Effect Of Hydrogen Sulfide On Apoptosis Of Osteoblasts Induced By Lipopolysaccharide

Objective:Chronic osteomyelitis is difficult to cure,which brings great pain and expensive economic burden to patients.The treatment of chronic osteomyelitis requires multiple operations and a long hospital stay,and many cases are difficult to completely cure,which will affect the quality of life of patients and economic development.Traditionally,Staphylococcus aureus was considered to be the main pathogenic bacteria in osteomyelitis.However,it can be found that the proportion of Gram-negative bacilli is continuous at present.Therefore,the specific mechanism of Gram-negative bacilli in the development of chronic osteomyelitis should be further studied to provide new ideas for targeted therapy.Enzymes that can metabolize organic sulfides are common in microorganisms.By released,hydrogen sulfide involves in many microorganisms physiological processes,such as antibiotic defense,signaling and energy metabolism.Similar to bacteria,there are many enzymes that metabolize hydrogen sulfide in human body.Hydrogen sulfide is considered to be the third endogenous signal gas after nitric oxide and carbon monoxide.However,the specific role of hydrogen sulfide in osteomyelitis infection is not clear.The first part of the study is to explore the release of hydrogen sulfide in vitro and in vivo of several common Gram-negative bacilli in chronic osteomyelitis,confirm the concentration of hydrogen sulfide released,and provide theoretical and experimental basis for the follow-up study.During osteomyelitis,bone loss is caused by decreased synthesis of bone matrix and increased apoptosis rate of osteoblasts.Extracellular bacterial components may reduce osteoblasts.The activation of cytoplasmic inflammatory body may cooperate with autocrine/paracrine death receptor ligand signal to induce cell death.Therefore,controlling the apoptotic pathway of infected bone cells may be an attractive new way to limit the injury of bone marrow inflammation.The main pathogen of Gram-negative bacteria is LPS.LPS promotes osteoblast apoptosis directly or indirectly.The increased expression of Caspase family proteins is an important sign of apoptosis.After the initiation of apoptosis,substrate division,chromatin concentration,DNA breakage and cell death were observed.In addition to the caspase family,which promotes apoptosis,the process of apoptosis is also affected by Bcl-2 family proteins.At present,the effective treatment for bacterial bone destruction is limited to antibiotic programs and surgical strategies to treat infectious bone diseases.Therefore,it is still the main goal to study the drugs that may restore the function of osteoblasts.In the second part,the effect of hydrogen sulfide produced by Gram-negative bacteria on osteoblasts apoptosis was studied in order to provide new ideas for new treatment.Apoptosis of cells is regulated by many kinds of signal pathways.In recent years,PI3K/AKT signaling pathway has been found to play an important role in the regulation of many physiological activities of cells,and AKT has been found to play an anti-apoptosis role in many cells.In addition,AKT pathway can also interact with other signaling pathways and play a role in inhibiting apoptosis.Downstream of AKT pathway involves multiple signal pathways.NF-?B signaling pathway is one of the important signaling pathways.Activated AKT can phosphorylate IKK,and then phosphorylate I?B linked to NF-?B.As a transcription factor,I?B can regulate a series of gene expression.The inhibition of AKT pathway can lead to the inhibition of NF-?B pathway,and then reduce the expression of survival factors in cells and promote apoptosis.Many experiments have found that hydrogen sulfide inhibits AKT phosphorylation.Therefore,the third part of the experiment is to verify whether hydrogen sulfide can promote the expression of apoptotic protein and accelerate the apoptosis of osteoblasts by inhibiting AKT phosphorylation and downstream NF-?B pathway.Methods:Three kinds of bacterial,Escherichia coli,Enterobacter cloacae and Klebsiella pneumoniae were cultured in NB medium in vitro.Three kinds of bacterial suspensions with 10~6CFU/mL concentration were prepared by plate counting method.The gradient concentration of NaHS was used as a standard to compare the amount of black precipitate produced by hydrogen sulfide on lead acetate test paper.The amount of hydrogen sulfide produced by the three kinds of bacteria in the medium was estimated by comparing the black precipitates produced on the test paper after 37?overnight in different bacterial solutions and NaHS groups.40 SD rats were randomly divided into control group and three bacterial experimental groups:Escherichia