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 explore clinical characteristics of multiple sclerosis(MS)patients in Suzhou,and to analyze main factors affecting their prognosis.Methods:General data,clinical symptoms,cerebrospinal fluid and imaging examinations of 51 MS patients admitted to Department of Neurology of the Second Hospital of Soochow University from July 31,2009 to July 31,2021 were retrospectively analyzed,and main factors affecting their prognosis were discussed.Results:Average age of onset of 51 MS patients was(43.3±15.6)years old,female accounted for 56.9%,male/female=1/1.3.Adult onset MS(AOMS)accounted for 62.8%,male/female=1/1.7;late onset MS(LOMS)accounted for 37.2%,male/female=1/0.9.Relapsing remitting MS(RRMS)accounted for 76.5%,and chronic onset accounted for 60.8%.Average annual recurrence rate was 8.8%.The first symptoms were numbness and weakness of limbs.Dizziness and numbness were more common in patients without recurrence after diagnosis of MS,and limb weak-ness and numbness were more common in patients with recurrence.Among lesions of MRI,62.7%(32/51)of periventricular involve-ment,52.9%(27/51)of spinal cord involvement,51.0%(26/51)of infratentorial involvement.Proportion of subtentorial and spinal cord(cervical,thoracic)involved were significantly higher in patients with recurrent MS than without recurrence.Values of albumin,IgG,IgA and IgM in cerebrospinal fluid increased with increase of recurrence times.EDSS score of male was higher than female,and LOMS score was higher than AOMS.MS patients without relapse had a low EDSS score,and median EDSS score at current follow-up was 0(0,1.00)score.MS score with relapse was relatively high,and median EDSS score at current follow-up was 2.75(0.25,7.25)score.Conclusion:MS patients with chronic onset are more common,with a high proportion of LOMS,and proportion of males increases with increasing age of onset.High EDSS score at first onset,cervical,thoracic and subtentorial lesions,increased values of cerebrospinal fluid albumin,IgG,IgA,IgM,age at first onset(50+years old),male associate with poor MS prognosis.

BACKGROUND:Titanium implants are widely used in clinical practice because of their high strength and good biocompatibility.However,during implantation,bacterial infection and tissue damage environment produce a large number of reactive oxygen species,which can easily lead to delayed tissue healing and surgical failure.Consequently,the development of titanium implants with antimicrobial and antioxidant properties becomes paramount. OBJECTIVE:Considering the potent antimicrobial attributes of copper ions and the remarkable antioxidant qualities of polyphenols,we proposed the fabrication of polyphenol-mediated copper ion coatings on titanium surfaces.These coatings were subsequently assessed for their in vitro antimicrobial and antioxidant properties. METHODS:Nanostructures were generated on the titanium surface using the alkali thermal method.The titanium was immersed in a solution containing tannic acid and copper ions to achieve polyphenol-mediated copper ion coatings.The surface morphology and water contact angle were detected.The loading and release of copper ions were examined using atomic absorption spectroscopy.Staphylococcus aureus was inoculated on the surface of pure titanium sheet(blank group),alkali heat treated titanium sheet(control group),and polyphenol mediated copper ion modified titanium sheet(experimental group)to observe the bacterial survival status.Osteoblast precursor cells MC3T3-E1 were co-cultivated on the surface of three groups of titanium sheets to assess their antioxidant properties and bioactivity. RESULTS AND CONCLUSION:(1)Scanning electron microscopy showed that the polyphenol-mediated copper ion modified titanium sheet had rod-like nanostructures and no cracks on the surface.The surface hydrophilicity of copper ion modified titanium sheet mediated by polyphenol was close to that of pure titanium sheet.Atomic absorption spectrometry results showed a 51%increase in the loading capacity of copper ions after polyphenol mediation,with a uniform release of copper ions.(2)The antibacterial rates of titanium sheets in the blank group,control group,and experimental group were 0%,21.65%,and 93.75%,respectively.The live/dead staining and CTC staining showed that the live bacteria on the surface of titanium plates in the blank group were the most,and the live bacteria on the surface of titanium plates in the experimental group were the least.(3)The results of live/dead staining and CCK-8 assay showed that the three groups of titanium sheets had good cytocompatibility,and the titanium sheets in the experimental group were more conducive to the proliferation of MC3T3-E1 cells.Active oxygen fluorescence probe detection exhibited that compared with the other two groups,the fluorescence intensity of active oxygen on the surface of the experimental group was significantly reduced.The results of alkaline phosphatase and alizarin red S staining showed that the osteogenic differentiation and extracellular matrix mineralization of MC3T3-E1 cells on the surface of titanium sheets in the experimental group were stronger than those in the other two groups.(4)These results show that the polyphenol-mediated copper ion coating has strong antibacterial and antioxidant properties and promotes osteogenic differentiation.

