Aim To investigate the dynamic changes of neuronal cells at different time points following acute cerebral ischemia-reperfusion injury by establishing a model of brain ischemia-reperfusion injury.Methods Thirty male Sprague-Dawley(SD)rats were ran-domly divided into six groups:sham group and cere-bral ischemia-reperfusion injury(IR)groups at differ-ent time points.Focal cerebral ischemia-reperfusion injury model was established using the middle cerebral artery occlusion(MCAO)technique.The Longa sco-ring method was used to assess neurobehavioral scores in rats.After successful model preparation,routine paraffin sections were made,and TUNEL staining and immunohistochemistry staining with NeuN antibody were performed to observe cell apoptosis and neuronal cell survival,respectively.Immunohistochemistry stai-ning was also performed to investigate the changes in glial fibrillary acidic protein(GFAP)as a marker for astrocytes,ionized calcium-binding adapter molecule 1(IBA-1)as a marker for microglia,and CD31 as a marker for endothelial cells at different time points.Results No significant changes were observed in neu-ronal cells of the sham group at different time points.In the cerebral ischemia-reperfusion injury groups,cell apoptosis was activated at IR3h and increased in quan-tity with morphological damage as time progressed.Ne-uN+neurons showed signs of ischemic injury after IR3h,with abnormal cell morphology.From 12 h,Ne-uN+neurons decreased in a time-dependent manner and reached their peak severity at 24 h.GFAP+astro-cytes decreased significantly after IR3h,while poorly labeled GFAP+astrocytes increased at IR 6 h and al-most disappeared in the infarcted area at 24 h and 48 h.The number of IBA-1+microglia-positive cells de-creased at IR3h,and their volume increased at IR6h.Microglial cell death was observed in the infarcted area at IR12h.CD31+endothelial cells around the infarc-ted cortex and striatum increased significantly after IR3h and persisted until 48 h.Conclusions After cerebral ischemia-reperfusion injury,the number of ap-optotic cells increases with the prolongation of time,and NeuN+neurons exhibit the most severe damage at 24 h.GFAP+astrocytes and microglial cells gradually die over time.The number of CD31+endothelial cells increases significantly around the infarcted cortex and striatum after 3 h of reperfusion and persists until 48 h.

This study aimed to investigate the therapeutic effects of Morinda officinalis iridoid glycosides (MOIG) on bone loss of rheumatoid arthritis (RA) rats, and the mechanism of osteoclast function and activity induced by lipopolysaccharide (LPS). RA rats were established by injecting bovin type II collagen. The Bio-ethic Committee of Zhejiang Chinese Medical University approved all experimental protocols associated with this study (IACUC-20180410-03). The collagen-induced arthritis (CIA) rats were administered drug by gavage for 8 weeks; the femoral trabecular micro-structure changes were observed in CIA rats by micro-CT; the LPS-induced osteoclasts model further observed the effect and mechanism of anti-inflammatory osteoporosis in vitro. The results indicated that MOIG could markedly increase bone mineral density (BMD) in CIA rats, improve trabecular micro-structure. In vitro studies demonstrated that MOIG could significantly inhibit osteoclastogensis and differentiation, suppress tartrate resistant acid phosphatase (TRAP) activity, F-actin ring formation, TNF receptor associated factor 6 (TRAF6) recruitment, and inhibitor of nuclear factor kappa-Bα (IκBα) degradation as well as p65 phosphorylation, thereby repressing nuclear factor kappa-B (NF-κB) signaling pathway activation. Subsequently, MOIG effectively inhibited osteoclast nuclear factor of activated T-cells c1 (NFATc1) and cellular oncogene Fos (c-Fos) expression, as well as bone resorption related protein activity including matrix metalloprotein 9 (MMP-9) and cathepsin K (CtsK). Meanwhile, MOIG also repressed the phosphorylation expression of Janus activating kinase 2 (JAK2) and signal transducer and activator of transcription 3 (STAT3), thereby inhibiting JAK2/STAT3 signaling pathway activation. Moreover, further studies found that MOIG could suppress glycogen synthase kinase-3β (GSK-3β) activity, and GSK-3β gene silencing could markedly inhibit oetsoclast F-actin ring formation as well as the phosphorylation expression of p65 and STAT3. Of note, compared with GSK-3β gene silencing group, there was no significant difference in the group treated with both MOIG with GSK-3β gene silencing simultaneously. Thus, the results suggested that MOIG may inhibit NF-κB signaling pathway and JAK2/STAT3 signaling pathway activation via regulating GSK-3β, thereby alleviating bone destruction in RA.

