OBJECTIVE: To investigate the effects of sodium valproate (VPA) in inhibiting Erastin-induced ferroptosis in bone marrow mesenchymal stem cells (BMSCs) and its underlying mechanisms. METHODS: BMSCs were isolated from bone marrow of 8-week-old Spragur Dawley rats and identified [cell surface antigens CD90, CD44, and CD45 were analyzed by flow cytometry, and osteogenic and adipogenic differentiation abilities were assessed by alizarin red S (ARS) and oil red O staining, respectively]. Cells of passage 3 were used for the Erastin-induced ferroptosis model, with different concentrations of VPA for intervention. The optimal drug concentration was determined using the cell counting kit 8 assay. The experiment was divided into 4 groups: group A, cells were cultured in osteogenic induction medium for 24 hours; group B, cells were cultured in osteogenic induction medium containing optimal concentration Erastin for 24 hours; group C, cells were cultured in osteogenic induction medium containing optimal concentration Erastin and VPA for 24 hours; group D, cells were cultured in osteogenic induction medium containing optimal concentration Erastin and VPA, and 8 μmol/L EX527 for 24 hours. The mitochondrial state of the cells was evaluated, including the levels of malondialdehyde (MDA), glutathione (GSH), and reactive oxygen species (ROS). Osteogenic capacity was assessed by alkaline phosphatase (ALP) activity and ARS staining. Western blot analysis was performed to detect the expressions of osteogenic-related proteins [Runt-related transcription factor 2 (RUNX2) and osteopontin (OPN)], ferroptosis-related proteins [glutathione peroxidase 4 (GPX4), ferritin heavy chain 1 (FTH1), and solute carrier family 7 member 11 (SLC7A11)], and pathway-related proteins [adenosine monophosphate-activated protein kinase (AMPK) and Sirtuin 1 (SIRT1)]. RESULTS: The cultured cells were identified as BMSCs. VPA inhibited Erastin-induced ferroptosis and the decline of osteogenic ability in BMSCs, acting through the activation of the AMPK/SIRT1 pathway. VPA significantly reduced the levels of ROS and MDA in Erastin-treated BMSCs and significantly increased GSH levels. Additionally, the expression levels of ferroptosis-related proteins (GPX4, FTH1, and SLC7A11) significantly decreased. VPA also upregulated the expressions of osteogenic-related proteins (RUNX2 and OPN), enhanced mineralization and osteogenic differentiation, and increased the expressions of pathway-related proteins (AMPK and SIRT1). These effects could be reversed by the SIRT1 inhibitor EX527. CONCLUSION VPA inhibits ferroptosis in BMSCs through the AMPK/SIRT1 axis and promotes osteogenesis.
Mesenchymal Stem Cells/metabolism* ; Ferroptosis/drug effects* ; Animals ; Valproic Acid/pharmacology* ; Rats ; Rats, Sprague-Dawley ; Sirtuin 1/metabolism* ; Cell Differentiation/drug effects* ; Cells, Cultured ; AMP-Activated Protein Kinases/metabolism* ; Osteogenesis/drug effects* ; Piperazines/pharmacology* ; Bone Marrow Cells/cytology* ; Reactive Oxygen Species/metabolism* ; Signal Transduction/drug effects*

Objective To verify the change of the anxiety and depression of patients anteriorposterior treatment at paraurethral duct dilitation after gonococcal infection of paraurethral duct.Methods Twenty-eight patients of paraurethral duct dilatation after male gonococcal infection of paraurethral duct were enrolled,and the patients were treated in the Department of Dermatology and Venerology,the First People's Hospital of Changshu City.The anxiety and depression of patients anteriorposterior treatment were assessed and compared with Hamilton depression scale and Hamilton anxiety scale.Results The total scores of Hamilton depression scale and Hamilton anxiety scale before treatment were positively correlated with course of disease (r =0.481,P =0.0095;r =0.549,P =0.0025).The total scores of Hamilton depression and anxiety before treatment were from 1 to 31 (18.9±7.5) and from 4 to 30 (14.9±6.8).The symptoms of depression and anxiety appeared in 11 patients and 15 patients respectively.The total scores of Hamilton depression andanxiety after treatment were from 0 to 8 (4.3±1.7) and from 0 to 8 (3.3±1.7).There was no symptom of depression and anxiety.The difference of the total score of Hamilton depression and anxiety before and after treatment had statistical significance (t =12.270,P＜0.001,t =10.626,P＜0.001).The difference of positive rate of depressive and anxious symptom before and after treatment had statistical significance too (x2 =9.05,P=0.003;x2 =13.06,P=0.000).Conclusions The sequelae of gonococcal paraurethral duct infection in male paraurethral duct dilatation can result in the anxiety and depression of patients,which should be treated.Effective therapy can relieve or cure the anxiety and depression of patients without the assistance of psychotropic drugs.

