Effect Of N-WASP Knockdown On Inflammatory Cytokines Expression In Human Gingival Fibroblasts And The Underneath Mechanism

ObjectivesWiskott-aldrich syndrome(WAS)is an X-linked disease characterized by eczema,thrombocytopenia,immunodeficiency and autoimmunity.The appearance of WAS is associated with various genes mutation in Wiskott-aldrich syndrome protein(WASP).N-WASP is a vital component of WASP family involved in actin cytoskeletal reorganization.N-WASP knockout caused immunodeficiency and chronic skin inflammation in mice,mainly as psoriasis.Psoriasis is a common chronic inflammatory multisystem disease,mainly manifested by the skin and joints,and its etiology is very complex,mainly related to changes of T lymphocyte-mediated immunity.Studies have shown that the onset of chronic periodontitis is also associated with changes of T lymphocyte-mediated immunityChronic periodontitis is a chronic inflammatory disease characterized by the destruction of supporting tissues of the teeth,which eventually leads to alveolar bone resorption and tooth loss.It is well known that the occurrence of periodontitis is caused by a complex and diverse microbial biofilm formed on the teeth.Both chronic periodontitis and psoriasis are chronic inflammatory diseases.Psoriasis has the same inflammatory process with periodontitis.N-WASP is associated with psoriasis,and psoriasis has the same risk factors and inflammatory processes as periodontitis.Therefore,we speculate that N-WASP may play a role in the pathogenesis of periodontitis,and there is no report on this aspect.In consequence,this present study aimed to evaluate the effect of N-WASP knockdown on the expression of inflammatory cytokines in human gingival fibroblasts(HGFs),as well as the underneath mechanisms.MethodsIn vivo:Seven-week-old mice were selected and divided into a negative control(NC)group and an experimental group in which N-WASP was knocked out using Crisper-Cas9.After the mice were sacrificed,the gingival tissue of the mice was selected.Gingival inflammatory condition of N-WASP knockout mice was evaluated by H&E staining.In vitro:Healthy gingival tissues of the impacted teeth were removed from healthy donors,and HGFs were obtained by enzymatic digestion.Passages 3-5 were used for the following experiments.HGFs were then transfected with 30nmol/L siRNA using lipofectamine 2000 to determine the transfection efficiency.The optimal knockdown sequence was obtained by quantitative real-time polymerase chain reaction(qRT-PCR).Then qRT-PCR was used to detect the expression of interleukin(IL)-6,IL-8,C-C motif ligand 2(CCL2),superoxide dismutase 2(SOD2)and prostaglandin endoperoxide synthase 2(PTGS2)in HGFs after N-WASP knockdown.Enzyme-linked immunosorbent assay(ELISA)was used to measure the secretion of IL-6,IL-8 and SOD2 in protein levels.Western blot(WB)was used to detect the activation of nuclear factor-?B(NF-?B)and mitogen-activated protein kinase(MAPK)after N-WASP knockdown.At last,qRT-PCR was used to verify the changes in the expression of inflammatory factors at the gene levels after 10?M c-Jun N-terminal kinase(JNK)inhibitor(SP60012),10?M extracellular signal-regulated kinase(ERK)inhibitor(U0126),10?M p38 inhibitor(SB203580)and 5?M p65 inhibitor(BAY 11-7082)were added separately.Results1.The result of in vivo experiments showed that gingival tissue in N-WASP knockout mice displayed histopathological signs of inflammation,such as significant lymphocytic infiltration,epithelial spike elongation,loss of acinar morphology and acinus were replaced by lymphocytes2.N-WASP knockdown upregulated the expression of inflammatory factors in HGFs.The result of qRT-PCR revealed that N-WASP knockdown upregulated the expression of SOD2(P<0.01),IL-6(P<0.001),IL-8(P<0.001),CCL2(P<0.001)and PTGS2(P<0.001)in gene levels compared with NC.As for ELISA result,the production of IL-6(P<0.01),IL-8(P<0.001)and CCL2(P<0.001)were promoted in protein levels after N-WASP knockdown.3.N-WASP knockdown upregulated the expression of inflammatory factors through NF-?B and MAPK signaling pathways.The result of WB showed that N-WASP knockdown significantly enhanced the expression of p65,and p38,JNK and ERK.The result of qRT-PCR showed that N-WASP knockdown enhanced the expression of IL-6,IL-8,CCL2,PTGS2 and SOD2 in mRNA levels(P<0.001),but SP60012,U0126,SB203580 and BAY11-7082 were significantly blocked the upregulation of IL-6,IL-8,CCL2,PTGS2 and SOD2 in mRNA levels after N-WASP knockdown(P<0.001).Conclusions1.The results of this study indicated that N-WASP may played a role in the pathogenesis of periodontitis,and its mechanism was that N-WASP knockdown promoted the expression of inflammatory factors in HGFs.2.N-WASP deficiency upregulated inflammatory cytokines expression via NF-?B and MAPK signaling pathways in HGFs.