急性肠损伤大鼠体内Ghrelin及受体的表达和电针干预作用的关联

发布时间：2018-03-05 11:20

本文选题：性肠损伤　切入点：盲肠结扎穿孔术　出处：《浙江中医药大学》2014年博士论文　论文类型：学位论文

【摘要】：目的探讨急性肠损伤大鼠体内Ghrelin及其受体的表达以及电针足三里穴的干预作用。方法①建立大鼠急性肠损伤(ABI)动物模型通过盲肠结扎穿孔术导致的腹腔感染来诱发急性肠损伤。将32只Wistar雄性大鼠分为两个实验组：急性肠损伤组(CLP组)和假手术对照组(Sham组),各16只。CLP组采用盲肠结扎穿孔术(CLP)的方法建立,取正中切口开腹,暴露盲肠并结扎根部,用16号针贯通穿刺盲肠4次形成肠瘘,再逐层缝合腹壁切口;假手术对照组以相同的方式暴露盲肠后回纳并缝合腹壁切口,并不行盲肠结扎和穿孔。于术后12h麻醉下留取血样本后处死,留取肠组织,测定大鼠血清TNF-α、HMGB1和肠组织含水率、肠组织髓过氧化物酶(MPO)活性和二胺氧化酶(DAO)活性,肠组织的大体及镜下病理组织学变化来确定ABI的形成。②急性肠损伤大鼠体内ghrelin及其受体的表达和电针足三里穴干预作用的关联将48只Wistar大鼠随机分为：假手术对照组(Sham组)、急性肠损伤组(CLP组)、电针治疗组(CLP+EA组)及ghrelin受体阻断剂组(CLP+GHSRA+EA组),每组各12只。假手术对照组取腹正中切口、暴露盲肠后回纳、缝合腹腔切口,急性肠损伤组采用盲肠结扎穿孔术(CLP)的方法建立,电针治疗组于建立大鼠ABI模型后取双侧足三里穴电针仪持续针刺30min, ghrelin受体阻断剂组于建立大鼠ABI模型后在尾静脉注射ghrelin受体阻断剂[D-Arg1, D-Phe5, D-Trp7.9,Leu11] substance P、再行双侧足三里穴电针仪持续针刺30min。于术后12h麻醉下留取血样本后处死,留取肠组织。测定血清ghrelin、TNF-α、HMGB1和肠组织含水率、肠组织髓过氧化物酶(MPO)活性和二胺氧化酶(DAO)活性,并通过western blot测定肠组织ghrelin及其受体、HMGB1蛋白表达,免疫组化测定肠组织ghrelin及其受体阳性的细胞数。结果①CLP组肠组织结构明显破坏,肠组织含水率和病理评分明显高于假手术对照组(P0.05)。与Sham组相比,ABI模型组血清TNF-α、HMGB1两者均较对照组高,有统计学意义(P0.05)。CLP组肠组织含水率和髓过氧化物酶(MPO)活性明显高于Sham组、而肠组织二胺氧化酶(DAO)活性则低于Sham组,两者有统计学意义(P0.05)。②CLP组血清ghrelin水平、肠组织ghrelin阳性细胞数和蛋白表达量均低于Sham组(P0.05),肠组织ghrelin受体蛋白表达量在CLP组明显低于假手术对照组,有统计学意义(P0.05)。③CLP+EA组血清TNF-α、HMGB1水平以及肠组织MPO活性均较CLP组低,而肠组织DAO活性则高于CLP组,均有统计学意义(P0.05),与CLP组相比,CLP+EA组肠组织含水率和病理评分均有下降(P0.05)。④CLP+EA组血清ghrelin水平、肠组织ghrelin阳性细胞数和蛋白表达量均高于CLP组(P0.05),与CLP组相比,CLP+EA组肠组织ghrelin受体蛋白表达量明显升高,有统计学意义(P0.05)。⑤CLP+GHSRA+EA组血清TNF-α、HMGB1水平以及肠组织MPO活性均较CLP+EA组高,而肠组织DAO活性则低于CLP+EA组,均有统计学意义(P0.05),与CLP+EA相比,CLP+GHSRA+EA组肠组织含水率和病理评分均升高(P0.05)。⑥血液中Ghrelin水平和血清HMGB1水平呈负相关(r=-0.664,p0.001),血液中Ghrelin水平和肠组织DAO活性呈负相关(r=-0.423,p=0.003,血液中Ghrelin水平和肠组织MPO活性呈负相关(r=-0.404,p=0.004)。结论①盲肠结扎穿孔术能诱发大鼠急性肠损伤模型。②急性肠损伤大鼠体内Ghrelin及其受体的血清水平和肠组织蛋白表达是下降的。③电针足三里穴能抑制急性肠损伤大鼠体内促炎因子(TNF-α、HMGB1)产生和改善肠粘膜屏障功能。④电针足三里穴能促进急性肠损伤大鼠体内Ghrelin的分泌和肠组织Ghrelin受体的表达。⑤Ghrelin受体阻断后,电针足三里穴就失去对急性肠损伤的保护作用,说明电针足三里穴对急性肠损伤的影响与大鼠体内Ghrelin及其受体的作用有关。
[Abstract]:Objective to investigate the expression of Ghrelin and its receptor in rats with acute intestinal injury and the intervention of Zusanli (Zusanli).
Methods to establish acute intestinal injury in rats (ABI) were caused by perforation of animal model of abdominal infection to acute intestinal injury induced by cecal ligation. By 32 Wistar male rats were divided into two groups: acute intestinal injury group (CLP group) and sham operation group (Sham group), 16 rats in each.CLP group by cecal ligation and puncture (CLP) method was established, the incision, exposure and ligation of the cecum root, with 16 needle puncture of the cecum through 4 times the formation of intestinal fistula, abdominal incision and then sutured; sham operation group in the same way after returning and exposed the cecum and abdominal wall incision suture. No cecal ligation and puncture. After 12h anesthesia, blood samples were collected, intestinal tissue, determination of serum TNF- in rats, HMGB1 water and intestine rate, intestinal myeloperoxidase (MPO) activity and two amine oxidase (DAO) activity, intestine gross and microscopic pathology group The histological changes to determine the formation of ABI. The expression and correlation of electro acupuncture intervention in ghrelin and its receptor in rat acute intestinal injury in 48 Wistar rats were randomly divided into sham operation group (group Sham), acute intestinal injury group (CLP group), treatment group (CLP+EA group) and ghrelin receptor antagonist group (CLP+GHSRA+EA group), 12 rats in each group. The sham operation control group median abdominal incision, exposed after cecal retraction, abdominal incision suture, acute intestinal injury group by cecal ligation and puncture (CLP) method was established, electroacupuncture group in a rat model of ABI after bilateral Zusanli electroacupuncture instrument for acupuncture 