| BackgroundInflammatory Bowel Disease（IBD）includes Ulcerative Colitis（UC）and Crohn’s Disease（CD）,in which UC lesions mainly involve the colonic mucosa and submucosa and are characterized by diffuse,superficial and remission,and the clinical symptoms are mainly recurrent abdominal pain,diarrhea and mucopurulent stools.At present,5-Aminosalicylic acid（5-ASA）,glucocorticoids,immunosuppressive agents and biologics have been used to induce treatment and maintain remission,but there is still lack of effective methods to prevent recurrence of intestinal inflammation.The incidence of UC is increasing year by year worldwide,and the economic burden and poor quality of life have become a global public health problem.the development of UC is associated with various factors such as genetic susceptibility,immune disorders,microbial imbalance,environmental factors,and defective intestinal mucosal barrier function.As the first barrier between the body and the outside,the dysfunction of intestinal mucosal barrier is closely related to the occurrence and progression of UC,but its cellular and molecular mechanisms are not fully understood.Therefore,the need to explore more effective treatment options and therapeutic targets has become a major challenge.Discoidin domain receptor 1（DDR1）is a member of the transmembrane RTK superfamily,which is widely expressed in epithelial cells,fibroblasts,and oligodendrocytes.Studies have shown that abnormal expression of DDR1 is closely related to inflammation,fibrosis,and tumors.Previous studies have reported that inhibition of DDR1 could alleviate BBB disruption after focal cerebral ischemia in rats.Considering the similarity of the physiological structures and functions of BBB and intestinal mucosal barrier,we hypothesized that DDR1 may be involved in the development of UC through regulating the function of intestinal mucosal barrier.Objective To investigate the role of DDR1 in the maintenance of intestinal mucosal barrier and its possible molecular mechanism,and to provide a potential target for the treatment of UC.Methods 1.The expression of DDR1 in the intestinal tissues of acute IBD patients and colitis induced by different concentrations of Dextran Sulfate Sodium（DSS）was detected by western blot.2.DDR1 knock-out C57/BL6 male mice(DDR1-/-)and wild-type C57/BL6 male mice（WT）were used to induced acute UC model,Eyeball blood was taken after anesthesia on day 8,colon and spleen of mice were taken after execution.（1）general conditions,stool properties and rectal prolapse of the mice were recorded daily,and daily body weight and Disease Activity Index（DAI）were recorded.（2）The length of colon was recorded and HE staining was performed to observe the changes of colon tissue in each group of mice,and the spleen index was recorded.（3）FITC-dextran,D-LA,and DAO in serum were detected to evaluate the function of intestinal mucosal barrier.（4）q RT-PCR was performed to detect the expression of inflammatory factors in the colon tissue.（5）the apoptosis in colonic tissues of each group was detected by TUNEL.（6）the expression of apoptosis-related protein in colonic tissues of each group was detected by western blot and q RT-PCR.（7）the expression of TJ proteins in each group of colon tissues was detected by western blot,q RT-PCR,and immunohistochemistry.3.HIEC and Caco2 were cultured in vitro to establish a monolayer barrier of intestinal epithelial cells mimicking the intestinal mucosal barrier,intestinal epithelial barrier injury was induced by TNF-αand IFN-γ（T/I）incubation to cell monolayers transfected with PCDH-DDR1 or p LKO.1-sh-DDR1-1 plasmids.（1）the expression of DDR1 in HIEC and Caco2 cells was evaluated by western blot,the formation of monolayer intestinal epithelial barrier in HIEC and Caco2 cells was observed by measuring the Trans-epithelial electrical resistance（TEER）.（2）The effect of DDR1 lentivirus was evaluated by western blot and q RT-PCR,and the effect of DDR1 knockdown or overexpression on the growth of HIEC and Caco2 cells was detected by CCK8.（3）TEER and FITC-dextran were used to assess the barrier function of intestinal epithelial cells monolayers.（4）the effect of DDR1 on the expression of inflammatory factors by epithelium was detected by q RT-PCR.（5）the effect of DDR1on cell apoptosis were evaluated by flow cytometry.（6）we examined the effect of DDR1 on the Bcl2/Bax-dependent apoptosis axis by western blot and q RT-PCR.（7）we measured the expression of TJ proteins using western blot and q RT-PCR.（8）the effect of DDR1 on NF-κB-MLCK-p-MLC2 signaling pathway was measured by western blot.