BACKGROUND:Psoralen has a strong anti-osteoporotic activity and may have a restorative effect on chemotherapy-induced osteoporosis. OBJECTIVE:To explore the restorative effect of psoralen on the osteogenic differentiation of bone marrow mesenchymal stem cells in mice inhibited by cyclophosphamide and its mechanism. METHODS:C57BL/6 mouse bone marrow mesenchymal stem cells were isolated and cultured.Effect of psoralen on viability of bone marrow mesenchymal stem cells was detected by MTT assay.Osteogenic induction combined with alkaline phosphatase staining was used to determine the optimal dose of psoralen to restore the osteogenic differentiation of bone marrow mesenchymal stem cells inhibited by cyclophosphamide.The mRNA expression levels of Runx2,alkaline phosphatase,Osteocalcin,osteoprotegerin,and Wnt/β-catenin signaling pathway-related genes Wnt1,Wnt4,Wnt10b,β-catenin,and c-MYC were measured by RT-qPCR at different time points under the intervention with psoralen.The protein expression of osteogenic specific transcription factor Runx2 and Wnt/β-catenin signaling pathway related genes Active β-catenin,DKK1,c-MYC,and Cyclin D1 was determined by western blot assay at different time points under the intervention with psoralen. RESULTS AND CONCLUSION:(1)There was no significant effect of different concentrations of psoralen on the viability of bone marrow mesenchymal stem cells.The best recovery of the inhibition of osteogenic differentiation of bone marrow mesenchymal stem cells caused by cyclophosphamide was under the intervention of psoralen at a concentration of 200 μmol/L.(2)Psoralen reversed the reduction in osteogenic differentiation marker genes Runx2,alkaline phosphatase,Osteocalcin and osteoprotegerin mRNA expression and Runx2 protein expression in bone marrow mesenchymal stem cells caused by cyclophosphamide conditioned medium.(3)Psoralen reversed the decrease in Wnt/β-catenin pathway-related genes Wnt4,β-catenin,c-MYC mRNA and Active β-catenin,c-MYC,and Cyclin D1 protein expression and the increase in DKK1 protein expression in bone marrow mesenchymal stem cells caused by cyclophosphamide conditioned medium.(4)The results showed that cyclophosphamide inhibited osteogenic differentiation of bone marrow mesenchymal stem cells in mice,and psoralen had a restorative effect on it.The best intervention effect was achieved at a concentration of 200 μmol/L psoralen,and this protective effect might be related to the activation of Wnt4/β-catenin signaling pathway by psoralen.

Objective: To analyze the disease burden of chronic obstructive pulmonary disease (COPD) and changes in its risk factors among residents in Zhejiang Province from 1990 to 2021, so as to identify key priorities for COPD prevention and control. Methods: Data on COPD mortality and disability-adjusted life years (DALY) for residents in Zhejiang Province from 1990 to 2021 were collected from the Global Burden of Disease (GBD) 2021 database. Standardized mortality and standardized DALY rate were calculated using the GBD 2021 world population standard structure. Premature mortality was computed via the life table method. The average annual percent change (AAPC) was applied to analyze trends in COPD mortality, DALY rate, and premature mortality. Changes in deaths of COPD risk factors were evaluated using population attributable fraction (PAF). Results: From 1990 to 2021, the standardized COPD mortality in Zhejiang Province decreased from 272.40/100 000 to 70.56/100 000 (AAPC=-4.395%), and the standardized DALY rate declined from 4 167.37/100 000 to 1 071.89/100 000 (AAPC=-4.396%). Similar downward trends were observed in both males (AAPC=-3.933%, -4.173%) and females (AAPC=-4.785%, -4.480%), all P<0.05. Crude mortality and DALY rates increased with age, and the crude mortality and DALY rates of various age groups in Zhejiang Province showed decreasing trends from 1990 to 2021 (all P<0.05). The premature mortality declined from 4.37% to 0.60% from 1990 to 2021 (AAPC=- 6.206%), with consistent trends across males and females (AAPC=- 6.144%, - 6.379%, all P<0.05). From 1990 to 2021, particulate matter pollution showed the largest reduction in PAF (- 56.76%), while ambient ozone pollution had the largest increase (103.07%) in Zhejiang Province. By 2021, smoking became the leading risk factor for deaths of COPD (PAF=43.32%). Conclusions The standardized mortality, standardized DALY rate, and premature mortality for COPD show consistent declining trends in Zhejiang Province from 1990 to 2021. However, risk factors such as smoking and ambient ozone pollution require intensified focus to further reduce disease burden of COPD.

