Acute Intestinal Damage Associated With In Vivo Rat Ghrelin And Its Receptor Expression And Acupuncture Interventions

Objective To study the levels of ghrelin and ghrelin receptor density in serum and intestinal tissue and the influence of electroacupuncture in the acute bowl injury (ABI) rats model.Methods①To establish the rats model of acute bowl injury Intra-abdominal infection leads to acute intestinal injury induced by cecal ligation and puncture (CLP).The study was performed on32Wistar male rats.Two experimental groups composed12rats were established:acute bowl injury(ABI)group(CLP group) was established by using cecal ligation and puncture (CLP):a2-cm ventral midline abdominal incision was performed, the cecum was then exposed, ligated just distal to the ileocecal valve,punctured four times with an16-gauge needle, and returned to the abdominal cavity,the incisionwas then closed in layers. Sham-operated group (Sham group) underwent the same procedure with the exception that the cecum was neither ligated nor punctured. Twele animals in each group were sacrificed after taking the blood sample,the intestines at the time of12h after CLP surgery.To detect the level of serum TNF-α and high mobility group box1(HMGB1),take intestinal tissue to detect the rate of water content、the activity of myeloperoxidase (MPO) and diamine oxidase (DAO), and to evaluate the existence of pathological changes of intestines to evaluate the existence of acute bowl injury.②To study on changes of ghrelin and ghrelin receptor density in serum and intestinal tissue and the influence of electroacupuncture in the ABI rats model to investigate the it’s mechanism of treatment on acute bowl injury whether is associated with ghrelin Forty-eight Wister male rats were randomly divided into four groups:Sham operation group(Sham group),acute bowl injury group(CLP group), electroacupuncture treatment group (CLP+EA group)and ghrelin receptor antagonist group(CLP+GHSRA+EA group),twele rats were in each group. CLP group was established by using cecal ligation and puncture (CLP):a2-cm ventral midline abdominal incision was performed, the cecum was then exposed, ligated just distal to the ileocecal valve,punctured four times with an16-gauge needle, and returned to the abdominal cavity,the incisionwas then closed in layers. Sham group underwent the same procedure with the exception that the cecum was neither ligated nor punctured. An electroacupuncture at Zusanli point (ST36) was performed at constant voltage immediately for30minutes after CLP surgery in CLP+EA group.In CLP+GHSRA+EA group,ghrelin receptor antagonist([D-Arg1,D-Phe5,D-Trp7.9,Leu11]substance P) was injected tail vein,and then electr-oacupuncture at Zusanli point (ST36) was performed at constant voltage immediately for30minutes, each group was taken the blood from the heart at the time of12h after operation,the-n,was killed and intestines was taken from the body.So,we can detect the level of TNF-α、 HMGB1and ghrelin in serum, and detect the rate of water content、the activity of myeloperoxidase (MPO) and diamine oxidase (DAO), and using western blot to detect the expression of ghrelin and ghrelin receptor protein, using immunohistochemistry to detect the positive cells of ghrelin and ghrelin receptor.Results①CLP led to the development of acute bowl injury.The model rats intestines structure was destroyed.The pathological scores of the CLP groups were significantly higher than the Sham groups(P<0.05).The levels of TNF-a and HMGB1of serum in CLP groups markedly higher than the Sham groups(P<0.05).The rates of water content and the activity of MPO in intestines in CLP groups were significantly higher than those in the Sham groups,while the activity of DAO in CLP groups was markedly lower than that in Sham groups(all P<0.05).②The ghrelin density of serum in CLP group is abviously lower than that in Sham group (P<0.05).The ghrelin and ghrelin receptor poteins are expressed in intestinal tissue,the expression quantity of them in CLP group were significantly lower than those in sham group(P<0.05).Ghrelin immunopositive cells and ghrelin receptor immunopositive cells are found in intestinal tissue,the numbers of them in CLP group were significantly lower than those in Sham group(P<0.05).③The levels of TNF-a and HMGB1of serum in CLP+EA group markedly lower than t CLP group (P<0.05).The rates of water content and the activity of MPO in intestines in CLP+EA group were significantly lower than those in CLP group,while the activity of DAO in CLP+EA group was markedly higher than that in in CLP group(all P<0.05). The model rats intestines structure was destroyed.The pathological scores of CLP+EA group were significantly lower than that of CLP group (P<0.05).④The ghrelin density of serum,the expressions quantity of ghrelin and ghrelin receptor poteins, and the numbers of ghrelin and ghrelin receptor immunopositive cells in intestinal tissue in the CLP+EA group were significantly higher than those in CLP group (all P<0.05).⑤The levels of TNF-α and HMGB1of serum, the rates of water content and the activity of MPO in intestines,and The pathological scores in CLP+GHSRA+EA group were significantly higher than those in CLP+EA group,while the activity of DAO in CLP+GHSRA+EA group was markedly lower than that in CLP+EA group (all P<0.05).⑥The level of HMGB1was in serum negatively correlated with ghrelin (r=-0.664, p<0.001) in serum.the activity of MPO in intestines was negatively correlated with ghrelin (r=-0.404, p=0.004) in serum, the activity of DAO in intestines was negatively correlated with ghrelin (r=-0.423, p=0.003) in serum.Conclusion①The acute intestinal injury model of rats can be induced by cecal ligation and puncture.②The level of ghrelin of serum in ABI was decrease, the expressions quantity of ghrelin and ghrelin receptor poteins,and the numbers of ghrelin and ghrelin receptor immunopositive cells in intestinal tissue were very low in ABI.③Electroacupuncture at Zusanli point (ST36) can inhibit the production of proinflammatory cytokines(TNF-α, HMGB1) in acute bowl injury in rats,and can ameliorate the intestinal mucosal barrier.④Ele-ctroacupuncture at Zusanli point (ST36) can increase the level of serum ghrelin and promote the expression of ghrelin and ghrelin receptor in intestinal in acute intestinal injury.⑤When the ghrelin receptor is blocked, Electroacupuncture at Zusanli point (ST36) can’t ameliorate the intestinal mucosal barrier.So the effect of electroacupuncture at Zusanli point (ST36) on acute bowl injury is realized by promoting the expression of ghrelin and ghrelin receptor in intestinal.