（9）The cells were pretreated with the NF-κB inhibitor JSH-23,the expression of NF-κB-MLCK-p-MLC2-related pathway proteins and TJ proteins in each group of cells was detected by western blot after T/I incubation.Results 1.The DDR1 protein levels in colonic tissues were markedly decreased in both patients with CD and patients with UC compared with paired non-inflamed colonic tissues,Similar to observations in human IBD,DDR1 protein levels decreased significantly in the colon tissues of DSS-treated acute colitis compared with WT mice.Consistently,DDR1 expression decreased during the development of DSS-induced acute colitis in mice.However,DDR1 expression is elevated in DSS-induced chronic colitis in mice.2.DDR1 knock-out male mice(DDR1-/-)was constructed by CRISPR/Cas9 were generally similar to WT male mice,DDR1 knock-out does not affect the morphology,structure and permeability of intestinal mucosal barrier under normal conditions.After inducd by 4%DSS,（1）the body-weight loss,DAI of the DSS(DDR1-/-)were significantly lower than the DSS（WT）mice.（2）spleen index,and colon length of the DSS(DDR1-/-)were significantly lower than the DSS（WT）mice.DSS treatment induced substantially less epithelial damage and crypt architecture disruption in DDR1-/-mice compared with the WT littermates.（3）the serum levels of FITC dextran,D-LA and DAO in DSS(DDR1-/-)group were significantly decreased compared with DSS（WT）.（4）DDR1 knock-out attenuated the expression of the mucosal proinflammatory cytokine.（5）A large number of TUNEL-positive cells were found in DSS-treated WT mice,but the colon tissue of DDR1-/-mice had significantly reduced TUNEL-positive signals.（6）the expression of Cleaved-caspase3 and Bax was dramatically suppressed while Bcl2 was upregulated in DDR1-/-mice compared with the WT mice after DSS induction.（7）DDR1 deficiency prevented the DSS-induced reduction in ZO-1 and Occludin expression compared with DSS（WT）.3.（1）HIEC and Caco2 cells stably expressed DDR1,and the TEER of HIEC and Caco2 cells showed a gradual increase during monolayer barrier formation,with HIEC cells stabilizing at 9-10 days and Caco2 cells at 19-21 days.（2）DDR1 inhibition was found to suppress the proliferation of HIEC and Caco2 cells,while DDR1 over-expression significantly promoted the proliferation of cells,the knockdown and overexpression of DDR1 did not affect the function of the monolayer intestinal epithelial barrier without T/I stimulation compared with the corresponding control lentivirus.（3）DDR1 inhibition significantly alleviated the TEER reduction by T/I compared with the monolayers transfected with control lentiviruses.The FITC-dextran permeability measurements were consistent with the TEER measurements,featuring further-decreased FITC-dextran after incubation with T/I in intestinal epithelial cell monolayers.However,DDR1 over-expression aggravated the barrier dysfunction after incubation with T/I,manifesting lower TEER and higher FITC-dextran flux compared with those transfected with control lentiviruses.（4）DDR1 knockdown significantly decreased the level of inflammatory factors,and DDR1 overexpression significantly increased the level of inflammatory factors after T/I incubation.（5）DDR1 inhibition decreased the apoptosis of cells,while DDR1 over-expression significantly promoted the apoptosis of intestinal epithelial cells after T/I incubation.（6）DDR1 over-expression promotes T/I induced Bcl2/Bax axis activation,whereas DDR1 inhibition suppresses those processes in HIEC and Caco2 cell monolayers.（7）after incubation with T/I,we found inhibiting the DDR1 expression restored the ZO-1 and Occludin expression significantly.Conversely,DDR1 over-expression significantly promoted the degradation of ZO-1 and Occludin.（8）After T/I incubation,DDR1 knockdown significantly inhibited the activation of NF-κB-MLCK-p-MLC2 signaling pathway,and conversely,DDR1 overexpression promoted the activation of NF-κB-MLCK-p-MLC2signaling pathway.（9）After JSH-23 pretreatment followed by T/I stimulation,the activation of NF-κB-MLCK-p-MLC2 signaling pathway was significantly reduced,but DDR1 knockdown still inhibited the activation of NF-κB-MLCK-p-MLC2 signaling pathway to some extent and suppressed the decrease of TJ protein,on the contrary,DDR1 overexpression still promoted the activation of NF-κB-MLCK-p-MLC2signaling pathway and promoted the degradation of TJ proteins ZO-1 and Occludin.Conclusion 1.DDR1 is decreased in acute colitis while is upregulated in chronic colitis.2.DDR1 knockout alleviates the development of acute experimental colitis and attenuates intestinal mucosal barrier damage.3.DDR1 aggravates intestinal mucosal barrier damage by promoting intestinal epithelial cell apoptosis and TJ protein degradation.4.the NF-κB-MLCK-p-MLC2 pathway may be a downstream molecular mechanism for DDR1 to regulate intestinal mucosal barrier disruption under inflammatory conditions. |
