| Aims Tissue remodeling such as muscular thickening and collagen deposition can be observed in the esophageal wall of patients with achalasia,while the role of remodeling in the pathogenesis of achalasia is still unknown.In this study,the degree of eosinophilic infiltration and expression levels of remodeling factors were evaluated in esophageal specimens of achalasia patients and animal models,and the correlation between them was discussed.Then mechanism of eosinophils induced remodeling of esophageal smooth muscle cells were conducted in vitro.And finally investigated the role of anti-eosinophil drugs in esophageal tissue remodeling and development of achalasia,with a view to understanding the pathogenesis and finding new therapeutic target of achalasia from the perspective of esophageal smooth muscle remodeling.Methods(1)Histopathological study: Forty patients with achalasia and ten patients with esophageal carcinoma were enrolled as the experimental group and control group,respectively.The tissue specimens of LES were obtained from all the patients during POEM or surgery.All the specimens were stained with HE firstly to evaluate the infiltration of eosinophils in the esophageal smooth muscle.Masson staining was used to evaluate the degree of fibrosis in esophageal smooth muscle.And to observe the morphological structure and microstructure of esophageal smooth muscle cell bundles,immunofluorescence staining and transmission electron microscopy were used.In addition,expression levels of remodeling factors,such as transforming growth factor-β1(TGF-β1),α-smooth muscle actin(α-SMA),collagen I and collagen Ⅲ were evaluated through immunohistochemistry and Western Blot.Finally,to assess the symptoms and esophageal motility,Eckardt score and clinical examinations were performed before and 3 months,6 months,1 year,and 2 years after POEM,respectively.Furthermore,the correlations among count of eosinophils and expression levels of remodeling factors and clinical parameters were analyzed also.Study on the thickness of esophageal smooth muscle: Ten patients diagnosed as achalasia and treated with POEM were included in the experimental group,another 10 patients with gastric antrum protrusion lesion were selected as the control group.Patients in both groups performed esophageal emptying test and ultrasound endoscopy.Maximum width of the esophagus,the height of barium in different time points,and thickness of lamina propria at LES and 5cm,10 cm and 15cm above LES were measured,respectively.What’s more,the index above were compared between the AC patients before and after POEM,and between the experimental group and control group.(2)Eighty Balb/c mice were randomly divided into model groups(mechanical obstruction group,eosinophilic infiltration group,mechanical obstruction combined with eosinophilic infiltration group)and control group.The differences of esophageal motility between groups were assessed by the esophageal manometry and esophageal emptying test.Further more,histopathological examination(the count of eosinophil and expression levels of TGF-β 1,α-SMA,collagen Ⅰ and collagen Ⅲ)of esophageal samples collected from different groups were assessed by HE staining,Masson staining,immunohistochemical staining and Western Blot.Further study was performed using the mice of mechanical obstruction combined with eosinophilic infiltration model group which met the clinical and pathological changes of AC patients best.The esophageal smooth muscle strips were obtained from the mice in mechanical obstruction combined with eosinophilic infiltration model group and control group to measure isometric contractions with electric stimulation.(3)Five SD rats were used to separate esophageal smooth muscle cells and eosinophils for co-cultured in vitro.And set the following groups,control group,eosinophils combined treatment group A,B and C.MTT assay was used to evaluate the proliferation of esophageal smooth muscle cells at different time points of co-culture.Western blot was used to detect the protein levels of TGF-β1,α-SMA,collagen I,Ⅲ and the signal molecule Smad2/3 and phosphorylated Smad 2/3.