| Background and purposeSjogren’s syndrome,SS is a systemic autoimmune disease with exocrine gland damage and multiple organ system involvement.In recent years,the incidence of SS has increased significantly.The prevalence rate of Sjogren’s syndrome in Chinese population is 0.33%-0.77%,second only to rheumatoid arthritis.90%of the patients are female,and the peak age of onset is about 50 years old.The most common symptom in patients with SS is keratoconjunctivitis sicca and xerostomia,which is closely related to the functional damage of salivary and lacrimal glands,but the mechanism of injury is not completely clear.Clinically,the pathological diagnosis of labial gland biopsy in patients with SS plays an important role.However,the pathological diagnostic criteria of labial gland biopsy can not fully reflect the degree of gland injury,and lack of judgment on the degree of lesion,stage and prognosis of patients.It is necessary to explore a practical,effective and simple pathological diagnosis that can reflect the degree of injury of acinar secretory function,so as to assist clinical rheumatologists to make a clear diagnosis and predict the prognosis of patients.Therefore,on the basis of the accumulation of previous studies in our group,by observing the sections of labial glands and trying to use Alcian blue staining,that is,AB staining,a histochemical staining method showing mucus secretion,and using E-Cad IHC staining to show the integrity of cell membrane,combined with E-Cad+AB staining,to describe and analyze the status of inflammatory epithelial injury of labial glands.Then the artificial intelligence computer AI technology was applied to quantitatively analyze the mucus secretion area of the labial gland lesions after doublestaining by the method of full-section digital scanning image analysis.Pyroptosis is a new way of cell death.It is also an inflammatory programmed cell death.It mainly depends on the membrane pores formed by members of the Gasdermin protein family,which is usually completed by the activation of inflammatory caspase-1.Cell pyrolysis is closely related to innate immune response.Under the stimulation of a variety of pathogen-related molecular models(PAMPs)and damage-associated molecular patterns(DAMPs),cell pyrogenesis can occur in myeloid cells such as macrophages,dendritic cells and other cells,which further leads to inflammatory response.In our routine clinicopathological observation,we found that the acinar epithelium of labial glands in patients with SS atrophied,decreased and disappeared,and we speculated that the disappearance of cells may be reduced by cell death,so we selected three kinds of scorch related proteins NLRP3,Caspase-1 and GSDMD to detect the expression of labial glands in patients with SS by IHC method,and analyzed the correlation with clinical data and autoantibodies.The purpose of this study is to explore whether there is a mechanism of cell pyrogenesis in the decrease of glandular mucus secretion.Materials and methods1.Samples:the labial gland biopsy samples and corresponding clinical data of 100 patients with suspected Sj?gren’s syndrome who were anti-SSA or SSA/Ro positive in Shantou Central Hospital from 2013 to 2021 and 10 normal labial gland controls were selected.2.H&E staining:The morphological changes of tissue specimens were observed under light microscope.According to the current classification standard of ACR-EULAR primary Sjogren’s syndrome in 2016,the samples were classified into pathological types.More than one lymphocyte infiltration focus in each 4mm2 was diagnosed as positive and divided into FS≥1 group(60 cases,mainly including FLS and FSLS)and FS<1 group(40 cases,mainly NSCS and SCS).3.E-cad+AB staining:The labial acinar epithelial cell membrane was labeled by E-cadherin IHC staining,and then the mucus in the labial acinar epithelium was stained with AB mucus staining to observe the epithelial secretion and mucus overflow after epithelial injury.4.AI image analysis:use 3DHISTECH scanner to scan 100pieces of dye sections 40 times,export digital slices to group archive,label tissue area by ASAP software,save the result as XML file,and use Multiresolutionimageinterface to analyze ASAP label file.Use Openslide to read the slice file and generate the tissue mask.Finally,the inRange method of Opencv was used for image processing,and the percentage of mucus secretion area of each slice to the area of labial gland tissue was calculated.5.Immunohistochemical staining:Labial glands were stained with NLRP3,Caspase-1 and GSDMD antibodies by Envision two-step method.We used QuantaCenter image analysis system to evaluate and analyze the expression of pyroptosis-related proteins in inflammatory epithelium of labial glands.6.Statistical analysis:SPSS(statistic25.0)was used for data analysis,t-test,one-way ANOVA,nonparametric rank sum test and Pearson correlation analysis were used.Result1.E-Cad+AB overlay staining to observe the injury of acinar epithelium(1)E-Cad staining of normal mucous glands showed continuous and complete cell membranes with sharp and uniform linear structures,AB staining of mucous gland epithelial cytoplasm in a more uniform bright blue color,and tiny blue secretory vesicles were visible in the glandular vesicles.