Objective To establish a dexamethasone(Dex)-induced zebrafish model of glucocorticoid-induced osteoporosis(GIOP)combined with glucocorticoid-induced hypertension(GIHT),and to validate the model by the systematic evaluation of both the phenotypic manifestations and molecular mechanisms.Methods Zebrafish larvae at 3 or 4 d post-fertilization(dpf)were divided randomly into a control group(0.1％dimethyl sulfoxide)and a model group(10 μmol/L Dex).Osteogenic parameters and vessel diameter were assessed at 0,48,and 96 h post-administration(n=10).Bone mineralization and density were determined by the total area and sum brightness after Alizarin red staining.Vessel diameter was measured by detecting blood flow in the dorsal aorta.After confirming the optimal administration time,expression levels of bone-formation-related proteins(protein kinase B(Akt),glycogen synthase kinase(GSK)-3β,β-catenin)and angiogenesis-related proteins(AMP-activated protein kinase(AMPK),nuclear factor(NF)-κB)were detected by Western Blot to verify the molecular effectiveness of the model.Results Exposure to Dex for 96 h reduced bone mineralization and density in zebrafish larvae compared with the control group,and statistical analysis identified 4 dpf zebrafish and Dex administration for 96 h as the optimal modeling times for the GIOP model.Blood vessel diameter was significantly decreased in the model group compared with the control group(P＜0.05),and the difference became more pronounced with longer administration time and was particularly evident at 4 dpf and treatment for 96 h.Western Blot analysis showed that Dex significantly decreased protein expression levels of Akt,β-catenin,and NF-κB(P＜0.05)and significantly increased the expression of GSK-3β and AMPK(P＜0.05),suggesting that Dex effectively inhibited bone formation and angiogenesis after 96 hours treatment in 4 dpf zebrafish.Conclusions Treatment of 4 dpf zebrafish larvae with 10 μmol/L Dex rapidly established a reliable zebrafish model of GIOP combined with GIHT,providing an ideal animal model for further studies of the common mechanisms of the two diseases and for screening new drugs.

Objective:To evaluate the effect of cathepsin B(CTSB)/NOD-like receptor pyrin domain containing 3(NLRP3)pathway on the polarization of macrophages induced by LPS.Methods:The well-growing RAW264.7 mouse mononuclear macrophage lines were cultured in vitro and divided into 3 groups(n=6)according to the random number table method:control group(C group),LPS group(L group)and LPS+CA074-me(CTSB inhibitors)group(B group).C group was cultured normally for 24 h,L group was cultured with LPS concentration of 1 μg/ml medium for 24 h.B group was pretreated with CTSB inhibitor CA074-me 30 μmol/L for 1 h before LPS induction,and co-cultured with LPS concentration of 1 μg/ml medium for 24 h.After 24 hours,the morphological changes of the cells were observed by microscope,the concentrations of IL-1β and IL-18 in the supernatant were determined by ELISA.The ex-pressions of cathepsin B precursor(pro-CTSB),mature cathepsin B(mature-CTSB),NLRP3,apoptosis-related speck protein(ASC)and apoptosis-related speck protein-1(caspase-1)were detected by Western blot.The mRNA expression levels of CD32,inducible ni-tric oxide synthase(iNOS),arginase 1(Arg-1)and CD206 were detected by qRT-PCR.The positive expression rates of M1 macro-phage surface marker CD86 and M2 macrophage surface marker CD206 were detected by flow cytometry.Results:Compared with group C,the morphology of cells in groups L and B became larger and pseudopodia appeared.The concentrations of IL-1β and IL-18 in cell supernatant were increased,the expressions of pro-CTSB,mature-CTSB,NLRP3,ASC and caspase-1 were increased,and the expressions of CD32,iNOS mRNA were up-regulated and the positive rates of CD86 and CD206 were increased(P<0.01).Arg-1 and CD206 mRNA in group B were up-regulated(P<0.01).Compared with group L,the pseudopodia of group B were reduced,and the morphology was closer to group C.The concentration of IL-1β and IL-18 in the supernatant,the expression of mature-CTSB,NLRP3,ASC and caspase-1,CD32 and iNOS mRNA and the positive rate of CD86 were down-regulated in group B.The expression of pro-CTSB,Arg-1 and CD206 mRNA and the positive rate of CD206 were increased(P<0.01).Conclusion:Inhibition of CTSB/NLRP3 pathway can reduce the inflammatory response,reduce the LPS-induced polarization of RAW264.7 cells to M1 macrophages,and pro-mote their polarization to M2 macrophages.

