Effect Of Bisphosphontes On Macrophagic Differentiation

[Objective]Bisphosphonates(BPs)is a kind of bone resorption inhibitor,have been widely used in clinic for the treatment of various diseases,such as osteoporosis,multiple myeloma,Paget's disease,malignant bone metastasis etc.Bisphosphonate-related osteonecrosis of the jaw(BRONJ)is a serious complication by long-term use of bisphosphonates.Since 2003,a number of studies have sought to explore the pathogenesis,but so far the pathogenesis of BRONJ has not been fully understood.Recently,local immune dysfunction has been concerned as a mechanism of BRONJ.Macrophage is an important component of innate immunity and adaptive immunity.As the precursor of osteoclasts,macrophages are the major target cells of BPs.Macrophages have a strong plasticity,and can be polarized into classical activated macrophages(M1)and selective activation of macrophages(M2),Ml macrophage polarization induced by LPS and TNF-?,play a role in tissue destructive inflammation.M2 macrophage induced by IL-4 and IL-13 is anti-inflammatory,and can to promote tissue reeonstruction and wound healing.BPs can repolarize M2 macrophages to M1 type,and change the balance of macrophage polarization.KLF4 plays a key role in the proeess of macrophage polarization.KLF4 is a nuclear transcription factor containing zinc finger structure,which is involved in cell proliferation,growth,differentiation and other physiological activities.KLF4 could promote the polarization of M2 macrophages,and inhibit the polarization of M1 macrophages.However,we don't know that whether the imbalance of macrophage polarization is related to the change of KLF4 expression in BRONJ.In this paper,we would like to research the effect of BPs on the differentiation observe whether the expression of KLF4 in the tissues of patients with BRONJ has been changed,and whether the expression of KLF4 in the macrophages of BPs can be changed by in vitro experiments.[Methods]The expression of KLF4 in gingiva is detected by immunohistochemistry in 5 patients with BRONJ and 3 patients with periodontitis and 2 patients with normal gingiva.CCK8 was used to test the cell toxicity of zoledronic acid(ZOL)in RAW264.7.ELISA was used to detect IL-6 expression level.Flow cytometry was used to detect CD86 and CD206.Real-time quantitative PCR was used to detect TNF-?,IL-10 mRNA levels and KLF4 mRNA level in each group.WB was used to detect the expression of KLF4.[Results]The expression of KLF4 in gingival tissues of patients with BRONJ was significantly lower than that of normal gingival and periodontitis inflammatory gingival.The concentration of ZOL was less than or equal to 2.5?g/ml,which had no toxic effect on RAW264.7 cells.When ZOL was used alone in RAW264.7 cells,the level of IL-6 detected by ELISA was not significantly different from the control.M1 macrophage specific marker gene TNF-? mRNA detected by QPCR had no significant difference from the control in statistics.The level of CD86 detected by flow cytometry was not significantly different from the control.M2 macrophage specific marker gene expression of IL-10 mRNA had no difference from the control in statistics too.The level of CD206 detected by flow cytometry was not significantly different from the control.In cells induced by LPS alone,the expression of IL-6,TNF-?,CD86 was significantly higher,suggesting that macrophage undergo M1 activation stimulated by LPS.In cells induced by 11-4,the increased expression of IL-10 mRNA had significant difference from the control,suggesting that macrophage undergo M2 activation stimulated by IL-4.The level of IL-6,TNF-?,CD86 detected in disposed by ZOL and LPS was significantly higher when compared with LPS alone.In RAW264.7 cells exposed to ZOL and LPS,the expression of TNF-? was increased compared to LPS induced alone.In RAW264.7 cells exposed to ZOL and IL-4,the expression of IL-10 mRNA was significantly lower than that of IL-4 alone.In RAW264.7 cells exposed to ZOL alone,the expression of KLF4 mRNA compared with the control group had statistically significant,while LPS alone in the cell,the reduced expression levels of KLF4 mRNA and the control group had statistically significant.The results showed that exposed to LPS alone,RAW264.7 polarized to Ml macrophages and KLF4 reduced expression.Compared with the control group,the expression of KLF4 mRNA was significantly increased in RAW264.7 cells when exposed to IL-4 alone,which indicated that the expression of KLF4 increased in IL4 induced M2 macrophages.WB analysis showed that exposed to LPS alone,the reduced expression of KLF4 and the control had statistically significant and exposed to IL-4 alone,the expression of KLF4 was significantly increased when compared with the control.[Conclusion]After stimulation with ZOL alone in RAW264.7 cells,macrophages may not undergo any activation.The polarization of M1 macrophages stimulated by LPS enhanced,and the polarization of M2 macrophages stimulated by IL-4 inhibited when pretreated with ZOL.In vitro,the expression of KLF4 in RAW264.7 stimulated by ZOL alone increased,which is in conflict with BRONJ patients with low expression in gingival.Therefore,we can be inferred that KLF4 in macrophages is related to BRONJ.