Intestinal Mucosal Barrier Is Injured By BMP2/4 After Ischemic Reperfusion

BackgroundIntestinal ischemic reperfusion (I/R) injury is a common pathophysiological process, the intestinal ischemic reperfusion injury were occured during the course of many diseases at different levels, such as mesenteric artery embolism,strngulted herni,congestive heart failure,severe trauma,hemorrhagic shock and so on. Some operation and clinical operation could also lead to intestinal ischemia-reperfusion injury,such as major vascular surgery, small bowel transplantation, cardiopulmonary bypass, hemodialysis.Acute intestinal ischemia is a life-threatening vascular emergency,and its inhospital mortality rate remains high at approximately 60%-80%.In recent years, the incidence of intestinal ischemia showed a rising trend, therefore understanding the mechanisms of intestinal ischemia-reperfusion injury mechanism, help to reduce ischemia-reperfusion injury. Although many studies have focused on I/R, the mechanisms have not been fully elucidated, some researchers have made a variety of theory,including oxygen free radical injury, granulocytes adhesion and endothelial cells injury,cytokines and inflammatory mediators, cell apoptosis and so on.These theories have important contact with nuclear factor-kappa B (NF-κB) signaling pathway. Intestinal ischemic reperfusion could lead the activation of NF-κB resulted in the release of many kinds of damage factors.NF-κB is involved in the transcription of inflammatory genes (such as adhesion molecules, cytokines, enzymes),cell proliferation and differentiation, and cell apoptosis,oxidative stress,The damge of intestinal mucous membrane barrier not only include the increased permeability of intestinal epithelial cells caused by the various inflammatory mediators, but also contain the increased interstitial space caused by the rupture of tight junction proteins All these factors resulted in bacterial translocation, endotoxemia and severe systemic inflammatory response syndrome. This paper mal some researches on the mechanism of the activation of NF-κB during the ischer reperfusion injury.methods1. establish the cell injury model by hypoxia and animal model of rat with intestine ischemia-reperfusioninjury,Extraction of proteins from IEC-6 cells after 6 h of hypoxia,The cells were washed twice with phosphate-bufered saline (PBS) before lysis in cold RIPA buffer, the rats were fasted for 12 h,their abdomens were opened via a midline incision under sodium pentobarbital anesthesia, the superior mesenteric artery (SMA) was occluded for 20 minutes followed by 1 h of reperfusion, the tissue was cut along the longitudinal axis, washed in physiological saline, and immediately frozen in liquid nitrogen and stored at -70℃ for future use. Then we used western blot and IF to detect the change of BMP signal pathway.2. The IEC-6 was put into cell cultured plate, exogenous BMP2,BMP4 and BMP-specific antagonist noggin were added to the medium, Western-bolt and immunofluorescence detect the effect of BMP2 and BMP4 on the activation of NF-κB signaling pathway. For animal experiments,the rats were received intraperitoneal injection of 4 ug/kg noggin 30 min before I/R, the Sham group and the I/R group were served as the controlgroup,immunofluorescence detect the change of NF-κB signal caused by ischemic reperfusion..3. RT-PCR was used to detect the expression of TNF-α mRNA and IL-6 mRNA in IEC-6 cells after treatment with BMP2,BMP4 and BMP-specific antagonist noggin for hour.4. Western-bolt detect the change of AKT signaling pathway and occludin after treatment the IEC-6s with exogenous BMP2 or BMP4 for 24 hour.Results1. The expression level of BMP2 and BMP4 was upregulated in IEC-6 cells after 6 h of hypoxia,and the main BMP receptors BMPRII,BMPRIa also increased after 6 h of hypoxia.Meanwhile immunofluorescence analysis showed BMP2,BMP4,BMPRII,BMPRIa were also significantly increased in intestinal villus after 1 h of I/R compared to the Sham group.2. Exogenous BMP2 and BMP4 could directly activate the NF-κB IEC-6,immunofluorescence analysis showed that the NF-κB was translocated to nuclears after treatment the IEC-6 with BMP2 and BMP4,and the noggin completely reversed the nuclear localization of NF-κB induced by BMP2 and BMP4. Meanwhile, the in vivo results showed that NF-κB signaling was obviously activated in intestinal epithelial cells after 1 h of I/R treatment. In contrast,after intraperitonealinjection of 4 ug/kg noggin 30 min before I/R, the expression of NF-κB was significantly inhibited.3. Treatment of IEC-6 cells with BMP2 and BMP4 caused the level of TNF-α mRNA and IL-6 mRNA significantly increased compare to the control group,the noggin could decrease the release of TNF-α and IL-6.4. The phosphorylation of AKT progressively increased from 30 min to 120min after treatment of IEC-6 cells with BMP2 and BMP4. The tightjunction protein occludin decreased after after treatment the IEC-6 cells with BMP2 and BMP4 for 24 hour, both noggin and PDTC could reverse the decrease in TJ protein.Conclusion:1. hypoxia and ischemia-reperfusion colud induce the expression of BMP and BMP4 intestinal epithelial cells, and upregulated the expression of BMPRIa,BMPRII.The expression of BMP2 and BMP4 are concentrated in the mid-to-distal villus,while the increase in the crypt and mesenchyme was weaker.2. BMP2 and BMP4 could atviate the NF-κB signaling pathway in IEC-6 cells, resulted in the release of TNF-α and IL-6,and increased permeability of intestinal mucous membrane barrier.3. BMP2 and BMP4 could decrese the expression of occluding through the activation of AKT signaling pathway, damaged the integrity of intestinal mucous membrane barrier.