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 explore clinical characteristics of multiple sclerosis(MS)patients in Suzhou,and to analyze main factors affecting their prognosis.Methods:General data,clinical symptoms,cerebrospinal fluid and imaging examinations of 51 MS patients admitted to Department of Neurology of the Second Hospital of Soochow University from July 31,2009 to July 31,2021 were retrospectively analyzed,and main factors affecting their prognosis were discussed.Results:Average age of onset of 51 MS patients was(43.3±15.6)years old,female accounted for 56.9%,male/female=1/1.3.Adult onset MS(AOMS)accounted for 62.8%,male/female=1/1.7;late onset MS(LOMS)accounted for 37.2%,male/female=1/0.9.Relapsing remitting MS(RRMS)accounted for 76.5%,and chronic onset accounted for 60.8%.Average annual recurrence rate was 8.8%.The first symptoms were numbness and weakness of limbs.Dizziness and numbness were more common in patients without recurrence after diagnosis of MS,and limb weak-ness and numbness were more common in patients with recurrence.Among lesions of MRI,62.7%(32/51)of periventricular involve-ment,52.9%(27/51)of spinal cord involvement,51.0%(26/51)of infratentorial involvement.Proportion of subtentorial and spinal cord(cervical,thoracic)involved were significantly higher in patients with recurrent MS than without recurrence.Values of albumin,IgG,IgA and IgM in cerebrospinal fluid increased with increase of recurrence times.EDSS score of male was higher than female,and LOMS score was higher than AOMS.MS patients without relapse had a low EDSS score,and median EDSS score at current follow-up was 0(0,1.00)score.MS score with relapse was relatively high,and median EDSS score at current follow-up was 2.75(0.25,7.25)score.Conclusion:MS patients with chronic onset are more common,with a high proportion of LOMS,and proportion of males increases with increasing age of onset.High EDSS score at first onset,cervical,thoracic and subtentorial lesions,increased values of cerebrospinal fluid albumin,IgG,IgA,IgM,age at first onset(50+years old),male associate with poor MS prognosis.

Objective To analyze the effects of activating transcription factor 3(ATF3)pathway mediated by circSLC8Al on oxidative stress and iron activity of epileptic cells.Methods An epileptic cell model was established using human neuronal-hippocampal cells through Mg2+-free method.The expression levels of circSLC8A1 and ATF3 in healthy control group and model group were detected.Plasmid transfection was used to establish circSLC8A1 knockout group,ATF3 knockout group,circSLC8A1 knockout+ATF3 overexpression group,and ATF3 knockout+circSLC8A1 overexpression group.After 6h transfection,cells were cultured in normal medium for 48h.The cell viability,iron activity,reactive oxygen species(ROS),lactate dehydrogenase(LDH)and glutathione(GSH)of the different intervention groups were detected and compared.Results The expression levels of circSLC8A1,ATF3,ROS,LDH and iron activity in model group were significantly higher than those in healthy control group,while cell activity and GSH expression were significantly lower than those in healthy control group(P＜0.05).Knocking out circSLC8A1 can significantly reduce the expression of circSLC8A1 in epileptic model cells,while knocking out ATF3 can significantly reduce the expression of ATF3 in epileptic model cells(P＜0.05).Knocking out circSLC8A1 or ATF3 will increase the cell viability,decrease the iron activity and relieve the oxidative stress in epileptic model cells.Knocking out circSLC8A1 and overexpressing ATF3 can reverse the above trend,but knocking out ATF3 and overexpressing circSLC8A1 will not lead to the above phenomenon.Conclusion circSLC8A1 can influence the cell activity,oxidative stress and iron activity process of epileptic model cells by mediating ATF3 pathway,which provides some reference for the mechanism of epilepsy and its targeted therapy.

Objective To compare the value of three-dimensional tomography ultrasound imaging(3D-TUI)and MRI for diagnosng early cesarean scar pregnancy(CSP).Methods Seventy-six patients who had gestational week not more than 12 weeks and with clinically suspected CSP were retrospectively enrolled.According to surgical and postoperative pathological findings,the patients were categorized into CSP group(n=65)and non-CSP group(n=11).The diagnostic performance of 3D-TUI,MRI and the combination of these two for CSP,also the depiction of CSP characteristics of 3D-TUI and MRI were compared.Results The sensitivity,specificity,accuracy,positive predictive value and negative predictive value of combination of 3D-TUI and MRI for diagnosing CSP was 96.92％,90.91％,96.05％,98.44％and 83.33％,respectively,with the accuracy higher than that of 3D-TUI alone(P＜0.05).No significant difference of diagnostic efficacy was found between 3D-TUI and MRI(all P＞0.05).3D-TUI showed a higher accuracy rate than MRI for displaying yolk sac,embryo,villi invasion into local scars and implantation of villi into muscle layer(all P＜0.05),whereas MRI was more accurate for diagnosing intrauterine bleeding(P＜0.05).Conclusion 3D-TUI could be used as the preferred initial imaging examination for early CSP.Combining with MRI could further improve its diagnostic accuracy.

