A Study Of The Role And Mechanism Of P2X7Receptors In The Therapeutic Effect Of Estrogen Receptor P In Inflammatory Bowel Disease (IBD)

【Objective】Inflammatory bowel disease (IBD) is a kind of chronic and recurrent intestinalinflammation, which results from different causes and mediated by abnormal immunalresponses. IBD has a trend of lifelong recurrence. It has two major types, namelyulcerative colitis (UC）and Crohn disease (CD).[1]In recent years, the incidence rate ofIBD in China has been increasing year by year, which mechenisms may be the interactionsof diverse factors, including environment, genetics, infection and imunnity.[1]The specificclinical syndromes are recurrent diarrhea, abdominal pain, loss of body weight and bloodystools.[1]However, there hasn’t been any radical treatment in the clinic. Therefore, how tocure IBD thoroughly has become a medical problem to be solved and ugently needs morefoundamental researches to explore its pathogenesis, thus looking for a novel therapeuticschedule to improve the life quality of IBD patients.Rencent researches found that during colitis, selective activation of estrogen receptorβ (ERβ) could significantly attenuate the symptoms of mice, while reducing the expressionof ERβ by gene knocked-out could further exacerbate the symptoms of colitis, suggestingthat activation of ERβ could play an anti-inflammatory and anti-nociceptive role in IBD.[2-4]In addition, studies also found that, in the models of colon cancer induced by AOM or DSS,ERβ could also play an anti-tumorous role.[5-7]Although activation of ERβ has exerted amarked therapeutic effect in colitis and related cancers, its mechanism by which ERβactivation could play such a role hasn’t been clear by far, thus in need of further studies.On the other hand, there’s another kind of proteins that is in close relationship withthe initiation and development of IBD, namely P2receptors. So far, many studies havedemonstrated that P2X7receptor, an isotype of P2X receptors, plays a crucial role in IBD.During the development of IBD, the expression of colorectal P2X7receptors wassignificantly increased, which could not only induce some immunocytes, like mast cells,macrophages, helper T cells and so on, to release large amount of inflammatory mediators,including interleukins, leukotriene, TNF-α and so on[8-11]; but also result in the damages ordeaths of colorectal neurons, thus accelerate the exacerbation of colitis.[12]To futher investigate the relationship between estrogens and P2X receptors in theinitiation and development of IBD, we used animal behavior test, western blotting, Elisaand immunohistochemistry to study the role of estrogen receptor β activation in IBD andfocused mainly on its relationship with P2X7receptors. 【Methods】1. Induction of colitis in rats.Experimental colitis was induced by colorectal injection of30%trinitrobenzenesulfonic acid (TNBS,40mg/kg) in ethanol. Under slight anesthesia ofpentobarbital sodium (2%,2mg/kg), a plastic tube, which diameter is6-Fr, was insertedinto the rectocolon of rats until its anterior extremity was8cm far from the anus, andTNBS solution was perfused. Through observation of rats’ body weight, DAI scores,colorectal HE staining and MPO concentration, the severity of rats’ colitis was assessed.On the third or fourth day after TNBS or saline was injected, saline, PPT, BBG, DPN orERB-041was subcutaneously injected for successive4days.2. HistologyRats were sacrificed and normal or inflamed colons were obtained. After cleaning insaline, colons were fixed in4%paraformaldehyde for24-48h and then placed into30%sucrose for dehydration until colons sank to the bottom. Colons were then taken out andcut into frozen sections. Accoding to the instruction in the kit of eosin-hematoxylinstaining, colonic sections were stained and taken photos under microscope.3. Western BlottingRats were sacrificed, then rectocolon and DRG were obtained. The supernatant afterhigh speed homogenation and centrifugation was just protein extracts. The concentration oftotal protein was measured by BCA method.10%separation gel and4%concentrated gumwere made. After electrophoresis (100mV,100min), transmembrane (300mA,90min) andblock in non-fat milk, the membrane was incubated in primary antibodies (antibodies forP2X7receptor, estrogen receptor β and β-actin)4℃overnight. After fast washing in TBST,the membrane was placed in secondary antibodies (room temperature,1h) and washedagain. Finally, the photos were taken and the grey scales were measured.4. Construction of ERβ mRNA plasmid and lentivirus transfection.Firstly, ERβ mRNA was synthesized according to its sequence (gene ID:25149), then,insert the above mRNA into plasmids, and sequence. Transfect the high-purity recombinantplasmids and lentivirus expression vector (LV-5-EF1a-GFP/Puro) into293T cells, thenpackage and produce virus, extract virus liquid, concentrate and purify. Transfect the293Tcells with high-quality virus liquid, and measure the virus titre by QT-PCR for furtherexperiments. On the day before TNBS injection, control virus (20μl) and virus containingERβ (20μl) were injected into the colorectal wall of rats directly through abdomen. 