(4)Thirty Blab/c mice were randomly divided into three groups,mechanical obstruction combined with eosinophilic infiltration group,anti-Siglec-F antibody group and control group.The differences of esophageal motility between the three groups were assessed by the esophageal manometry and esophageal emptying test.HE staining and immunohistochemical staining were also used to compare the degree of eosinophilic infiltration and expression levels of TGF-β1,α-SMA,collagen Ⅰ and collagen Ⅲ in esophageal smooth muscle.Results(1)Histopathological study: The results showed that eosinophils had infiltrated in the smooth muscle of LES in 17/40(42.5%) achalasia patients,which were much larger than control(P<0.05).In 32/40(80%)achalasia patients,fibrotic tissue extension to intersmooth muscle bundles were seen,which was more serious than the control group(P <0.05).Tissue specimens of achalasia patients showed that the shape of smooth muscle cell bundles were significantly thicker and disordered than those in the control group.Collagen fibers were observed in bundles and increased significantly in the tissue samples,in addition,myofibroblasts were observed in the cytoplasm of achalasia patients under transmission electron microscopy.Expression levels of remodeling factors(TGF-β 1,α-SMA,collagen Ⅰ and collagen Ⅲ)of esophageal smooth muscle in LES were increased compared with the control group(all P <0.05).Furthermore,after 2 years of POEM,it was found that the esophagus peristaltic function were not restored in all the achalasia patients,and the count of eosinophils and expression levels of remodeling factors was positively correlated with Eckard score postoperatively.Study on the thickness of esophageal smooth muscle: The maximum width of esophagus and the height of barium at different time points in achalasia patients were significantly higher than those in the control group(all P<0.05).After 2 years of POEM,the maximum width of esophagus was similar with that before treatment(P>0.05),while the barium height at 1,3,and 5 minutes was lower than before,which were similar with control group(all P> 0.05).Before POEM,the thickness of circular muscle of LES,5,10 and 15 cm above LES were thicker than those in the control group(all P<0.05)significantly,and the circular muscle at the LES was the thickest,while the thickness of longitudinal muscle in each part was similar with the control group.It was seen that the muscular layer was still thickened 2 years after POEM treatment,and there was no significant difference compared with before treatment(P> 0.05).(2)The weight of each model group was less than that of the control group(all P<0.05),and the LES pressure were higher and the emptying time of the esophagus body and cardia were longer significantly(all P<0.05)compared with the control group.Comparison between the model groups found that in the eosinophilic infiltration group and mechanical obstruction combined with eosinophil infiltration group,more eosinophils infiltration,higher expression levels of remodeling factors and more serious fibrosis were observed(all P<0.05).Moreover,the mechanical obstruction combined with eosinophilic infiltration group was selected for the next study.In addition,the expression levels of remodeling factors in the esophageal smooth muscle of achalasia model mice were significantly higher than those in the control group(all P<0.05).Furthermore,under certain parameters of electrical stimulation,the contraction of esophageal muscle strips in the model group was lower than that in the control group(P<0.05).(3)The survival rate of esophageal smooth muscle cells of eosinophil combined treatment group B was higher than that of other groups at different time points(all P<0.05).After 48 hours of co-cultivation,the expressions of remodeling factor proteins in eosinophil combined treatment group B were detected.The amount was significantly higher than the other groups(all P<0.05),after co-culture for 48 h,the expression level of phosphorylated Smad2/3 protein in eosinophil combined treatment group B was much more higher than that in the other groups(all P<0.05).(4)After anti Sigec-F antibody intervention,the results of esophageal dynamics of the mice were better than those of the model group.And the pathological indicators were not completely improved(P>0.05),but the count of eosinophilic infiltration and expression of some remodeling factors were significantly lower than that in the model group(all P<0.05),and there was no significant difference between anti Sigec-F antibody group and control group(P>0.05).Conclusion The infiltration of eosinophils and expression levels of remodeling factors in esophageal smooth muscle of achalasia patients is related to prognosis.Mechanical obstruction combined with eosinophil infiltration animal models could mimic the pathology of achalasia patients.Eosinophils may induce the esophageal tissue remodeling through TGF-β / Smad dependent signaling,and further lead to esophageal dysmotility,which promotes the development of achalasia.In addition,eosinophils maybe a new treatment target of achalasia accompanied by esophageal tissue remodeling. |
PDF Full Text Request