Mechanosensitive Ion Channel PIEZO1 Promotes Intestinal Inflammation Through NLRP3 Pathway In Crohn’s Disease

Background Crohn’s disease(CD),a main type of inflammatory bowel disease(IBD),is a non-specific chronic intestinal inflammation that occurs mainly in young and middle-aged patients.The disease involves entire digestive tract from oral cavity,stomach,small intestine to the anus.Unfortunately,there is no effective therapy making it a lifelong incurable disease with remission and recurrence alternation.The pathogenesis of Crohn’s disease is currently unknown.Persistent intestinal inflammation is one of the hallmarks of CD and a high risk factor of disease recurrence,complications,and cancer.Intestinal stricture is an important outcome in the progression of CD,resulting in gut obstruction and hypertension in intestinal lumen,which are the adverse results in CD.Mechanical force widely exists in the digestive tract,the digestive tract produces and responds to mechanical force,and mechanical pressure is sensed by mechanoreceptors as feedback signals and physiological stimuli,and then converted into electrical signals to play a series of functions.During pathological changes such as disorders,inflammation and stricture,these mechanical forces can have adverse effects on the biological behavior of intestinal epithelial cells,enterochromaffin cells and other cells.The mechanosensitive cation channel PIEZO1 is a representative of mechanoreceptors.Previous studies have shown that PIEZO1 plays an important role in immune response and epithelial cell repair,but whether it plays a similar role in CD and its mechanism have not been reported.Therefore,this study intends to explore the correlation between the expression of PIEZO1 and intestinal inflammation in intestinal specimens of CD patients,and to further clarify the role of PIEZO1 in intestinal inflammation and the specific mechanism in vitro,in the hope to provide possible research targets for key links in occurrence and development of CD intestinal inflammation.MethodsThe methods are divided into two parts: 1.Clinical observation study: collecting paraffin specimens of patients undergoing small intestine surgery from January 2018 to December 2021 in the Department of General Surgery,First Affiliated Hospital of Anhui Medical University.Paraffin sections were taken for hematoxylin-eosin staining(HE)and immunohistochemistry(IHC)to clarify the distribution of PIEZO1 in intestinal tissue and its correlation with intestinal histology inflammation.In addition,collecting 30 biopsies with confirmed CD patients who underwent double balloon enteroscopy in the Department of Gastroenterology,First Affiliated Hospital of Anhui Medical University from June 2021 to October 2021.Real-time quantitative polymerase chain reaction(q RT-PCR)and Western Blot(WB)were used to detect the expression of PIEZO1 in the intestinal tissues of patients with active CD.At the same time,the patient demographics,clinical symptoms,laboratory tests were collected and analyzed with the correlation of PIEZO1 expression.2.Mechanism research: combined with PIEZO1 agonists and inhibitors,calcium ion influx and reactive oxygen species(ROS)were observed by calcium ion imaging,flow cytometry,changes in mitochondrial membrane potential was observed by laser confocal microscopy imaging and flow cytometry.Small interfering RNA(small interfering RNA,si RNA)was used to knock down the expression of PIEZO1 and NOD-like receptor 3(NLRP3)in HT29 cells,using q RT-PCR,WB and enzyme linked immunosorbent assay(ELISA)to detect key factors in NLRP3 signaling pathway.Results1.The postoperative wax blocks of CD patients were collected for HE and IHC staining,and it was found the expression of PIEZO1 in the intestinal tissues of CD patients were significantly increased.According to the classification of inflammation,it was found that the expression of PIEZO1 increased with the degree of inflammation,and was positively correlated with Crohn’s disease activity index(CDAI)and histological scores.2.The m RNA and protein levels of PIEZO1 in the intestinal tissues of patients with active CD were significantly increased.According to CDAI,the intestinal inflammation of patients was divided into remission,mild,moderate and severe cases,PIEZO1 expression increased with disease severity and was positively correlated with CDAI,fecal calprotectin,platelet count,and hematocrit.3.The expression of PIEZO1 in HT29 cells was successfully knocked down by si RNA.After stimulation with 10 μg/ml lipopolysaccharide(LPS)for 24 hours,pro-inflammatory factors such as tumor necrosis factor-α(TNF-α),leukocytes Interleukin(IL)-6 was significantly decreased,and pro-inflammatory factors increased after PIEZO1 was activated.4.After activating PIEZO1,the intracellular calcium concentration significantly increased and the mitochondrial membrane potential decreased,while after PIEZO1 activity was inhibited,the calcium concentration was significantly reduced and the mitochondrial membrane potential increased.After knock-down PIEZO1 by si RNA,the calcium influx cannot be induced by Yoda1,an agonist of PIEZO1.5.After HT29 cells were induced by LPS for 24 hours,q RT-PCR,WB and ELISA were used to detect NOD-like receptor 3(NLRP3)key molecules in NLRP3 inflammasome pathway.The expression of NLRP3 IL-1β,IL-18 was significantly increased,and cleavages of gasdermin D and caspase1 were observed.After knock-down of PIEZO1,NLRP3,IL-1β,and IL-18 decreased,and gasdermin D and caspase1 were not activated.6.q RT-PCR,WB and ELISA were used to detect NLRP3 signaling pathway-related molecules in the intestinal tissues of active CD patients.It was found that the NLRP3 signaling pathway was activated in CD patients’ intestine,and the correlation analysis indicated that the expression of PIEZO1 was positively related to NLRP3,IL-1β and IL-18.7.After NLRP3 knock-down in HT29 cells by si RNA,the interleukin-1β,interleukin-18,TNF-α,and interleukin-6 in cells and culture medium were significantly decreased induced by LPS whether PIEZO1 was activated or not.Conclusions(1)The mechanosensitive ion channel PIEZO1 is widely expressed,both in epithelial cells and immune cells of CD patients’ small intestinal tissues;(2)The expression of PIEZO1 in the small intestinal tissues of active Crohn’s disease were significantly increased,and it was positively correlated with inflammatory markers such as fecal calprotectin,platelet count,hematocrit,CDAI,histological score;(3)In intestinal epithelial cells,up-regulation of PIEZO1 promotes the secretion of interleukin-1β,interleukin-6,interleukin-18 and tumor necrosis factor-α.Inflammatory cytokines were decreased after down-regulation of PIEZO1;(4)PIEZO1 regulates calcium influx and mitochondrial membrane potential,then activates the NLRP3 inflammasome,up regulated key molucules in this signaling pathway to aggravate gut inflammation.