| Gastroesophageal reflux disease(GERD)is a common refractory digestive disease in clinic.Non-erosive reflux disease(NERD)is the most common clinical subtype of GERD with high prevalence,complex and recurrent symptoms.The unbalance of anti-reflux defense mechanism and toxic refluxates is the primary cause of the disease.The intact esophageal mucosal barrier is one of the main defensive factors,and abnormal reflux stimulation and cytokine-mediated inflammation are main damage factors.Some patients do not respond to or depend on proton pump inhibitors for a long time because of the complexity of the pathogenesis,thus increasing the risk of Clostridium difficile-related diarrhea,gastric polyp and osteoporosis.Traditional Chinese medicine has a significant effect on the treatment of GERD,especially NERD.This study explores the mechanism of esophageal mucosal barrier injury based on the negative regulation of Nrf2/keapl on NF-κB signaling pathway,and the therapeutic effects of Jianpi Qinghua granule on NERD with spleen deficiency and damp-heat syndrome.Clinical Research Part One:Characteristics of Inflammation and Esophageal Mucosal Barrier Function in Patients with NERDObjectiveTo evaluate the esophageal mucosal barrier function of NERD patients with the expression of E-cadherin and explore the expression of Nrf2 and inflammatory factors in esophageal epithelium of patients with NERD.MethodsNERD patients and healthy volunteers who attended the Xiyuan Hospital of China Academy of Chinese Medical Sciences from September 2016 to April 2019 were randomly included.Serum and esophageal mucosal tissues were collected.Serum NTFs,IL-1β and IL-4 levels were measured by Elisa.Real-time PCR was used to detect the expression of E-cadherin,Occludin,Claudin1 and Claudin4 mRNA in esophageal tissues.HE staining was used to observe the pathological changes in esophagus.The distribution and expression of E-cadherin and Nrf2 in esophageal tissue were observed by enzyme-linked immunohistochemistry.Results32 patients were included in NERD group and 24 healthy volunteers were included in control group.Compared with the healthy control group,serum levels of NTFs and IL-1β in patients with NERD increased(P<0.05).The relative expression of Claudin1 mRNA in esophageal epithelium decreaseed(P<0.05)while E-cadherin mRNA increased(P<0.01)compared with healthy control group.HE staining showed no erosion or ulcers in the esophageal mucosa of the healthy control group and the NERD group.The morphology of esophageal tissue in the healthy control group was norma1.The esophageal tissue in the NERD group showed basal cell proliferation,papillary elongation,ballooning degeneration of squamous epithelial cells,dilated intercellular spaces and inflammatory cell infiltration Immunohistochemistry showed that Nrf2 was expressed in the cytoplasm of esophageal mucosa cells in healthy volunteers and in the nucleus and cytoplasm in patients with NERD,E-cadherin was expressed in full layer of esophageal epithelial membrane of healthy volunteers and in the surface esophageal membrane of NERD patients.Compared with the healthy control group,the average optical density value of E-cadherin in esophageal epithelium of patients with NERD decreased(P<0.01)while the average optical density value of Nrf2 increased(P<0.01).Conclusion1.NERD patients have esophageal mucosal barrier damage.The expression of E-cadherin and its degradation product NTFs can reflect the integrity of the esophageal epithelial barrier to a certain extent.2.Nrf2 may protect esophageal mucosal barrier by inhibiting cytokine-mediated inflammationIn Vivo Study Part One:Establishment and Evaluation of Rat Model of NERD with Spleen Deficiency and Damp-heat SyndromeObjectiveTo establish and evaluate of rat model of NERD with spleen deficiency and damp-heat syndrome.Methods60 SD rats were randomly divided into sham group,NERD model group,NERD and syndrome model group.The modified esophagogastroduodenal anastomosis(EGDA)was