Objective:To understand the clinical characteristics of hypophysitis due to nivolumab and provide reference for the safe use of nivolumab in clinic.Methods:Relevant databases at home and abroad (up to May 31, 2024) were searched and case reports of hypophysitis caused by nivolumab were collected. Relevant information of patients (gender, age, primary disease), single and combined use of nivolumab, occurrence of hypophysitis, causality evaluation, intervention measures and outcomes were extracted and analyzed descriptively and statistically.Results:A total of 56 case reports, 53 in English and 3 in Chinese, were enrolled in the analysis, involving 59 patients, with 40 males and 19 females. Their ages ranged from 26 to 84 years, with an average age of 61 years. The primary diseases were malignant melanoma in 20 cases, renal cell carcinoma in 19 cases, lung cancer in 11 cases, glossopharyngeal cancer in 4 cases, esophageal cancer in 2 cases, and gastric cancer, colon cancer and pleural mesothelioma in 1 case each. Among the 59 patients, 33 were treated with nivolumab monotherapy, 24 with nivolumab and ipilimumab, 1 with nivolumab and anlotinib and 1 with nivolumab and platinum. The average time of hypophysitis caused by nivolumab alone was 25.6 weeks after treatment, and that caused by the combination therapy was 9.9 weeks after treatment. The main clinical manifestations of hypophysitis caused by nivolumab were fatigue, anorexia, headache, and nausea. Among the 59 patients, 56 patients discontinued nivolumab and received glucocorticoid, 20 of whom resumed nivolumab treatment after improvement of clinical symptoms; 3 patients′ situation was not described clearly. The outcomes of the 59 patients were as follows. Four were recovered, 46 were relieved, 2 patients′ pituitary function was not recovered, 4 died, 1 developed secondary autoimmune vasculitis, 1 developed secondary autoimmune hepatitis, and it was unknown in one patient.Conclusions:The average occurrence time of hypophysitis caused by nivolumab monotherapy is longer than that caused by combination therapy. The main clinical manifestations are fatigue, anorexia, headache, etc. After timely discontinuation of medication and symptomatic treatments with glucocorticoid, most patients have a good prognosis, but it can lead to death in severe cases.

Objective To establish a dexamethasone(Dex)-induced zebrafish model of glucocorticoid-induced osteoporosis(GIOP)combined with glucocorticoid-induced hypertension(GIHT),and to validate the model by the systematic evaluation of both the phenotypic manifestations and molecular mechanisms.Methods Zebrafish larvae at 3 or 4 d post-fertilization(dpf)were divided randomly into a control group(0.1％dimethyl sulfoxide)and a model group(10 μmol/L Dex).Osteogenic parameters and vessel diameter were assessed at 0,48,and 96 h post-administration(n=10).Bone mineralization and density were determined by the total area and sum brightness after Alizarin red staining.Vessel diameter was measured by detecting blood flow in the dorsal aorta.After confirming the optimal administration time,expression levels of bone-formation-related proteins(protein kinase B(Akt),glycogen synthase kinase(GSK)-3β,β-catenin)and angiogenesis-related proteins(AMP-activated protein kinase(AMPK),nuclear factor(NF)-κB)were detected by Western Blot to verify the molecular effectiveness of the model.Results Exposure to Dex for 96 h reduced bone mineralization and density in zebrafish larvae compared with the control group,and statistical analysis identified 4 dpf zebrafish and Dex administration for 96 h as the optimal modeling times for the GIOP model.Blood vessel diameter was significantly decreased in the model group compared with the control group(P＜0.05),and the difference became more pronounced with longer administration time and was particularly evident at 4 dpf and treatment for 96 h.Western Blot analysis showed that Dex significantly decreased protein expression levels of Akt,β-catenin,and NF-κB(P＜0.05)and significantly increased the expression of GSK-3β and AMPK(P＜0.05),suggesting that Dex effectively inhibited bone formation and angiogenesis after 96 hours treatment in 4 dpf zebrafish.Conclusions Treatment of 4 dpf zebrafish larvae with 10 μmol/L Dex rapidly established a reliable zebrafish model of GIOP combined with GIHT,providing an ideal animal model for further studies of the common mechanisms of the two diseases and for screening new drugs.