Objective To introduce a new minimally invasive lumbar positioning system and its positioning method as well as conduct a prospective control study on the differences in positioning time,positioning frequency and radiation exposure dose between the new positioning method and the traditional positioning method.Methods 121 patients with lumbar disc herniation combined with lumbar instability and patients with thoracolumbar vertebral fracture admitted in our hospital from May 2010 to February 2013 were randomized into two groups.68 patients in Group A had undergone the traditional positioning method before and during operation.Among the 68 patients,41 were with single-segment lesion and 27 were with two-segment lesion.53 patients in Group B had undergone the new minimally invasive lumbar positioning system before and during operation.Among the 53 patients,34 were with single-segment lesion and 19 were with two-segment lesion.There were no significant differences in gender,age,course of disease and other general information between the two groups.The positioning time,positioning frequency and radiation dose of the two groups before and during operation were compared and statistically analyzed.Results The positioning time of Group A and Group B were 8.26±3.44 min and 3.51±1.82 min respectively; the positioning frequencies were 3.57 and 1.22; the accuracy were 60.8％ and 96.2％.For the patients with single-segment lesion,the positioning time before implanting two percutaneous pedicle screws during operation were 15.12±4.69 min and 5.51±1.32 min respectively and the positioning frequency were 6.47 and 2.45.For the patients with two-segment lesion,the positioning time before implanting three percutaneous pedicle screws during operation were 24.91±7.43 min and 8.84±2.32 min respectively and the positioning frequency were 11.72 and 3.69.Moreover,the radiation dose detected at neck,chest and wrist of the surgical staff in Group A were 3.09±0.24 Gy,4.23±0.71 Gy and 5.17±0.62 Gy and that detected in Group B were 1.38±0.47 Gy,2.69±0.33 Gy and 3.21±1.05 Gy.There were significant differences in positioning time,positioning frequency and radiation dose between Group A and Group B.Conclusion The minimally invasive spine positioning system and positioning method can simplify the operative procedures and largely reduce radiation exposure,which is characterized by high positioning accuracy,short positioning time,low fluoroscopy frequency and X-ray radiation dose.

[Objective] To observe the effects of autophagy induced by different doses of ultraviolet B (UVB) irradiation on the apeptosis in human skin fibroblasts.[Methods] Skin fibroblasts were isolated from the circumcision specimen of a 23-year-old healthy male,and subjected to a primary culture.After 3 to 10 passages,the cells were collected and applied in the following experiment.Methyl thiazolyl tetrazolium (MTT) assay was performed to evaluate the proliferation of some fibroblasts treated with different concentrations (0,0.5,2.0,5.0and 10.0 mmol/L) of 3-methyladenine (3-MA).To qualitatively and quantitatively detect the autophagy in fibroblasts treated with different concentrations of 3-MA and in fibroblasts treated with 3-MA of 0.5 mmol/Lfollowing UVB irradiation,monodansylcadaverine (MDC) staining was carried out,and immunofluorescence was used to detect the expression of microtubule-associated protein 1 light chain (LC3).Some fibroblasts were classified into 8 groups to remain untreated,be irradiated with UVB of 30,50 and 100 mJ/cm2 alone,treated with 3-MA of 0.5 mmol/L alone,or treated with 0.5 mmol/L 3-MA following irradiation with UVB of 30,50 and 100 mJ/cm2,respectively,then,cell apoptosis was qualitatively detected by Hoechst and propidium iodide (PI)staining,and quantitatively detected by flow cytometry with annexin V and PI.[Results] The percentage of autophagic cells was (63.037 ± 5.876) ％ in fibroblasts treated with starvation condition,significantly decreased to (34.425 ± 5.183) ％ in fibroblasts treated with 3-MA of 0.5 mmol/L.The expression of LC3 showed a gradually increasing trend from untreated fibroblasts,to fibroblasts irradiated with UVB of 30,50 and 100 mJ/cm2,while the increase was attenuated by the 4-hour treatment with 3-MA immediately after the irradiation.Compared with the other concentrations,the 3-MA of 0.5 mmol/L showed the least influence on the viability of fibroblasts.The addition of 3-MA of 0.5 mmol/L increased the percentage of cells both positive for Hoechst and PI staining in fibroblasts irradiated with UVB of 50 mJ/cm2,but decreased that in fibroblasts irradiated with UVB of 100 mJ/cm2.Similarly,the percentage of middle and late apoptotic cells was significantly higher in fibroblasts irradiated with UVB of 50 mJ/cm2 followed by treatment with 3-MA of 0.5 mmol/L than in those irradiated with UVB of 50 mJ/cm2alone ((10.933 ± 0.839) ％ vs.(7.267 ± 0.473) ％,t =5.20,P＜ 0.05),but lower in fibroblasts irradiated with UVB of 100 mJ/cm2 followed by treatment with 3-MA of 0.5 mmol/L than in those irradiated with UVB of 100 mJ/cm2alone ( (7.100 ± 0.781 ) ％ vs.( 1 0.133 ± 0.681 ) ％,t =6.29,P ＜ 0.05 ).[Conclusion]s The irradiation with UVB of 50 mJ/cm2 may protect fibroblasts by inducing autophagy and suppressing apoptosis,while the high level of autophagy induced by UVB of 100 mJ/cm2 may lead to autophagic cell death in fibroblasts.

