化物酶(GSH-Px)和超氧化物歧化酶(SOD)水平;采用苏木精-伊红染色观察回盲部肠组织的病理学变化,并采用Khailova标准进行回盲部肠组织损伤评分;采用免疫组化染色检测回盲部肠组织中半胱氨酸天冬氨酸特异性蛋白酶-3(caspase-3)的表达。结果与模型组相比,低、中、高剂量组新生大鼠回盲部肠组织匀浆中的丙二醛水平均明显降低,而CAT、GSH-Px和SOD水平均明显升高(均P<0.05)。低、中、高剂量组新生大鼠伴有肠绒毛轻中度充血水肿及回盲部肠组织黏膜固有层轻中度肿胀分离,但程度均不及模型组,且低、中、高剂量组回盲部肠组织损伤评分[(1.91±0.33)分、(1.56±0.31)分、(1.35±0.26)分]均明显低于模型组[(3.21±0.43)分],差异均具有统计学意义(均P<0.05)。与模型组(9.34±0.98)相比,低、中、高剂量组回盲部肠组织中的caspase-3蛋白相对表达量(5.51±0.57、4.46±0.45、2.15±0.21)均明显降低,差异均具有统计学意义(均P<0.05)。结论川芎嗪对缺氧冷应激联合配方奶侵袭性喂养所致NEC新生大鼠具有较好的保护作用,可有效抑制肠细胞的凋亡。

Objective To explore the protective effect of tetramethylpyrazine on newborn rats with necrotizing enterocolitis(NEC)caused by hypoxic cold stress combined with formula milk invasive feeding.Methods According to the random number table methods,50 newborn SD rats were randomly divided into five groups,including blank group,model group,low dose group,middle dose group and high dose group.Except for the blank group,newborn rats in all other groups were treated with hypoxic cold stress combined with formula milk invasive feeding to establish NEC models.The rats in the low,medium and high dose group were given tetramethylpyrazine by 2.09,5.18 and 10.36 mg/kg respectively for 3 consecutive days.The rats in the model group was given equal volume of sodium.In the blank group,no treatment was given,and the lactating mothers were used to substitute milk.Twelve hours after the last artificial administration,the rats in each group were sacrificed by cervical dislocation method,the small intestine ileocecal tissue was taken,and the levels of malondialdehyde,catalase(CAT),glutathione peroxidase(GSH-Px)and superoxide dismutase(SOD)in ileocecal intestinal homogenate was detected.Hematoxylin eosin staining was used to observe the pathological changes of ileocecal intestinal tissue,and Khailova standard was used to evaluate ileocecal intestinal tissue injury.The expression of cysteine-containing aspartate-specific proteases-3(caspase-3)was detected by immunohistochemical staining.Results Compared with the model group,the levels of malondialdehyde in the treatment groups were significantly lower,and the levels of CAT,GSH-Px and SOD were significantly higher(all P<0.05).The newborn rats in the low,medium and high dose groups were accompanied by mild and moderate intestinal villi hyperemia and edema and the ileocecal mucosa lamina propria swelling were separated,but the degree was not as high as that in the model group.Compared with the model group(3.21±0.43),the ileocecal tissue injury scores of the low,medium and high dose groups(1.91±0.33,1.56±0.31 and 1.35±0.26)were significantly lower,and the differences were statistically significant(all P<0.05).Compared with the model group(9.34±0.98),the positive expression of caspase-3 was significantly relieved in the low,medium and high dose groups(5.51±0.57,4.46±0.45 and 2.15±0.21),and the differences were statistically significant(all P<0.05).Conclusions Tetramethylpyrazine has good protective effects in the hypoxic cold stress combined with formula milk invasive feeding induced necrotizing enterocolitis in newborn rats.