(2)E-Cad+AB staining in inflammatory damaged lacrimal glands:blue mucus showed that the glandular epithelial cells first appeared mildly swollen,with large and small vacuoles in the lumen;then the internal cytosolic membrane of the gland was partially or completely broken,and the mucus fused into a luminal mucus lake,and if the basal part of the mucous glandular epithelial cytosolic membrane ruptured,the mucus overflowed to form an extra-luminal mucus lake;in the final stage of the injury,the glandular epithelial cells atrophied and disappeared,and mucus secretion was absent.E-Cad positive tan markers show the linear structure of the cell membrane due to cell swelling by pressure thinning,deformation to the linear structure rough,delamination,interruption,mutilation or even disappearance buried in the mucus lake.2.AI automatic analysis of mucus volume(%)results:(1)the whole labial gland sample mucus volume(%)was obtained by AI automatic analysis.Statistical analysis showed that the mucus volume(%)of FS<1 group was significantly higher than that of FS≥1 group.It is suggested that the labial gland without focal lymphocyte infiltration may be in the early stage of inflammatory injury,and the mucus secretion can be maintained at a high level.It is suggested that the epithelium before focal cell aggregation may be mainly humoral immune injury.However,the existing analysis methods can not show the morphological quantitative index of acinar epithelial injury,which needs to be further studied.(2)Combined with the clinical serological data,it was found that there was a negative correlation between the amount of mucus(%)and the titer of anti-SSB antibody(r=0.216～0.035),that is,the higher the titer of anti-SSB antibody,the less mucus(%)secreted by the labial gland.3.The score of glandular injury was observed by E-Cad+AB staining:(1)after observing and summarizing the injury morphology of labial gland after E-Cad+AB staining,the edema of glandular epithelial cells was 1,the formation of large and small vacuoles was 2,the rupture of cell membrane was 3,the atrophy of glandular epithelium and the loss of secretion were 4,which were described in part 2.2.3 of the text.(2)according to the injury score,the labial glands were divided into four groups:[1-2),[2-3),[3-4)and 4″.The[1-2)scores were mainly edema and a small number of intracavitary vacuoles.The[2-3)scores were mainly caused by the formation of large and small vacuoles in the cavity and a small amount of mucous lakes,and[3-4)were mainly caused by rupture of the epithelial membrane of mucous glands.It can be accompanied by a small amount of acinar atrophy and loss of secretion,in which the formation of the mucus lake in the cavity indicates the rupture of the cell membrane on the cavity surface and side of the mucous gland epithelial cell,while the extra-cavity mucus lake represents the rupture of the cell membrane on the basal surface of the mucous gland epithelial cell.4 points represent almost complete destruction of glands and loss of mucus secretion.Thus it can be seen that the higher the injury score is,the higher the injury degree of the corresponding mucous gland is.(3)according to the statistics,the amount of mucus in the injury score[3-4)group was significantly lower than that in the injury score[2-3)group(P<0.05),suggesting that the injury mainly caused by the formation of mucus lake has belonged to a more serious injury stage.that is,the mucus secretion function of labial gland which is mainly caused by rupture of glandular epithelial cell membrane has been significantly decreased.The mucus volume of labial gland with injury score of 4 was significantly lower than that of the other three groups(P<0.05),which suggested that the labial gland of this group had severe mucus glandular epithelial atrophy,decrease,loss of secretion and final destruction.(4)combined with the clinical data,it was found that there was a significant positive correlation between the injury degree score and the serum anti-SSA antibody titer(P<0.05),that is,the higher the anti-SSA antibody titer,the higher the labial gland injury score of the corresponding cases,suggesting that anti-SSA antibody is also involved in the damage mechanism of glandular epithelial cell membrane rupture and final secretion loss.4.The results of expression analysis of cell death-related proteins:(1)NLRP3,Caspase-1 and GSDMD were localized in the cytoplasm of epithelial cells.There was no positive expression of NLRP3 protein in the cytoplasm of mucous gland in normal labial gland tissue,while a little granular positive expression could be seen in the cytoplasm of mucous gland in Caspase-1 and GSDMD.(2)The expression of NLRP3,Caspase-1 and GSDMD were all positive in the labial glands with inflammatory injury,and the positive expression was the strongest in the glandular epithelial cells with glandular atrophy and loss of secretion.Statistical analysis showed that there was a significant positive correlation among the expression scores of NLRP3,Caspase-1 and GSDMD.This suggests that the classic scorch death pathway of NLRP3 inflammatory bodies-Caspase-1-GSDMD may be involved in the loss of epithelial cells after inflammatory injury of SS acinar epithelium.(3)According to statistical analysis,the IHC expression score of NLRP3 and Caspase-1 in the group with injury score>2 was significantly higher than that in the group with injury score ≤2,which suggested that the expression of NLRP3 and Caspase-1 in labial glands with rupture of glandular epithelium,glandular atrophy and loss of secretion was significantly higher than that in labial glands with edema and intracavitary vacuoles.(4)There was a significant positive correlation between the expression of NLRP3,Caspase-1 and GSDMD and the score of injury degree,and the correlation coefficients were 0.327,0.288 and 0.243,respectively,suggesting that no matter whether there was lymphocyte infiltration in the labial gland or not.The higher the degree of injury,the higher the positive expression of scorch death-related proteins in labial glands.Conclusion1.E-Cad+AB overstaining method was used to evaluate epithelial injury and mucus overflow for the first time.This method can be used to evaluate the inflammatory injury of labial gland epithelium.2.The automatic detection of mucus secretion by AI is helpful to comprehensively and accurately evaluate the degree of mucus secretion of glands.3.The expressions of NLRP3,Caspase-1 and GSDMD were significantly increased in acinar epithelium with decreased secretion and non-secretion,suggesting that there is a mechanism of cell death in the occurrence and development of labial gland injury in Sj?gren’s syndrome. |
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