Objective To explore clinical and radiographic outcomes of unstable distal clavicle fractures (Neer ⅡB) fixed with lateral clavicle anatomic locking compression plate (LCP).Methods Between January 2009 and October 2010,eleven consecutive patients with unstable fractures of the distal clavicle (Neer ⅡB) were treated using lateral clavicle anatomic LCP.There were 9 men and 2 women,with the mean age of 37.2 years (range,23-43 years).The right shoulder was involved in 6 patients and the left in 5 patients.The interval between injuries to operation was 24-72 h (mean,48 h).After fracture reduction,the plate was place on superior of the distal clavicle.According to the distal fragment length,3 to 6 locking screws were carefully inserted,3 locking screws were used to fix proximal fractures.Coracoclavicular ligament was not repaired.Functional recovery of the shoulder joint was assessed using the American Shoulder and Elbow Surgeons (ASES) rating scale score.Plain radiographs of clavicles were used to assess bony union.Results All the patients were followed up for 9 to 12 months (mean,10.3 months).Solid bony union was eventually achieved in all patients.The mean ASES scores were 89.1 (range,84-91) on the injured side versus 96.2 (range,94-100) on the contralateral side.No implant-related fracture,fixation failure and rotator cuff injury occurred.Conclusion Lateral clavicle anatomic LCP fixation in the treatment of distal clavicular fractures is a reliable and simple technique.

OBJECTIVETo study the effects of Chrysanthemum morifolium based on functional diversity in soil microbial community with different cropping system and fertilization and offer scientific basis for the establishment of the reasonable planting patterns.

METHODCombined yield and quality of Ch. morifolium, 8 treatments of different cropping system and fertilization on functional diversity in soil microbial community of Ch. morifolium were investigated by the Biolog.

RESULTThe AWCD of Ch. morifolium paddy-dryland rotation was higher than that of the continuous cropping, the AWCD of organic fertilizer and compound NPK > single organic fertilizer > single compound NPK > no fertilizer. The principal component analysis about Ch. morifolium soil microbial carbon source use showed that the contribution rate of principal component 1 was 45.5% and principal component 2 was 12.1%, which could explain most information about the variation. Different cropping system of Ch. morfolium differentiated in principal component 1 axis, different fertilization differentiated in principal component 2 axis. The yield of Ch. morifolium and volatile oil content paddy-dryland rotation was significantly higher than that of continuous cropping.

CONCLUSIONThe Ch. morifolium should be cultivated with organic fertilizer and compound NPK by paddy-dryland rotation patters.

