Study On The Anti-inflammatory Mechanism Of Huanglong Decoction Combined With Meropenem On CLP Model Rats

Purpose:To observe the anti-inflammatory effect of Huanglongtang combined with meropenem on sepsis rat model,the rats in TLR4,ileocecal ileum tissue MyD88,PI3 K,NFkappa B,Akt,p-Akt protein expression level detection,to investigate the regulatory effect of Huanglongtang combined with meropenem on sepsis rats ileum TLR4/NF-K B and PI3K/Akt signaling pathway,describes the anti-inflammatory mechanism of Huanglong decoction combined with meropenem on rat model of sepsis.Materials and methods: SPF male SD 70 rats,200 + 20 g,purchased from Liaoning Changsheng Biotechnology Co.Ltd.,animal production license number: SCXK(Liaoning)2010-0001.experimental animal acquired after feeding in the animal SPF experimental animal center of Liaoning University of Traditional Chinese Medicine laboratory,license No.: SYXK(Liaoning)2013-0009.routine granular feed,free water,12 hour circadian rhythm.The rats were acquired after one week of adaptive feeding,were in good condition,fully familiar with the surrounding environment,good activity,began formal experiments.Huanglongtang of Chinese herbal medicine,purchased from the Affiliated Hospital of Liaoning University of Traditional Chinese Medicine pharmacy.Meropenem for Injection,CHINO(Shijiazhuang Pharmaceutical Group Pharmaceutical Shijiazhuang)Limited production of.70 rats,10 rats were randomly selected as sham operation group,the remaining 60 rats underwent cecal ligation and puncture,after the model was replicated,again randomly divided into model group,western medicine group,Huanglong decoction combined with western medicine group,a total of four groups.Rats weighed after intraperitoneal injection of 10% chloral hydrate,3ml/kg body weight,when rats after deep anesthesia,the left abdomen 3-4 lumbar 0.8cm at the shearing,prone position fixed on the operating table,to the hair at the iodophor after routine disinfection,longitudinal incision 1cm step by step,blunt separation,until the peritoneal tissue forceps,exploration,careful to find the cecal tip,incision,distance of cecal tip 1cm cecal ligation in the cecal tip,and the No.7 needle pierced the cecum repeated 3 times,the other a little squeeze the cecum,cecum overflow,cecum homing,sutured muscle layer and skin layer,iodophor disinfection.Rats in the sham operation group type as above,but not the cecal ligation and perforation.24 hours after the surgery,in the sham rats with cecal ligation and puncture were randomly selected in 2 rats only,broken marrow is put to death,take the ileocecal junction at 3cm ileum.Soaked in 4% poly Formaldehyde Solution is fixed,the removal of paraffin sections were prepared to observe the pathological changes of ileum,HE staining method,to determine whether the model was successfully established.Sham operation group: normal feeding without treatment.After surgery the next day,when the model was established successfully,the rats in the sham operation group of normal feeding,no treatment;model group rats by mouth distilled water,2ml/ and-1,at the same time by intraperitoneal injection of saline,1ml/,-1,2 times daily for 4 days;the western medicine group rats by intraperitoneal injection of meropenem injection,1ml/,-1,2 times daily for 4 consecutive days;Huanglong Decoction combined with western medicine group rats by gavage of Huanglongtang,2ml/ and-1,while intraperitoneal injection of meropenem injection,1ml/,-1,2 times daily for 4 days.One hour after the last administration,the rats were anesthetized with 10% chloral hydrate by intraperitoneal injection,the abdominal cavity was opened,the whole blood 5ml was collected from abdominal aorta puncture into the coagulant tube,and the rats were kept at room temperature for 1 hour or 2000 rpm,and centrifuged for 10 minutes.The supernatant was used to detect the content of IL-8 in IL-1 ?-TNF-?.The ileum tissue was cut off at the proximal ileocecal 3cm for 2 cm after blood was taken from the rats in each group.After washed with the precooled PBS buffer solution,it was frozen in the 2ml cryopreservation tube to detect the content of IL-1 ?-TNF-? TNF-?,IL-6 IL-8 and the expression level of TLR4-MyD88NF-kappa B in the ileum tissue.The ileum tissues were also taken for 2 cm and immersed in 4% paraformaldehyde solution to detect the expression of PI3KP-AktP-Akt in ileum tissue by immunohistochemical method.After the CLP model was established,the general state of the rats was observed daily.After the last administration,the serum and ileal tissue levels of IL-1 ?