Effects Of Sodium Butyrate On The Autophagy Of RAW264.7 Cells And Osteoclast Differentiation

The gut microbiota plays a key role in hosts' gastrointestinal tract,immune and nervous systems,and can even regulate BMD by affecting the immune system.The intestinal flora in the cecum and large intestine produces SCFAs by fermenting dietary fibers that can not be digested in the small intestine,and participate in the regulation of human metabolism,with the widest SB researches.In addition to providing energy to colonic epithelial cells,SB is the first discovered histone deacetylase inhibitor that controls gene expression and transcriptional regulation.It regulates energy intake and utilization and controls body weight.They also plays an important role in cancer by affecting the proliferation and apoptosis of tumor cells.Osteoporosis is common in the hip,femur and lumbar spine and is inseparable from bone remodeling.It is one of the main causes of disability and death in the elderly.Once the balance of bone remodeling in osteoporosis is broken,the formation and function of osteoclasts is increased,and bone resorption is greater than bone formation.There is a complex and close relationship between intestinal flora and bone health.SB affects bone remodeling due to its action as a histone deacetylase inhibitor,which increases bone sialoprotein and osteoprotegerin levels to stimulate bone formation.SBcan also increase the expression of osteopontin in osteoblasts to inhibit the differentiation of osteoclasts in early time.Macrophage is a kind of very important immune cell,which is widely present in various tissues and organs of the human body.Osteoclasts are differentiated from blood mononuclear / macrophages.RAW264.7 cell is derived from mouse mononuclear / macrophage cell strain widely used in macrophage research.This experiment investigated the effects of different concentrations of SB on autophagy and osteoclast differentiation in RAW264.7 cells,CCK-8 kit was used to detect the effect of SB on the activity of RAW264.7 cells,Hoechst33342 kit was used to detect the effect of SB on the nucleus of RAW264.7 cells.The effect of SB on ROS and ATP level in RAW264.7 cells was detected too.Cyto-ID autophagy detection kit was used to detect the effect of SB on autophagosome production.Western blotting was used to detect related proteins' expression.Fluorescence microplate reader,fluorescence microscope,PCR instrument and flow cytometry were used to test the experimental results.The results showed that SB dose-dependently affected cell viability,cell activity was significantly reduced at 1 m M,and the nucleus showed obvious fragmentation.The intracellular ROS increased with increasing SB concentration.While ATP level showed a downward trend with the increase of SB concentration.Western blotting showed that 1 m M SB reduced phosphorylation of the rapamycin target protein(m TOR),activated the autophagyassociated protein Unc-51-like kinase 1(ULK1)and Beclin-1,and reduced the phosphorylation of DRP1.With the increase of SB treatment concentrations,the acetylation level of histone H3(Histone H3)increased,the content of p65 protein in the nucleus decreased,the Bcl-2 like protein 4(Bax)and cleaved-caspase3 are increased.SB's researches on macrophages were mainly focused on the inhibition of inflammation.Our experiment proved that 1 m M SB can reduce the expression of interleukin-6 and interleukin-1? m RNA level,indicating that SB may inhibit the activation of intestinal macrophages.For bloodline macrophages,SB may inhibit the release of growth factors which promote osteoclast differentiation and reduce osteoclastogenesis.This paper uses RANKL to induce the fusion of RAW264.7 cells to differentiate into mature osteoclasts,studying the effect of 1 m M SB on the differentiation and function of osteoclasts and changes in the skeleton.The induced osteoclasts were stained with TRAP and fluorescent staining of the cytoskeleton,analyzing the bone resorption.The results showed that 1 m M SB significantly reduced the number of osteoclasts and scars formed by bone resorption,the cytoskeleton is altered and the F-actin loop is destroyed.RANKL induces osteoclast differentiation by activating the NF-?B signaling pathway.We hypothesized that SB may reduce the number of mature osteoclasts by inhibiting the NF-?B signaling pathway,thereby reducing bone resorption.Inhibition of NF-?B signaling by SB also caused apoptosis of cells,and the apoptosis of precursor cell lines was also responsible for the decrease of osteoclasts.In addition,the increase of SB-induced autophagy did not play a positive role in the differentiation of osteoclasts.The effect of autophagy on osteoclasts remains to be further studied.Studies in this paper have shown that SB can affect macrophages and mature osteoclasts.Intestinal flora can not only affect bone mass by affecting the intestinal immune system,but also use metabolites to affect bone cells.The close relationship between intestinal flora and bone tissue provides new ideas for the prevention and treatment of osteoporosis.