30min, ghrelin receptor antagonist group in a rat model of ABI blocking agent [D-Arg1, the intravenous injection of ghrelin receptor D-Phe5, D-Trp7.9 Leu11], substance P, and then underwent bilateral Zusanli electroacupuncture instrument for acupuncture 30min. on postoperative 12h Ma Drunk blood samples were collected in intestinal tissue. Determination of serum ghrelin, TNF- alpha, HMGB1 water and intestine rate, intestinal myeloperoxidase (MPO) activity and two amine oxidase (DAO) activity, and through the Western blot determination of ghrelin and its receptor in intestinal tissue, the expression of HMGB1 protein was detected by immunohistochemistry. Intestinal tissue ghrelin and its receptor positive cell number.
Results CLP group structure was damaged, water content and pathological score of intestinal tissue was significantly higher than that of the sham operation control group (P0.05). Compared with Sham group, ABI model group, serum TNF- alpha, HMGB1 both were higher than the control group, there was statistical significance (P0.05).CLP group water content and myeloperoxidase (MPO) the activity was significantly higher than Sham group, and two in intestinal tissue monoamine oxidase (DAO) activity was significantly lower than that in Sham group, which was statistically significant (P0.05). In CLP group the serum level of ghrelin, number and protein expression in intestinal tissue of ghrelin positive cells were lower than that of Sham group (P0.05), in CLP group were significantly lower than those in sham operation group ghrelin receptor protein expression in intestinal tissues had statistical significance (P0.05). Serum TNF- group CLP+EA alpha, HMGB1 level and MPO activity in intestinal tissue were lower than group CLP, but the activity of DAO in intestinal tissue was higher than that of CLP group, there was statistically significant (P0.05), compared with the CLP group, CLP+EA group Intestinal tissue water content and pathological score decreased in CLP+EA group (P0.05). The serum level of ghrelin, number and protein expression in intestinal tissue of ghrelin positive cells were higher than CLP group (P0.05), compared with the CLP group, increased ghrelin receptor protein expression in intestinal tissue of CLP+EA group had statistical significance (P0.05). The serum TNF- of group CLP+GHSRA+EA alpha, HMGB1 level and MPO activity in intestinal tissue were higher than CLP+EA group, but the activity of DAO in intestinal tissue was lower than that of group CLP+EA, were statistically significant (P0.05), compared with CLP+EA, water content and pathological score of CLP+GHSRA+EA group were significantly increased (P0.05). The level of Ghrelin and HMGB1 in serum the blood in the negative correlation (r=-0.664, p0.001), the Ghrelin level in the blood and intestinal tissue DAO activity was negatively correlated (r=-0.423, p=0.003, MPO activity and Ghrelin level in the blood and intestinal tissue were negatively correlated (r=-0.404, p=0.004).
Conclusion the cecal ligation and puncture can induce acute intestinal injury model of rats. Serum and intestinal tissue protein acute intestinal injury in rats Ghrelin and its receptor expression is decreased. The electroacupuncture can inhibit the acute intestinal injury in rats in vivo proinflammatory cytokines (TNF- alpha, HMGB1) and improve the intestinal mucosal barrier the function of electro acupuncture can promote the expression of acute intestinal injury in rats Ghrelin secretion and intestinal tissue. The Ghrelin receptor blocking Ghrelin receptor after electroacupuncture lose protective effect on acute intestinal injury, the effect of electro acupuncture in three foot acupoints on acute intestinal injury in rats with Ghrelin and its receptor role.

【学位授予单位】：浙江中医药大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R245

【相似文献】