-TNF-? and IL-6 IL-8 were detected by Elisa,and the expression of TLR4,MyD8NF-? B in ileum was detected by western-blot method.Immunohistochemical method was used to detect the expression level of PI3KP-Aktna-Akt in ileum tissue.Result:1.model evaluation: sham operation group after CLP rats in ileocecal ileum tissue HE staining showed that the sham operation group rats intact mucosa layer,especially epithelial layer,no damage to change,submucosa no infiltration of inflammatory cells in.CLP rats with ileal HE staining showed cortical degeneration,fall off the intestinal villi,disorder,or even dissolve or missing,there are a lot of submucosal infiltration of inflammatory cells and the serosa is also visible inflammatory cells,focal necrosis was observed in general 2.general observation: after CLP surgery,3 rats died,fully awake after feeding until the next day,only 12 died during the next day,2 rats were randomly selected for the evaluation model,the residual CLP after operation 43 rats were randomly divided into three groups,15 rats in model control group,western medicine group 14,Huanglongtang the joint western medicine group 14.The intervention period,the rats in the model control group died 7,western medicine group and 6 rats died,Huanglong decoction combined with western medicine group and 5 death.Death was not found in sham operation group,2 rats were randomly selected for model evaluation.After the last injection,the rats in the sham operation group of wound healing,white coat color,rich luster,good activity,food and water.The rats in the model control group the worst state,hair withered,curled up in the corner of the cage cluster,the activity is few,the sharp reduction in the amount of water to eat.The western medicine group and Huanglong decoction combined with western medicine group rats were significantly better than the model control group,no rats cluster,but activity significantly compared with the sham group,the wound healing was acceptable.The serum and tissue of 3.rats in IL-1 beta,TNF-alpha,IL-6,IL-8 test results show that the content: compared with sham operation group,the other three rats serum and tissue IL-1 beta,TNF-alpha,IL-6,IL-8 were significantly increased(P < 0.01),there were significant differences in the control group;compared with the model group,western medicine and Huanglong decoction combined with western medicine group rats serum and tissue IL-1 beta,TNF-alpha,IL-6,IL-8 were significantly lower(P < 0.01),there was significant difference;compared with the western medicine group,Huanglong decoction combined with western medicine group rats serum and tissue IL-1 beta,TNF-alpha.IL-6,IL-8 content decreased significantly(P < 0.01),there was statistically significant difference.TLR4,in rat ileum 4.groups in MyD88,NF-kappa B expression level.The results showed that: compared with the sham group,TLR4 control group,rat ileum in the model MyD88,NFkappa B expression level was increased,there was significant difference(P < 0.01);control group and model group,Western Medicine.TLR4,ileum of rats Huanglong decoction combined with western medicine in MyD88,the expression level of NF-kappa B significantly decreased,there was significant difference(P < 0.01);compared with the western medicine group,TLR4 group,ileal Huanglong decoction combined with western medicine in MyD88,the expression level of NF-kappa B significantly decreased,there was significant difference(P < 0.01).PI3K,in rat ileum was 5.Akt,the expression level of p-Akt.The results showed that: compared with the sham group,PI3 K control group,rat ileum in the model of Akt,the expression level of p-Akt was significantly increased,there was significant difference(P < 0.01);control group,western medicine group and model,PI3 K,ileum rats in Huanglong decoction combined with western medicine in Akt,the expression level of p-Akt was significantly reduced,there were statistically significant differences(P < 0.01);compared with the western medicine group,PI3 K group,ileal Huanglong decoction combined with western medicine in Akt,the expression level of p-Akt was significantly reduced,there were statistically significant differences(P < 0.01).Conclusion:1.The cecal perforation of 1.rats can cause severe inflammatory reaction in the whole body,which is similar to the process of clinical sepsis.It can be used for the pathogenesis and drug efficacy observation of sepsis.2.Huanglong decoction combined with meropenem on serum IL-1 and CLP in ileal tissue in a rat model of beta,TNF-alpha,IL-6,IL-8 content.3.the effect of anti Huanglongtang combined with meropenem on CLP model rats through inhibition of serum and local infection of IL-1 beta,TNF-alpha,IL-6,and IL-8 levels.4.Huanglong decoction combined with meropenem can TLR4 down sepsis model in rat intestinal tissue MyD88,the expression level of NF-kappa B protein.5.the effect of anti Huanglongtang combined with meropenem on rat model of sepsis may be achieved by inhibiting the excessive activation of TLR4/NF-kappa B signaling pathway.6.Huanglong decoction combined with meropenem can PI3 K down sepsis model in rat intestinal tissue Akt,p-Akt protein expression levels.7.the effect of anti Huanglongtang combined with meropenem on rat model of sepsis may be achieved by inhibiting the activation of PI3K/Akt/NF-kappa B signaling pathway.