performed.The internal and external damp-heat intervention was performed from the 8th day after operation and lasted for 14 days.Samples were taken at the 21th and 35th day after operation.The pH values of gastric juice were recorded.The level of total bile acid and pepsin of gastric juice and serum AQP4 were measured by Elisa,and the pathological changes of esophagus were observed by HE staining.ResultsThe rats in sham group were in good condition,and the rats in NERD model group and NERD and syndrome model group were debilitated.The survival rates of the sham group,NERD model group,NERD and syndrome model group were 95%,75%and 75%respectively.Compared with sham group,the body weight,food intake,water intake,and grip of the rats in NERD and syndrome model group were decreased at the 3rd and 5th week after operation(P<0.01)while the rectal temperature was increased(P<0.01).Compared with NERD model group,the body weight,water intake,and grip of the rats in NERD and syndrome model group decreased at the 3rd and 5th week after operation(P<0.01)while the rectal temperature increased(P<0.01).At the 3rd and 5th week after surgery,the feces of rats in sham group and NERD model group were strip-shaped,and the rats in NERD and syndrome model group were yellow soft stools or thin stools with less faces water content compared with sham group(P<0.01)and NERD model group(P<0.01).Compared with sham group,the gastric juice pH of the rats in NERD and syndrome model group increased at the 3rd and 5th week after operation(P<0.01)as well as the content of total bile acid increased(P<0.05).Compared with NERD model group,the gastric juice pH of the rats in NERD and syndrome model group decreased(P<0.05)at the 3rd week after operation and the total bile acid content increased(P<0.05)at the 5th week after operation.The serum AQP4 content of rats in NERD and syndrome model group was higher than that of sham group and NERD model group at the 3rd and 5th week after operation(P<0.01).There was no obvious erosion and ulcer in the esophageal mucosa in each group.The anastomosis of the rats in NERD model group and NERD and syndrome model group were unblocked.The morphology of esophageal epithelial cells in sham group was normal,and the esophageal mucosa of NERD model group and NERD and syndrome model group showed basal cell proliferation,papillary elongation,dilated intercellular spaces and inflammatory cell infiltration at the 3rd and 5th week after surgery.Inflammatory cell infiltration and proliferation of basal cells were more obvious in NERD and syndrome model group than that in NERD model group.ConclusionEGDA combined internal and external damp-heat intervention is a reliable method to establish rat model of NERD with spleen deficiency and damp-heat syndrome.In Vivo Study Part Two:Role of Nrf2 in Mediating NF-κB Pathway and the Mechanism of Jianpi Qinghua G ranule in Regulating Esophageal Mucosal Inflammatory ImpairmentObjectiveTo investigate the role of Nrf2/keap1 mediating NF-κB signaling pathway in the esophageal mucosal,and elucidate the mechanism of Jianpi Qinghua granule in treating NERD.Methods96 SD rats were randomly divided into sham group,NERD group,NERD and syndrome group,Jianpi Qinghua low dose(JPQH-L)group,Jianpi Qinghua medium dose(JPQH-M)group,Jianpi Qinghua high dose(JPQH-H)group,omeprazole group and agonist TBHQ group.The rat model of NERD with spleen deficiency and damp-heat syndrome was established by EGDA combined internal and external damp-heat intervention,and the rats were treated for 14 days accordingly.The TEER value of esophageal mucosa was measured by mini-Ussing chamber.Histopathological changes of esophagus were observed by HE staining.Serum MMP3,IL-1β,TNF-α contents were measured by Elisa.Esophagus Nrf2,keapl,NF-κB and IKK mRNA expression were detected by real-time PCR.Western blot was used to detect esophageal Nrf2,keapl,NF-κB p65,IKKβ,p-IKBα,E-cadherin,ADAM-10 protein expressions.Distribution and expression