Objective:To evaluate the effect of cathepsin B(CTSB)/NOD-like receptor pyrin domain containing 3(NLRP3)pathway on the polarization of macrophages induced by LPS.Methods:The well-growing RAW264.7 mouse mononuclear macrophage lines were cultured in vitro and divided into 3 groups(n=6)according to the random number table method:control group(C group),LPS group(L group)and LPS+CA074-me(CTSB inhibitors)group(B group).C group was cultured normally for 24 h,L group was cultured with LPS concentration of 1 μg/ml medium for 24 h.B group was pretreated with CTSB inhibitor CA074-me 30 μmol/L for 1 h before LPS induction,and co-cultured with LPS concentration of 1 μg/ml medium for 24 h.After 24 hours,the morphological changes of the cells were observed by microscope,the concentrations of IL-1β and IL-18 in the supernatant were determined by ELISA.The ex-pressions of cathepsin B precursor(pro-CTSB),mature cathepsin B(mature-CTSB),NLRP3,apoptosis-related speck protein(ASC)and apoptosis-related speck protein-1(caspase-1)were detected by Western blot.The mRNA expression levels of CD32,inducible ni-tric oxide synthase(iNOS),arginase 1(Arg-1)and CD206 were detected by qRT-PCR.The positive expression rates of M1 macro-phage surface marker CD86 and M2 macrophage surface marker CD206 were detected by flow cytometry.Results:Compared with group C,the morphology of cells in groups L and B became larger and pseudopodia appeared.The concentrations of IL-1β and IL-18 in cell supernatant were increased,the expressions of pro-CTSB,mature-CTSB,NLRP3,ASC and caspase-1 were increased,and the expressions of CD32,iNOS mRNA were up-regulated and the positive rates of CD86 and CD206 were increased(P<0.01).Arg-1 and CD206 mRNA in group B were up-regulated(P<0.01).Compared with group L,the pseudopodia of group B were reduced,and the morphology was closer to group C.The concentration of IL-1β and IL-18 in the supernatant,the expression of mature-CTSB,NLRP3,ASC and caspase-1,CD32 and iNOS mRNA and the positive rate of CD86 were down-regulated in group B.The expression of pro-CTSB,Arg-1 and CD206 mRNA and the positive rate of CD206 were increased(P<0.01).Conclusion:Inhibition of CTSB/NLRP3 pathway can reduce the inflammatory response,reduce the LPS-induced polarization of RAW264.7 cells to M1 macrophages,and pro-mote their polarization to M2 macrophages.

Objective:To understand the clinical characteristics of hypophysitis due to nivolumab and provide reference for the safe use of nivolumab in clinic.Methods:Relevant databases at home and abroad (up to May 31, 2024) were searched and case reports of hypophysitis caused by nivolumab were collected. Relevant information of patients (gender, age, primary disease), single and combined use of nivolumab, occurrence of hypophysitis, causality evaluation, intervention measures and outcomes were extracted and analyzed descriptively and statistically.Results:A total of 56 case reports, 53 in English and 3 in Chinese, were enrolled in the analysis, involving 59 patients, with 40 males and 19 females. Their ages ranged from 26 to 84 years, with an average age of 61 years. The primary diseases were malignant melanoma in 20 cases, renal cell carcinoma in 19 cases, lung cancer in 11 cases, glossopharyngeal cancer in 4 cases, esophageal cancer in 2 cases, and gastric cancer, colon cancer and pleural mesothelioma in 1 case each. Among the 59 patients, 33 were treated with nivolumab monotherapy, 24 with nivolumab and ipilimumab, 1 with nivolumab and anlotinib and 1 with nivolumab and platinum. The average time of hypophysitis caused by nivolumab alone was 25.6 weeks after treatment, and that caused by the combination therapy was 9.9 weeks after treatment. The main clinical manifestations of hypophysitis caused by nivolumab were fatigue, anorexia, headache, and nausea. Among the 59 patients, 56 patients discontinued nivolumab and received glucocorticoid, 20 of whom resumed nivolumab treatment after improvement of clinical symptoms; 3 patients′ situation was not described clearly. The outcomes of the 59 patients were as follows. Four were recovered, 46 were relieved, 2 patients′ pituitary function was not recovered, 4 died, 1 developed secondary autoimmune vasculitis, 1 developed secondary autoimmune hepatitis, and it was unknown in one patient.Conclusions:The average occurrence time of hypophysitis caused by nivolumab monotherapy is longer than that caused by combination therapy. The main clinical manifestations are fatigue, anorexia, headache, etc. After timely discontinuation of medication and symptomatic treatments with glucocorticoid, most patients have a good prognosis, but it can lead to death in severe cases.

