Objective:To investigate the role of heme oxygenase-1(HO-1)regulating autophagy and its impact on the intracel-lular growth of Mycobacterium abscessus(M.abs).Methods:The standard strain of M.abs ATCC19977 was used to stimulate mouse RAW264.7 macrophages with a specified multiple of infection(MOI)for a specified time.Western blot analysis was performed to detect the expression levels of HO-1 protein and autophagy related proteins Atg5,LC3Ⅱ and p62.Macrophages were pretreated with HO-1 specific inducer CoPP and enzyme activity inhibitor SnPP for 12 h,and incubated with M.abs for 2 h.Amikacin removed extra-cellular bacteria and continued to culture until the specified time,and cell activity was detected by CCK-8 method.Western blot and LysoTracker Red were used to detect the regulatory relationship between HO-1 protein and autophagy related proteins.Bacterial survival and TNF-α secretion levels were detected by colony count and ELISA.Results:Compared with the control group,M.abs could induce increased expression of HO-1,Atg5,LC3Ⅱ and p62 proteins.Over-expression and inhibition of HO-1 were found to effectively regu-late the expressions of Atg5,LC3Ⅱ and p62 induced by M.abs.LysoTracker Red,colony count,and ELISA results demonstrated that SnPP promoted the increasement of intracellular lysosomes,significantly inhibited M.abs intracellular proliferation,and reduced TNF-α secretion.Conclusion:Inhibition of HO-1 can enhance the autophagy flow induced by M.abs and reduce the intracellular growth of M.abs,which provides scientific basis for the development of targeted drugs targeting HO-1.

BACKGROUND:Iron overload is an independent factor inducing osteoporosis,but the action mechanism is currently unclear.Therefore,exploring the effects of iron overload on osteoblast-related cells will help to deeply understand the pathogenesis of osteoporosis and provide potential strategies for osteoporosis treatment.OBJECTIVE:To explore the effects of iron overload environment on osteoblast precursor cell activity,ferroptosis,and osteogenic differentiation.METHODS:Osteoblast precursor cells(MC3T3-E1 cells)were divided into blank group,iron overload group,fer-1 group,and deferoxamine group.The iron overload group was treated with 300 μmol/L ammonium ferric citrate in the culture medium for 48 hours to simulate the iron overload microenvironment.The cells in fer-1 group and deferoxamine group were pretreated with 5 μmol/L antioxidant fer-1 and 5 μmol/L deferoxamine for 8 hours,respectively,and then added with 300 μmol/L ammonium ferric citrate for 48 hours.CCK-8 assay was used to determine the cell viability.Intracellular reactive oxygen species levels were detected employing a reactive oxygen species fluorescent probe.Changes in mitochondrial membrane potential were monitored with a mitochondrial membrane potential fluorescent probe.Mitochondrial morphology was observed employing transmission electron microscopy.Cellular glutathione levels were measured with a reduced glutathione colorimetric assay kit.Lipid peroxidation levels were assessed with a malondialdehyde colorimetric assay kit.Cellular ferrous ion levels were determined with a ferrous ion colorimetric assay kit.The osteogenic and mineralization capabilities of the cells were verified by alkaline phosphatase staining and alizarin red staining.Collagen secretion ability was detected using Sirius Red staining.The expression of osteogenic/ferroptosis-related genes and proteins was examined through reverse transcription quantitative polymerase chain reaction and western blot analysis.RESULTS AND CONCLUSION:(1)In an iron-overload environment,the mitochondrial membrane potential of cells decreased and their structure was compromised,with an elevation in intracellular lipid peroxidation levels and a downregulation of genes and proteins associated with ferroptosis resistance.However,pretreatment with fer-1 and deferoxamine led to an increase in mitochondrial membrane potential and partial restoration of morphology,a reduction in intracellular lipid peroxidation levels,and an upregulation of genes and proteins related to ferroptosis resistance.(2)In an iron-overload environment,the levels of cellular alkaline phosphatase,the formation of mineralized nodules,and the synthesis of collagen fibers were all found to be decreased.Pretreatment with fer-1 and deferoxamine was observed to upregulate the expression of osteogenic differentiation in cells.(3)In summary,iron overload could increase intracellular oxidative stress levels,mediate ferroptosis in MC3T3-E1 cells and inhibit osteogenic differentiation,thereby inducing osteoporosis.Therefore,maintaining iron homeostasis and inhibiting osteogenesis-related ferroptosis may be potential strategies to prevent or treat osteoporosis.