of Nrf2,keapl,NF-κB p65,IKKβ,E-cadherin,and ADAM-10 in esophagus were observed by enzyme-linked immunohistochemistry.ResultsCompared with sham group,the rats of NERD and syndrome group were debilitated with loose stools and turbid perianal while the weight,food intake,water intake,and grip decreased(P<0.05)and rectal temperature increased(P<0.01).Compared with NERD and syndrome group,JPQH-M and JPQH-H groups had improved mental status,better stool formation,increased body weight,food intake,water intake and grip(P<0.01),while rectal temperature decreased(P<0.01).No obvious erosion and ulcers were found in the esophageal mucosa in each group.The esophageal morphology of sham group was normal.Basal cell proliferation,papillary elongation,dilated intercellular spaces and inflammatory cell infiltration were observed in NERD group and NERD and syndrome group,and the above phenomenen were reduced to varying degrees in treatment groups.The TEER values of esophageal epithelium in NERD group and NERD and syndrome group were lower than that in sham group(P<0.01).The TEER values of esophageal epithelium in treatment groups increased compared with NERD and syndrome group(P<0.05),in which JPQH-M,JPQH-H and TBHQ groups were higher than JPQH-L group(P<0.01).Elisa showed that the levels of serum IL-1β,TNF-α,and MMP3 in NERD group and NERD and syndrome group were higher than sham group(P<0.01).The contents of IL-1β,TNF-α and MMP3 in JPQH-M and JPQH-H groups were lower than NERD and syndrome group(P<0.05).The contents of TNF-α and MMP3 in the omeprazole and TBHQ groups were lower than NERD and syndrome group(P<0.01).Real-time PCR showed that Nrf2 and keap1 mRNA levels were increased in JPQH-M,JPQH-H and TBHQ groups compared with NERD and syndrome group(P<0.05).The NF-κB and IKK mRNA levels in NERD and syndrome group were increased compared with sham group(P<0.01),and the NF-κB and IKK mRNA in JPQH-L,JPQH-M and JPQH-H groups were down-regulated compared with NERD and syndrome group(P<0.01).As western blot showed,compared with sham group and NERD group,the expressions of Nrf2,NF-κB p65,IKKβ,p-IκBα,and ADAM-10 protein in NERD and syndrome group were increased(P<0.01)while keapl and E-cadherin protein expression were decreased(P<0.01).The expressions of Nrf2,keapl,and E-cadherin were increased in each treatment group(P<0.01)while the expressions of NF-κB p65,IKKβ,p-IκBα,and ADAM-10 were decreased(P<0.01).Immunohistochemistry showed that Nrf2 was expressed in the cytoplasm of the esophageal epithelium cells in sham group and in cytoplasm and nucleus in NERD group,NERD and syndrome group and treatment groups.Keap1 was expressed in spinal layer and part of the basal layer cells.The mean optical density values of Nrf2 and keapl in NERD and syndrome group were higher than sham group(P<0.05),and JPQH-L,JPQH-M,JPQH-H and TBHQ groups were higher than model groups(P<0.05).NF-κB p65 and IKKβ are expressed in the cytoplasm and nucleus of esophageal epithelium cells.The mean optical density values of NF-κB p65 and IKKβ in NERD group were higher than sham group(P<0.01),and treatment groups were significantly lower than NERD and syndrome group(P<0.01).E-cadherin and ADAM-10 were expressed in the membrane of esophageal epithelial cells.The mean optical density value of E-cadherin decreased while AD AM-10 increased in NERD group and NERD and syndrome group compared with sham group(P<0.01).The mean optical density of E-cadherin in JPQH-L,JPQH-M and JPQH-H groups increased compared with NERD and syndrome group(P<0.05)and ADAM-10 decreased compared with NERD group(P<0.05).Conclusion1.Nrf2/keap1 inhibits NF-κB activation by down-regulating IKKβ,and alleviates inflammatory damage of esophageal mucosa by cytokines.2.Jianpi Qinghua granule reduces NERD esophageal mucosal inflammatory response and improve the esophageal mucosal barrier function by activating Nrf2/keap1 to negatively regulate NF-κB pathway. |
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