ObjectiveTo explore the therapeutic effect and mechanism of Guipitang on rats with myocardial ischemia. MethodFifty SD rats were divided into five groups： a control group， a model group， low and high-dose Guipitang （7.52， 15.04 g·kg^-1） groups， and a trimetazidine group （0.002 g·kg^-1）. By intragastric administration of vitamin D₃ and feeding rats with high-fat forage and injecting isoproterenol， the rat model of myocardial ischemia was established. After drug treatment of 15 d， an electrocardiogram （ECG） was performed to analyze the degree of myocardial injury. A fully automatic biochemical analyzer was used to detect the changes in the serum levels of total cholesterol （TC）， high-density lipoprotein cholesterol （HDL-C）， triglyceride （TG）， and low-density lipoprotein cholesterol （LDL-C）. Hematoxylin-eosin （HE） staining and Masson staining were used to observe myocardial histopathological changes. TdT-mediated dUTP nick end labeling （TUNEL） staining was used to detect cardiomyocyte apoptosis. Western blot was adopted to detect the protein levels of extracellular signal-regulated kinase 1/2 （ERK1/2）， phospho-ERK1/2 （p-ERK1/2）， p38 mitogen-activated protein kinase （p38 MAPK）， phospho-p38 MAPK （p-p38 MAPK）， B-cell lymphoma-2 （Bcl-2）-associated X （Bax）， Bcl-2， and cleaved cysteine aspartate proteolytic enzyme （cleaved Caspase-3）. ResultCompared with the control group， the ECG S-T segment decreased in the model group. The serum levels of TC， TG， and LDL-C were increased significantly （P<0.05）. The arrangement of myocardial tissue was disordered， and the proportion of cardiomyocyte apoptosis increased. The protein levels of cleaved Caspase-3， Bax， and p-p38 MAPK in the heart were increased， and the Bcl-2 expression was decreased （P<0.05）. Compared with the model group， the S-T segment downward shift was restored in the low and high-dose Guipitang groups and trimetazidine group， and the levels of TC， TG， and LDL-C were decreased. The protein expression of cleaved Caspase-3 and Bax in the heart dropped， and p-p38 MAPK and p-ERK1/2 protein expressions increased significantly （P<0.05）. The degree of myocardial injury was alleviated， and the proportion of cardiomyocyte apoptosis decreased. Bcl-2 protein expression was increased significantly in the low-dose Guipitang group （P<0.05）. ERK1/2 and p38 MAPK proteins had no significant difference among different groups. ConclusionGuipitang could alleviate myocardial injury and inhibit cardiomyocyte apoptosis in rats by activating the expression of ERK1/2 and p38 MAPK.

Objective:To investigate the impact of the birth of preterm infants on their family functioning, and to provide a reference for the formulation of targeted intervention programs.Methods:Based on the socio ecosystem theory, phenomenological research methods and objective sampling were used to conduct semi-structured interviews with 16 preterm infants in the neonatal intensive care unit (NICU) of Children′s Hospital of Soochow University from September 2023 to January 2024, who meet the inclusion and exclusion criteria, and the Colaizzi 7-step analysis method was used to analyze the interview data.Results:All 16 caregivers of the NICU preterm infants who participated in the interviews were the infants′ mothers, aged from 26 to 34 years. A total of 3 themes and 10 sub-themes were summarized. The micro system: physical and mental exhaustion coexisted with role growth of caregivers (physical and mental health was affected, positive experience after psychological adjustment, and adaptation to role change). The intermediate system: family relationship mode change (family satisfaction decreased, family difficulties affected family harmony, family communication content changed, family harmony and conflict coexisted, and family conflict resolution was actively resolved). The outer system: social support system needed to be improved (benefited from social support and not yet perceived external support).Conclusions:Medical staff can improve the multiple support system and provide continuous and personalized family support, so as to improve the family adjustment ability and improve the family functioning of preterm infants in the NICU.