The Study Of Mongersen’s Anti-inflammatory Effect In Periodontal Inflammatory Microenvironment In Vitro

Background:Periodontitis is a chronic inflammatory disease whose characteristics are periodontal connective tissue destruction and alveolar bone resorption.Its incidence rate is over 80%,and it can not only affect dental function,but also be the risk factor for diabetes,atherosclerosis,adverse pregnancy,rheumatoid arthritis,cancer,eye disease and liver disease,which do harm to overall health.As an important part of the innate immune system of tissues,macrophages are one of the cells that produce the initial immune response to invading bacteria and their products.On the one hand,they play a defensive role to maintain the stability of periodontal tissues.On the other hand,they can cause periodontal tissue destruction and alveolar bone resorption by releasing various pro-inflammatory factors.Smad7 is a downstream signal transduction protein of the TGF-βfamily and widely expressed in immune cells such as macrophages.By directly activating the IKK-β promoter in macrophages,Smad7 promotes inflammation by releasing a large number of inflammatory factors such as TNF-a,IL-1β and IL-6.At the same time,IKK-1β also promotes the expression of Smad7 protein,thus forming a positive feedback pathway,which further aggravates the immune inflammation response of periodontial tissues.Mongersen,as a new antisense oligonucleotide of Smad7,can inhibit inflammation by inhibiting the expression of Smad7 in macrophages.Therefore,it is speculated that Smad7 could play a key role in periodontal tissue destruction,and mongersen could regulate its downstream signaling pathway by inhibiting the expression of Smad7 protein and related pro-inflammatory cytokines,thus achieving the goal of treating periodontitis.Therefore,this study will open up a new way for the treatment of periodontitis.Methods:1.The effect of Pg-LPS on expression of Smad7 in macrophagesRAW264.7 cells were stimulated by Pg-LPS at different concentrations(1μg/mL,5μg/mL,10μg/mL)at different time(3h,6h,12h,24h)in its logarithmic growth period.Total mRNA of Smad7 was extracted from cell samples and detected by real-time quantitative PCR,then we detected the protein expression of Smad7 when gene expression is high.2.Mongersen inhibited the expression of Smad7 protein in macrophages(1)Mongersen with Cy-5 label was transfected into RAW264.7 by Lipo2000,and the optimal transfection ratio was determined by flow cytometry(FCM).(2)Total protein of Smad7 was extracted from cell samples and protein secretion of Smad7 was detected by Western Blot.3.The effect of Mongersen on inflammatory factors in macrophages(1)After the transfection of mongersen,the optimal concentration of Pg-LPS was used to stimulate RAW264.7.Total RNA was extracted from cell samples after 3,6,12 and 24 hours,and the expression levels of inflammatory factors,IL-1β,IL-6,TNF-a,iNOS,IL-10 and TGF-β1were detected by real-time quantitative PCR..(2)Method is the same as above.The cell culture supernatant was collected after 3,6,12 and 24 hours,and the protein secretion of the above inflammatory factors was determined by enzyme linked immunosorbent assay(ELISA).Results:1.After RAW264.7 cells line and different concentrations of Pg-LPS(1μg/mL,5μg/mL,10μg/mL)co-culturing for 3h,6h,12h and 24h,the mRNA expression of Smad7 was significantly increased when the concentration of Pg-LPS was 5μg/mL at 12h.There was no significant increase in the mRNA expression level of Smad7 under other conditions.Moreover,the protein expression of Smad7 was also significantly increased compared with the control group when the concentration of Pg-LPS was 5μg/mL at 12h.2.After transfecting mongersen into RAW264.7,the transfection efficiency could reach to 76.57%by flow cytometry.Western Blot showed that the expression of Smad7 protein in experimental group could be significantly reduced.It was confirmed that Lipo2000-loaded mongersen had a good transfection efficiency in RAW264.7 and could significantly inhibit the expression of Smad7 protein.3.After transfecting RAW264.7 with mongersen,the mRNA expression and protein secretion of IL-6 in the experimental group was significantly decreased at different time points when Pg-LPS and RAW 264.7 cells were co-cultured at different time.The mRNA expression and protein secretion of IL-1β,TNF-a and iNOS decreased only at 6 hours or 12 hours.The mRNA expression of anti-in:flammatory cytokine TGF-β1 decreased significantly at 6h and 12h,but the protein secretion did not change significantly.There was no significant change in mRNA expression and protein secretion of IL-10.Conclusions:1.Pg-LPS can increase gene and protein expression of Smad7 when stimulating macrophages.2.Mongersen could significantly inhibit the expression of Smad7 protein and pro-inflammatory factors such as IL-1β,IL-6 and TNF-a.