BACKGROUND:Iron overload is an independent factor inducing osteoporosis,but the action mechanism is currently unclear.Therefore,exploring the effects of iron overload on osteoblast-related cells will help to deeply understand the pathogenesis of osteoporosis and provide potential strategies for osteoporosis treatment.OBJECTIVE:To explore the effects of iron overload environment on osteoblast precursor cell activity,ferroptosis,and osteogenic differentiation.METHODS:Osteoblast precursor cells(MC3T3-E1 cells)were divided into blank group,iron overload group,fer-1 group,and deferoxamine group.The iron overload group was treated with 300 μmol/L ammonium ferric citrate in the culture medium for 48 hours to simulate the iron overload microenvironment.The cells in fer-1 group and deferoxamine group were pretreated with 5 μmol/L antioxidant fer-1 and 5 μmol/L deferoxamine for 8 hours,respectively,and then added with 300 μmol/L ammonium ferric citrate for 48 hours.CCK-8 assay was used to determine the cell viability.Intracellular reactive oxygen species levels were detected employing a reactive oxygen species fluorescent probe.Changes in mitochondrial membrane potential were monitored with a mitochondrial membrane potential fluorescent probe.Mitochondrial morphology was observed employing transmission electron microscopy.Cellular glutathione levels were measured with a reduced glutathione colorimetric assay kit.Lipid peroxidation levels were assessed with a malondialdehyde colorimetric assay kit.Cellular ferrous ion levels were determined with a ferrous ion colorimetric assay kit.The osteogenic and mineralization capabilities of the cells were verified by alkaline phosphatase staining and alizarin red staining.Collagen secretion ability was detected using Sirius Red staining.The expression of osteogenic/ferroptosis-related genes and proteins was examined through reverse transcription quantitative polymerase chain reaction and western blot analysis.RESULTS AND CONCLUSION:(1)In an iron-overload environment,the mitochondrial membrane potential of cells decreased and their structure was compromised,with an elevation in intracellular lipid peroxidation levels and a downregulation of genes and proteins associated with ferroptosis resistance.However,pretreatment with fer-1 and deferoxamine led to an increase in mitochondrial membrane potential and partial restoration of morphology,a reduction in intracellular lipid peroxidation levels,and an upregulation of genes and proteins related to ferroptosis resistance.(2)In an iron-overload environment,the levels of cellular alkaline phosphatase,the formation of mineralized nodules,and the synthesis of collagen fibers were all found to be decreased.Pretreatment with fer-1 and deferoxamine was observed to upregulate the expression of osteogenic differentiation in cells.(3)In summary,iron overload could increase intracellular oxidative stress levels,mediate ferroptosis in MC3T3-E1 cells and inhibit osteogenic differentiation,thereby inducing osteoporosis.Therefore,maintaining iron homeostasis and inhibiting osteogenesis-related ferroptosis may be potential strategies to prevent or treat osteoporosis.

