MANF Inhibits Sjogren’s Syndrome Submandibular Gland Epithelial Cell Apoptosis And Antigen Expression And Presentation Of SSA Through Endoplasmic Reticulum Stress/Autophagy Pathway

Primary Sjogren’s syndrome is an autoimmune disease characterized by lymphocyte infiltration and destruction of the salivary and lacrimal glands and the production of systemic Ro/SSA and La/SSB autoantibodies.Clinical manifestations caused by lymphocyte infiltration may involve other organs（such as kidney,lung,liver or thyroid）.SS is more common in women than men by a ratio of about 9 to 1.The pathogenesis of autoimmune diseases is thought to be multifactorial:genetic,hormonal,and environmental factors seem to play a role in their development.Studies have shown that in patients with SS disease,salivary gland epithelial cells endoplasmic reticulum expansion and function change,leading to chronic fatigue,life quality serious decline,it could be the result of endoplasmic reticulum dysfunction,the function disorder of endoplasmic reticulum（ER）is due to not folded and/or the misfolded proteins accumulation of the ER,protein synthesis imbalance,thus（ER stress,ERS）processes the correct folding of newly synthesized proteins.The ERS induced cellular stress response,known as unfolded protein reaction（UPR）,also induces autophagy.This network is initially designed to restore the protein homeostasis,but if prolonged or severe will eventually trigger apoptosis.Autophagy is a lysosomal dependent degradation pathway occurring in eukaryotic cells,which is the balance and survival mechanism of cells under harmful stress,Excessive autophagy may lead to abnormal function or even death in some cases,and ERS induced autophagy can promote cell survival.Studies have shown that the activated PI3K/AKT/mTOR pathway is related to various autoimmune diseases,and the AKT/mTOR pathway is involved in the pathogenesis of p SS,inhibits the phosphorylation of AKT and mTOR,increases autophagy and inhibits apoptosis.Mesencephalic astrocyte-derived neurotrophic factor（MANF）is a newly discovered small soluble ER resident protein,was secreted during ERS and can promote the survival of neurons and other target cells.MANF can also inhibit inflammatory responses by regulating inflammatory related pathways.In vitro cell culture studies have shown that MANF has an inhibitory effect on ERS induced cell death.In order to balance the harmful effects of ERS,the body activates UPR to stimulate the secretion of MANF to reduce the accumulation of misfolded proteins and restore the normal function of ER.Although some studies have shown that MANF can protect cells from ERS,the specific mechanism of MANF protecting cells is not clear,but the effect of MANF on autophagy and AKT/mTOR/LC3B signaling pathway in the development of Sjogren’s syndrome is not clear.In this study,we focused on the effect of MANF on HSGECs and ESS mice and its potential mechanism.We found that MANF can activate autophagy by inhibit the AKT/mTOR/LC3B signaling pathway,inhibit apoptosis and reduce the expression of SSA,so we speculated that MANF can affect AKT/mTOR/LC3B signaling pathway regulates HSGECs and alleviates disease progression of SS.OBJECTIVE:1.Observe the abnormal expression of ERS and AKT/mTOR/LC3B autophagy signaling pathway and autoantigen SSA during the pathogenesis of SS2.ERS can reduce autophagy by activating AKT/mTOR/LC3B pathway,promote SGEC apoptosis,and increase the expression and presentation of autoantigen SSA3.Clear that MANF promotes autophagy by regulating ERS and AKT/mTOR/LC3B signaling pathway,inhibits SGECs apoptosis,and reduces the expression and presentation of autoantigen SSAMETHODS:1.Collect samples of human lip gland tissue,immunohistochemistry was used to detect the expression of MANF and AKT/mTOR/LC3B signaling pathway proteins and ERS proteins GRP78、XBP1S、CHOP in human lip gland tissues.2.ESS mice induced by collagen protein were established and divided into Normal、Model、MANF and Pre group.The body condition of the mice was observed during the modeling process,and the body weight and saliva volume were recorded once a week.H＆E staining was used to observe the pathological changes of submandibular gland.Collect spleen、thymus and salivary glands to calculate organ index.Flow cytometry was used to determine the proportion of T cell subsets in salivary glands.Western blot and immunofluorescence were used to detect the protein expressions of MANF and AKT/mTOR/LC3B signaling pathway,as well as the expressions of GRP78、XBP1S、CHOP and Beclin-1 in submandibular gland of mice.3.Using IFN-αstimulation of HSGECs,divided into Control,IFN-α,MANF,and Rapa groups,Western blot and immunofluorescence was used to detect the expression of MANF and AKT/mTOR/LC3B signaling pathway proteins and ERS proteins GRP78、XBP1S、CHOP and autophagy protein Beclin-1.CO-IP was used to detect the co-expression of major histocompatibility complex II（MHCII）and SSA,immunofluorescence was used to observe the co-localization of MHCII and SSA,flow cytometry was used to detect apoptosis and MHCII expression of HSGECs.RESULTS:1.Compared with normal people,there was severe lymphocyte infiltration in the lip gland tissue of SS patients,ductal dilation and congestion,atrophy of acinar,and the levels of MANF and ERS proteins GRRP78,XBP1S,CHOP and AKT and mTOR phosphorylation in the lip gland tissues of SS patients were increased,and the expression of autophagic protein LC3B was decreased.2.In mice,compared with the normal group,the saliva secretion of the model group mice decreased,lymphocytes were infiltration in the submandibular gland tissue,local small blood vessels were dilated and congested,and the submandibular gland index and spleen index were increased;compared with the model group,the saliva flow of the mice in the MANF and Pre groups increased,and the degree of lymphocyte infiltration decreased,in the MANF group,the spleen index,thymus index and salivary gland index decreased,while the salivary gland index decreased in the Pre group;compared with the normal group,The model group MANF and ERS proteins GRRP78,XBP1S,CHOP and p-AKT,p-mTOR pathway proteins increased,the expression of LC3B and Beclin-1 decreased,and the proportion of CD4⁺T cell subsets increased;compared with the model group,MANF and Pre could reduce MANF and ERS proteins GRP78,XBP1S,CHOP and reduce the levels of AKT and mTOR phosphorylation in the submandibular gland tissues of ESS mice.Increased expression of autophagic proteins LC3B and Beclin-1,reduced proportion of CD4⁺T cell subsets.3.In HSGECs,compared with the Control group,the expression of MANF and ERS proteins GRRP78,XBP1S and CHOP in the IFN-αgroup increased,the phosphorylation level of AKT/mTOR pathway proteins increased,the expression of autophagic proteins LC3B and Beclin-1 decreased,and the expression of autoantigen SSA and MHCII on the membrane increased;compared with the IFN-αgroup,MANF and Rapa could reduce IFN-αafter induction The expression of MANF and ERS proteins GRP78,XBP1S and CHOP decreased the phosphorylation level of AKT and mTOR,increased the expression of autophagic proteins LC3B and Beclin-1,and reduced the expression of autoantigen SSA and MHCII on the membrane.CONCLUSIONS:1.ERS protein and AKT/mTOR/LC3B signaling pathway and autoantigen SSA protein are abnormally expressed in the lip gland tissue of patients with SS;2.ERS reduces autophagy by activating AKT/mTOR/LC3B pathway,promotes SGEC apoptosis,and increases the expression and presentation of SSA;3.MANF promotes autophagy by regulating ERS and AKT/mTOR/LC3B signaling pathway,inhibits SGEC apoptosis,and reduces the expression and presentation of autoantigen.SSA.