coli,Enterobacter cloacae and Klebsiella pneumoniae.After celiac anesthesia,tibia was drilled and injected with bacterial suspension or sterile NB medium according to groups.Three weeks after the operation,the patients were killed by intraperitoneal anesthesia.The tibia of rats were fixed and stained with HE method.The content of hydrogen sulfide in the tissues around the focus was measured.Two osteoblasts,MC3T3-E1 and hFOB,were cultured.MC3T3-E1 and hFOB were treated with gradient concentration of hydrogen sulfide donor without or with LPS.MTT assay was carried out after 2 days of culture.The effects of hydrogen sulfide treatment without or with LPS on cell proliferation were compared.The osteoblasts were divided into three groups:(1)control group;(2)LPS group and(3)LPS combined with hydrogen sulfide group.Flow cytometry,Hoechst staining and Western blot were used to observe the apoptosis of osteoblasts and the expression of apoptosis related proteins.After three groups of cells were treated,the osteoblast induction medium was replaced then ALP assay and qPCR were detected to observe the effect of different conditions on osteoblast osteogenic ability.Immunofluorescence and Western blot were carried out in the three groups to observe the expression of AKT/NF-?B pathway related protein in different conditions.After using AKT activator,flow cytometry,qPCR,ALP and alizarin red were used to detect the role of AKT pathway in the process of apoptosis and osteogenesis.Results:1.Gram negative bacteria release hydrogen sulfide in vitro.Three kinds of gram negative bacteria in this study:Escherichia coli,Enterobacter cloacae and Klebsiella pneumoniae produce hydrogen sulfide in NB medium,and the concentration of hydrogen sulfide is between 50-100?M.After HE staining of osteomyelitis tissue in rats,inflammatory cell infiltration and destruction of bone trabecular structure can be seen in the bone tissue infected by three kinds of bacteria under microscope,which indicates that the bacterial infection model is successful.The hydrogen sulfide assay results showed that the concentration of hydrogen sulfide in the local tissue of osteomyelitis increased significantly after infection with three kinds of bacteria.The results show that hydrogen sulfide can be produced in vivo and in vitro by E.coli,Enterobacter cloacae and Klebsiella pneumoniae.2.Hydrogen sulfide promoted the apoptosis of osteoblasts,and aggravated the inhibition of LPS on osteoblasts proliferation and osteogenic function.Two kinds of osteoblasts were treated with two hydrogen sulfide donors in different concentrations.Compared with the control group,the proliferation rate of the two kinds of osteoblasts was not significantly reduced in the range of 0-100?m hydrogen sulfide.However,when hydrogen sulfide and LPS act together,the proliferation rate in the two groups of cell lines decreased,and it was dose-dependent.It is suggested that hydrogen sulfide can aggravate the inhibitory effect of LPS on osteoblast proliferation.Compared with LPS group,the apoptosis rate decreased and the number of apoptotic cells increased in hydrogen sulfide and LPS group.All these results indicate that hydrogen sulfide can promote the apoptosis of osteoblasts.The results of qPCR and ALP showed that the expression of osteoblasts related genes and the amount of ALP production in hydrogen sulfide and LPS group were lower than those in LPS group.This suggests that hydrogen sulfide can enhance the inhibition of LPS on osteogenesis.After the application of FAS specific inhibitors,the apoptosis rate of osteoblasts and the expression of apoptosis related proteins decreased,indicating that hydrogen sulfide through FAS apoptosis pathway affects the apoptosis process of osteoblasts.3.Hydrogen sulfide affects the effect of LPS on osteoblasts by inhibiting AKT/NF-?B signaling pathway.Western blot and immunofluorescence showed that hydrogen sulfide inhibited the expression of pAKT and p65.After using pAKT activator,the expression of pAKT increased significantly.The apoptosis rate and osteogenic ability of osteoblasts are improved significantly.In addition,hydrogen sulfide inhibited the expression of COX-2,which may be related to the inhibition of hydrogen sulfide on osteogenesis.Conclusion:1.The gram-negative bacteria in chronic osteomyelitis produce hydrogen sulfide in vitro and in vivo,with the concentration of 50-100?M in vitro;2.Hydrogen sulfide promotes LPS inhibited effect of the proliferation and osteogenesis of osteoblasts;3.Hydrogen sulfide promotes LPS effect of apoptosis of osteoblasts;4.Hydrogen sulfide promote the apoptosis of osteoblasts through FAS apoptotic pathway.5.Hydrogen sulfide can induce apoptosis of osteoblasts by inhibiting AKT/NF-?B pathway.