Objective:To investigate the role of heme oxygenase-1(HO-1)regulating autophagy and its impact on the intracel-lular growth of Mycobacterium abscessus(M.abs).Methods:The standard strain of M.abs ATCC19977 was used to stimulate mouse RAW264.7 macrophages with a specified multiple of infection(MOI)for a specified time.Western blot analysis was performed to detect the expression levels of HO-1 protein and autophagy related proteins Atg5,LC3Ⅱ and p62.Macrophages were pretreated with HO-1 specific inducer CoPP and enzyme activity inhibitor SnPP for 12 h,and incubated with M.abs for 2 h.Amikacin removed extra-cellular bacteria and continued to culture until the specified time,and cell activity was detected by CCK-8 method.Western blot and LysoTracker Red were used to detect the regulatory relationship between HO-1 protein and autophagy related proteins.Bacterial survival and TNF-α secretion levels were detected by colony count and ELISA.Results:Compared with the control group,M.abs could induce increased expression of HO-1,Atg5,LC3Ⅱ and p62 proteins.Over-expression and inhibition of HO-1 were found to effectively regu-late the expressions of Atg5,LC3Ⅱ and p62 induced by M.abs.LysoTracker Red,colony count,and ELISA results demonstrated that SnPP promoted the increasement of intracellular lysosomes,significantly inhibited M.abs intracellular proliferation,and reduced TNF-α secretion.Conclusion:Inhibition of HO-1 can enhance the autophagy flow induced by M.abs and reduce the intracellular growth of M.abs,which provides scientific basis for the development of targeted drugs targeting HO-1.

National health standards involve all kinds of technical requirements formulated and numbered in accordance with the procedures and formats stipulated in the standardisation system for the implementation of national health and hygiene laws, regulations and policies, and the protection of human health. The establishment of health standards in China should align with our legal framework, including laws, regulations, departmental rules, and health and hygiene policies. During the development of these standards, a comprehensive approach is advocated, encompassing in-depth investigations, rigorous demonstrations, and extensive stakeholder engagement. However, the process of standard formulation may suffer from insufficient research evidence. The evidence-based concept emphasizes the significance of evidence. Therefore, integrating evidence-based concept into the process of developing health standards can enhance the quality and scientific basis of these standards. This article systematically elucidates the current status and development process of health standards in China, explores the necessity and feasibility of incorporating evidence-based concept into the development of these standards, analyzes the challenges and opportunities, and presents reflections and suggestions.

Objective To analyze the curative effect of neoadjuvant chemotherapy combined with limb salvage for osteosarcoma with pathologic fracture.Methods The cases were divided into combined group (68 cases of osteosarcoma with pathologic fracture who received limb salvage after neoadjuvant chemotherapy),non-chemotherapy group (22 cases of osteosarcoma with pathologic fracture who received operation without concurrent chemotherapy or chemotherapy only after operation),non-fracture group (48 cases of osteosarcoma without synchronous pathologic fracture who received limb salvage after neoadjuvant chemotherapy) and amputation group (32 cases of osteosarcoma combined with pathologic fracture who received amputation).Survival rate,local recurrence rate,metastasis rate and quality of life were compared between groups.Results Average estimated survival period of the combined group was obviously longer than that of the non-chemotherapy group (P ＜ 0.05),but had no significant difference from the non-fracture group or amputation group.Of the 5-year survival rate between groups,the combined group was evidently higher than the non-chemotherapy group (P ＜ 0.05),but without significant difference from the non-fracture group or amputation group.Whereas,non-chemotherapy group showed a lower 5-year survival rate than the amputation group or non-fracture group (P ＜ 0.05).Localized recurrence rate and metastasis rate of the combined group were markedly decreased when compared to the nonchemotherapy group (both P ＜ 0.01),but were not significantly different from the non-fracture group or the amputation group.The combined group showed a significantly higher score of social function than the amputation group (P ＜0.01),a significantly higher score of biological pofession than the non-chemotherapy group or the amputation group (P ＜ 0.05),and a significantly higher score of general health than the nonchemotherapy group (P ＜ 0.05).However,no significant differences were observed between groups regarding the scores for other items.Conclusion New adjuvant chemotherapy combined with limb salvage is a rational treatment of osteosarcoma with pathologic fracture for it saves the limb after the guarantee of survival rate without increasing the localized recurrence rate or metastasis rate,and improves quality of life.