Establishment And Validation Of Gnaq^-/-Chimeric Mice As A Spontaneous Animal Model For Primary Sj?gren’s Syndrome（pSS）and To Explore The Difference Of Deposition Of IgG And C3 In The Labial Biopsy From PSS Patients

Objective:First,to study whether Gnaq-/-chimeric mice can be used as a spontaneous animal model for pSS study,Second,to explore the levels of complement-related components,such as IgG,C3,and MAC(C5b9),in the submandibular gland tissues during pSS pathogenesis in this model.Methods:The Gnaq-/-chimeric mice spontaneous pSS model and Wild Type(WT)chimeric mice control groups were constructed.The transplantation chimerism in the Gnaq-/-chimeric mice was identified by comparing the salivary flow rates(ul/g × min),antinuclear antibody titers,and the concentration of anti-SSA/Ro60(IU/ml)in the serum from the Gnaq-/-and WT chimeric mice at months 0,3,6,and 9.The H&E staining,immunohistochemical staining of CD3,B220,and CD21,and the immunofluorescence staining of IgG,C3,and C5b-9 in the submandibular tissue sections for the Gnaq-/-and WT chimeric mice euthanized at each phase were compared.Mann-Whitney U was used to analyze the statistical differences.Generalized Estimation Equation was used for the repeated measurements.The model rate was then statistically analyzed to estimate the normal range of the cut-off value of the indicators in the mouse models above.p-value of less than 0.05 was considered statistically significant.Results:The decreased saliva flow rate,increased ANA titer,increased anti-SSA/Ro60 concentration,aggregated lymphoid foci,and the presence of CD3,B220(CD45R),and CD21 in the tissue sections of the Gnaq-/-chimerism mice suggested that the mice presented the common clinical manifestations of pSS.The pSS model had a formation rate of 77.7%at 9 months.At the same time,the immunofluorescence staining of frozen sections of the submandibular gland tissue in this model suggested that the immunofluorescence score of IgG,C3,and MAC(C5b9)increased as the mice aged.Conclusion:The Gnaq-/-chimeric mice present the common clinical manifestations of Primary Sj?gren’s syndrome and can be used as the disease model.The complement-related components,such as IgG,C3,and MAC(C5b-9),are deposited on the submandibular gland tissues during the pSS pathogenesis in this model.Objective:To explore the difference in the complement components in the labial biopsy from pSS patients.Immunofluorescence staining was used to investigate whether there was a difference in the complement pathological markers,IgG and C3,between the frozen sections of the pSS and non-pSS labial gland biopsy samples,according to the 2016 pSS classification criteria.Also,the potential difference in the level of IgG and C3 in the labial gland biopsy between the pSS patients without lymphoid foci and the non-pSS patients was investigated,according to the same criteria.In addition,the possible difference in the IgG and C3 levels in the labial gland biopsy between the pSS patients with lymphoid foci and those without lymphoid foci was examined using the same criteria.Methods:The study was designed as a single-center,prospective casecontrol study.From 2020 to 2021,labial gland biopsy was performed on the participants from the inpatient and outpatient departments of the First Affiliated Hospital of Xiamen University.The participants who met the 2016 pSS classification criteria were included in the pSS group.The participants who did not fulfill the 2016 pSS classification criteria were included in the control group.The study also excluded participants with other autoimmune diseases,malignant tumors,previous radiotherapy treatment,AIDS,hepatitis B or C virus infection,critical organ failure,severe infections,or other serious complications.The researchers established a patient registry and databases of patient response to questionnaires,follow-up patient information,and laboratory results.The labial gland biopsy samples were prepared into frozen sections.Hematoxylin-eosin staining and immunofluorescence staining for IgG and C3 were performed.The nonnormal distribution data were statistically analyzed for differences by Mann-Whitney U.The chi-square test was used to compare different pairs of groups.The Ridit analysis was used to compare the unidirectional ordered grade data between groups.p<0.05 was considered statistically significant.Result:From 2020 to 2021,133 participants were screened,108 eligible subjects were selected,and three cases of shedding were excluded.Among eligible subjects,52 were selected for the pSS group,and 56 patients were selected for the control group.In the pSS and control groups,the number of females was 39(75.00%)and 37(66.07%),respectively;the median age was 43.808 years(29,59.5)and 45.375 years(32,60.5),respectively.There were significant differences in the number of lymphoid foci,focus scores,IgG and C3 grade between the pSS,and the control groups(p<0.05);the difference was confirmed by the Ridit analysis.There were also significant differences in the number of lymphoid foci,focus scores,and IgG and C3 grades in the pSS with lymphoid foci group,pSS without lymphoid foci group,and the control group(p<0.05).The results were confirmed by the Ridit analysis.In addition,the Ridit subgroup analysis between the pSS with foci group and pSS without foci group demonstrated a significant difference in focus score(p<0.05),but no significant difference between the IgG and C3 grades.Moreover,the Ridit subgroup analysis between the pSS with foci group and control group showed a significant difference in the focus score and IgG and C3 grades(p<0.05).Lastly,the Ridit subgroup analysis between the pSS without foci group and the control group demonstrated a lack of statistical difference in the focus score,but the IgG and C3 grades in the two groups were significantly different(p<0.05).Conclusions:There was a significant difference in the level of complement pathological markers,IgG and C3,and between the pSS and non-pSS samples,according to the existing pSS classification criteria(p<0.05).Also,there was a significant difference in IgG and C3 levels between the pSS samples without lymphoid foci and the non-pSS samples(p<0.05).Thus,IgG and C3 grade may be used as potential pathological markers to distinguish the pSS group from the control group.There was no significant difference in IgG and C3 between the pSS with lymphoid foci and the pSS without lymphoid foci samples(p<0.05).Therefore,IgG and C3 grades may be used as pathological markers to improve the coverage of pSS pathological diagnosis.