Objective To investigate the effects of sirolimus and 3-methyladenine (3-MA) on the autophagy in,as well as matrix matalloproteinase (MMP)-1 and MMP-3 secretion by,human skin fibroblasts (HSFs).Methods HSFs were isolated from the circumcised foreskin of a healthy male,and subjected to primary culture.After 3 to 10 passages,HSFs were incubated with sirolimus of 20,50,100,250 nmol/L and 3-MA of 0.5,2.0,5.0,10.0 mmol/L respectively for 4 hours followed by the observation of autophagy and detection of MMP-1 and MMP-3 levels in the supernatant by enzyme linked immunosorbent assay (ELISA).Those HSFs remaining untreated or treated with dimethyl sulfoxide served as the control.Results The percentage of autophagy-positive cells was 59.075％ ± 6.884％,76.350％ ± 5.226％,85.063％ ± 6.002％,86.288％ ± 5.558％ and 96.825％ ± 1.500％ respectively in HSFs treated with sirolimus of 0,20,50,100 and 250 nmol/L; significant differences were observed between the 5 groups (P ＜ 0.01 ) but not between the cells treated with sirolimus of 50 and 100 nmol/L (P ＞ 0.05).After being treated with 3-MA of 0,0.5,2.0,5.0 and 10.0 mmol/L,the percentage of autophagy-positive cells in HSFs was 63.037％ ± 5.876％,34.425％ ± 5.183％,19.700％ ± 3.028％,12.900％ ± 3.334％ and 7.775％ ± 2.293％ respectively with a significant difference between these groups (all P ＜ 0.01 ).Elevated levels of MMP-1 and MMP-3 were observed in the supernatant of HSFs treated with sirolimus of 250 nmol/L and 3-MA of 10.0 mmol/L (all P ＜ 0.05).Conclusions The autophagy in HSFs can be upregulated by sirolimus,but downregulated by 3-MA.For the secretion of MMPs by HSFs,3-MA and high concentrations of sirolimus exert a promotive effect,and the effect of 3-MA is in a concentration-related manner,but the influences of sirolimus at lower concentrations remain unclear.

Objective To observe the changes of autophagy in human skin fibroblast (HSF) model for photoaging. Methods HSF model for photoaging was established through repeated exposure to ultraviolet B (UVB). Those HSFs receiving no irradiation served as the control. The degree of aging was evaluated by p-galactosidase assay, and autophagy level was detected. Results After repeated exposure to UVB, most pho-toaged HSFs were deformed and distorted, and some of them even died. The percentage of P-galactosidase-positive cells was 50.60% ± 5.04% and 14.58% ± 2.69%, respectively in photoaged and control HSFs (P< 0.01). Significant difference was also observed in the proportion of cytophagosome-positive cells between photoaged and control HSFs (14.91% ± 4.59% vs 68.45% ± 8.25%, P < 0.01). Conclusion The HSF model for photoaging shows obviously abnormal appearance and stagnant growth with increased degree of senescence and decreased autophagy compared with normal control HSFs.

Objective To compare the changes of skin thickness and collagen content in mouse models of scleroderma after irradiated with different doses of UVA1, so as to seek the suitablc irradiation dose in the treatment for scleroderma. Methods Sixty mice were randomly and equally divided into 6 groups: blank control group (no injection and no irradiation), model control group (injected only and killed 2 days after the last injection), high-dose (HD) UVA1 group (injected with bleomycin and irradiated with UVA1 of 100 J/cm2), medium-dose (MD) UVA1 group (injected with bleomycin and irradiated with UVA1 of 60 J/cm2), low-dose (LD) UVA1 group (injected with bleomycin and irradiated with UVA1 of 20 J/cm2), and negative control group (injected only and killed until the end of irradiation). Experimental mouse models of scleroderma were established by subcutaneous injection of 100 μL bleomycin (BLM) at 400 μg/mL into the back of BALB/c mouse once a day for 4 weeks. Phototherapy was performed 3 times weekly for 10 weeks. Thereafter, skin specimens were obtained from the injected or irradiated back of mice, and subjected to an observation on pathological changes of skin tissue and thickness, as well as the measurement of collagen content. Results There was statistical differences between blank control group and model control group in both the skin thickness (t= 4.945, P<0.001) and collagen content (t=3.712, P<0.01). UVAI phototherapy improved the skin sclerosis and reduced the thickness in mouse models, but significant effect was only observed with HD-UVA1 in both the parameters(both P<0.05). There was significant difference among the 3 groups receiving phototherapy in the changes of skin thickness (F=14.853, P<0.01) and collagen content (F= 6.317, P<0.01), and HD-UVAI was significantly more effective than MD-UVA1 and LD-UVA1. Conclusion High-dose UVAI irradiation could significantly reduce the changes in skin thickness and col- lagen content in mouse model of sclerosis induced by bleomycin,which may be related to its inhibition on collagen fiber proliferation and decrease in collagen content.