5. Analysis of colorectal MPO and inflammatory factorsPut standards and samples (100μl/well) into48-well plate, respectively. After washing,adding distilled water and primary antibody, washing, adding enzyme labelled antibody,washing, adding substrate and stopping solution, the light absorption value at450nm wasmeasured by microplate reader and analysed. According to the OD values, theconcentrations of MPO and different inflammatory factors were calculated.6. ImmunohistochemistryAfter deep anesthesia, rats were injected saline to clean their blood, and then injected4%paraformaldehyde of the same volume for fixation. Rectocolons were obtained as soonas possible and fixed in4%paraformaldehyde again for24-48h. After washing, therectocolons were placed into30%sucrose until they sank to the bottom. Cut therectocolons into10-μm-thick frozen sections. After washing, antigen exposure, washing,BSA blocking, washing, primary antibodies (antibodies for P2X7, ERβ, macrophage)incubation, washing, secondary antibodies incubation and washing, the photos were takenand preserved.【Results】1. Compared with saline group, rats with colitis induced by TNBS saw a significantdecrease in body weight, a significant increase in DAI scores and MPO concentrations. HEstaining showed that the rectocolons of colitis group had disordered walls and congestive,edematous intima. Western Blot showed that compared with saline group, the expressionsof ERβ in rectocolon and DRG neurons of colitis group were both significantly decreased;the expression of P2X7receptor in rectocolon was significantly increased, while in DRG,there was no change. Elisa showed that the levels of inflammatory factors like TNF-α,IL-1β and IL-6in the rectocolon of colitis group were significantly increased, while therewas no change in IL-10.2. After administration of specific antagonists of P2X7receptor, BBG, the bodyweight of colitis group was significantly reversed, DAI scores were significantly decreasedand HE staining showed attenuation in colorectal inflammatory responses.3. After administration of specific agonists of ERα, PPT, the body weight of colitisgroup was significantly lowered, DAI scores were significantly increased and HE stainingshowed excerbation in colorectal inflammatory responses.4. After administration of combinant lentivirus containing ERβ, the body weight ofcolitis group was significantly reversed, DAI scores were significantly decreased and HE staining showed improvement in colorectal inflammatory responses. Western Blot showedthat application of combinant lentivirus containing ERβ could significantly decreased theexpression of P2X7receptor in the rectocolon of colitis group, while in DRG neurons,there was no change.5. After administration of specific agonists of ERβ, DPN and ERB-041, the bodyweight of colitis group was significantly reversed, DAI scores were significantly decreasedand HE staining showed improvement in colorectal inflammatory responses. Western Blotshowed that application of DPN and ERB-041could significantly decreased the expressionof P2X7receptor in the rectocolon of colitis group, while in DRG neurons, there was nochange. Elisa showed that admistration of DPN and ERB-041could lower theinflammatory mediators in the rectocolon of colitis group, such as MPO, TNF-α, IL-1β andIL-6, while there was no change in IL-10.6. There was co-expression of ERβ and P2X7receptors in the rectocolon, and thisco-location mainly existed in the macrophages of rectocolon.【Conclusions】1. TNBS/ethanol solution can successfully induce colitis in rats.2. P2X7receptor is involved in the initiation and development of IBD. Inhibition ofP2X7receptor can exert a therapeutic role in IBD.3. ERα receptor also paticipates in the initiation and development of IBD. Activationof ERα is able to significantly enhance the inflammatory responses of rectocolon in IBD.4、Activation or overexpression of ERβ plays a markedly therapeutic role in IBD,which might be through down-regulation of P2X7receptor in the rectocolon and decreasein the release of inflammatory mediators.