Objective To compare the value of three-dimensional tomography ultrasound imaging(3D-TUI)and MRI for diagnosng early cesarean scar pregnancy(CSP).Methods Seventy-six patients who had gestational week not more than 12 weeks and with clinically suspected CSP were retrospectively enrolled.According to surgical and postoperative pathological findings,the patients were categorized into CSP group(n=65)and non-CSP group(n=11).The diagnostic performance of 3D-TUI,MRI and the combination of these two for CSP,also the depiction of CSP characteristics of 3D-TUI and MRI were compared.Results The sensitivity,specificity,accuracy,positive predictive value and negative predictive value of combination of 3D-TUI and MRI for diagnosing CSP was 96.92％,90.91％,96.05％,98.44％and 83.33％,respectively,with the accuracy higher than that of 3D-TUI alone(P＜0.05).No significant difference of diagnostic efficacy was found between 3D-TUI and MRI(all P＞0.05).3D-TUI showed a higher accuracy rate than MRI for displaying yolk sac,embryo,villi invasion into local scars and implantation of villi into muscle layer(all P＜0.05),whereas MRI was more accurate for diagnosing intrauterine bleeding(P＜0.05).Conclusion 3D-TUI could be used as the preferred initial imaging examination for early CSP.Combining with MRI could further improve its diagnostic accuracy.

Objective To analyze the effects of activating transcription factor 3(ATF3)pathway mediated by circSLC8Al on oxidative stress and iron activity of epileptic cells.Methods An epileptic cell model was established using human neuronal-hippocampal cells through Mg2+-free method.The expression levels of circSLC8A1 and ATF3 in healthy control group and model group were detected.Plasmid transfection was used to establish circSLC8A1 knockout group,ATF3 knockout group,circSLC8A1 knockout+ATF3 overexpression group,and ATF3 knockout+circSLC8A1 overexpression group.After 6h transfection,cells were cultured in normal medium for 48h.The cell viability,iron activity,reactive oxygen species(ROS),lactate dehydrogenase(LDH)and glutathione(GSH)of the different intervention groups were detected and compared.Results The expression levels of circSLC8A1,ATF3,ROS,LDH and iron activity in model group were significantly higher than those in healthy control group,while cell activity and GSH expression were significantly lower than those in healthy control group(P＜0.05).Knocking out circSLC8A1 can significantly reduce the expression of circSLC8A1 in epileptic model cells,while knocking out ATF3 can significantly reduce the expression of ATF3 in epileptic model cells(P＜0.05).Knocking out circSLC8A1 or ATF3 will increase the cell viability,decrease the iron activity and relieve the oxidative stress in epileptic model cells.Knocking out circSLC8A1 and overexpressing ATF3 can reverse the above trend,but knocking out ATF3 and overexpressing circSLC8A1 will not lead to the above phenomenon.Conclusion circSLC8A1 can influence the cell activity,oxidative stress and iron activity process of epileptic model cells by mediating ATF3 pathway,which provides some reference for the mechanism of epilepsy and its targeted therapy.

OBJECTIVE To study the effects of transferrin-targeting peptide T7 （7pep） on intracellular transportation of polyethylene glycol-polycaprolactone （PEG-PCL） micelles in human cervical cancer HeLa cells. METHODS Using coumarin-6 （C6） as fluorescent indicator probe， both coumarin-6 （C6）-loaded PEG-PCL （PEG-PCL-C6） micelles and 7pep-modified PEG- PCL （7pep-PEG-PCL-C6） micelles were prepared by film-dispersion method. The particle size， polydispersity index and appearance morphology were compared between two types of micelles； the real-time uptake of two types of micelles by HeLa cells was compared， and the colocalization of two types of micelles with early endosomes （EE）， endocytic recycling compartments （ERC） and late endosomes （LE） after entry into the cells was observed. RESULTS The particle sizes of PEG-PCL-C6 and 7pep-PEG-PCL- C6 micelles were（75.0±2.3）and（82.0±1.5）nm； the polymer dispersity indexes were 0.17±0.20 and 0.17±0.32， respectively， with a regular spherical appearance. The colocalization results showed that entry speed and amount of 7pep-PEG-PCL-C6 micelles were significantly faster/more than those of PEG-PCL-C6 micelles. 7pep-PEG-PCL-C6 micelles entered EE faster than PEG-PCL-C6 micelles， while PEG-PCL-C6 micelles entered ERC at a faster rate than 7pep-PEG-PCL-C6 micelles， and both PEG-PCL-C6 micelles and 7pep-PEG-PCL-C6 micelles tended to accumulate gradually in LE； Pearson coefficient， signal overlap ratio， and colocalization ratio of 7pep-PEG-PCL-C6 micelles with LE were significantly lower 60 minutes after entering the cell than those 30 minutes after entering the cell （P＜0.05 or P＜0.01）. CONCLUSIONS Targeting 7pep modification can increase the entry speed and amount of PEG-PCL-C6 micelles， and also alter their intracellular transportation behavior.