To investigate the associations of serum gamma-glutamyl transferase (GGT) levels with the risk of cardiovascular disease (CVD) and its subtypes in patients with type 2 diabetes mellitus (T2DM) in Jiangsu Province.Methods:The participants were enrolled in the Comprehensive Research project regarding 'Prevention and Control of Diabetes' in Jiangsu Province. The baseline survey was conducted from 2013 to 2014, and follow-up until December 31, 2021. After excluding the participants who self-reported with chronic liver disease/stroke/coronary heart disease at baseline survey and those with incomplete information on GGT, a total of 16 147 T2DM patients were included in the final analysis. Cox proportional hazard regression models were used to calculate the hazard ratio ( HR) and their 95% CI of GGT for CVD, myocardial infarction, and stroke. Restricted cubic spline models were applied to analyze the dose-response relationship between GGT and the risk of CVD and its subtypes. Results:During the median follow-up time of 8.02 years, 2 860 CVD cases were registered, including 196 cases of myocardial infarction and 2 730 cases of stroke. Multivariate Cox proportional risk regression model indicated that compared to the lowest serum GGT level group, the highest GGT level group had a 24% increased risk of CVD ( HR=1.24, 95% CI: 1.09-1.41) and a 23% increased risk of stroke ( HR=1.23, 95% CI: 1.08-1.40). The restricted cubic spline model showed a nonlinear dose-response relationship between GGT and the risk of CVD, myocardial infarction, and stroke in T2DM patients. Conclusions:High levels of GGT may be associated with an increased risk of cardiovascular disease in T2DM patients, which needs further exploration and validation in future clinical practice.

Objective:To analyze the causes of skin necrosis after penis lengthening surgery and corresponding treatment meas-ures,and observe the clinical effect of free skin graft repair in the treatment of penile skin defects.Methods:We retrospectively an-alyzed the clinical data on 12 cases of extensive penile skin necrosis and defect after penis lengthening surgery performed in our depart-ment from January 2017 to January 2022.The patients underwent free skin graft repair with medium-or full-thickness skin grafts from the thigh after wound preparation.Results:The skin grafts survived well in all the 12 patients and the incisions healed in the first stage without any complications.At 6 months after surgery,skin sensation was mostly recovered in the area of penis skin grafting,no obvious skin ulceration or edema was observed,and the appearance of the penis was satisfactory.The IIEF-5 scores,Erectile Hardness Scale(EHS)scores,and the results of penile hardness tests of the patients all indicated normal erectile function.Conclusion:Free skin graft repair with autologous medium-or full-thickness skin grafts is a safe and effective surgical option for extensive penile skin necrosis after penis lengthening surgery.

Aim To investigate the protective effects of different doses Gualou Xiebai Decoction (GXD) on type II cardiorenal syndrome (type II CRS) and explore its preliminary mechanisms. Methods The type II cardiorenal syndrome rat model was replicated by li-gating the left anterior descending coronary artery. After 10 weeks of intragastric administration, the cardiac function of the rats in each group was evaluated by echocardiography; serum were collected for biochemical testing; heart and kidney tissue samples were stained with HE and Masson to observe pathological changes. The hydroxyproline content in the heart and kidney was detected. The expression levels of endothelial/epitheli-al-to-mesenchymal transition (EndMT/EMT) related proteins in heart and kidney tissues were detecterd by immunofluorescence double staining ^Western blot. Results The index of heart and kidney organs of the low and high doses of GXD group significantly decreased (P<0. 05, P <0. 01), the cardiac function indexes were significantly improved (P < 0. 05, P < 0. 01). Serum characteristic indexes showed that heart and kidney functions were significantly improved (P < 0. 01). In addition, the pathological changes of the heart and kidney were significantly ameliorated. The hydroxyproline content in heart and kidney correspondingly declined (P < 0. 01). The expression levels of CD31 and E-cadherin were significantly up-regulated (P < 0. 05, P <0. 01), and the expression levels of Vimentin, a-SMA and TGF-f3l were down-regulated in heart and kidney tissues (P < 0. 05, P < 0. 01). Conclusions Therefore, GXD can significantly improve the heart and kidney function of type II CRS rats caused by ligation of the left anterior descending coronary artery, and delay the process of heart and kidney fibrosis. The mechanism may be related to the inhibition of EndMT/ EMT.

Objective: To investigate the association between long-term fasting blood glucose (FPG) variability and all-cause mortality in patients with type 2 diabetes. Methods: A total of 7 174 type 2 diabetic patients included in National Basic Public Health Service Program in Changshu of Jiangsu Province were recruited as participants. Long-term glucose variability was assessed using standard deviation (SD), coefficient of variation (CV), average real variability (ARV), and variability independent of the mean (VIM) across FPG measurements at the more than three visits. Death information were mainly obtained from the death registry system in Jiangsu. Then Cox proportional hazards regression models were used to estimate the associations of four variability indicators and all-cause mortality's hazard ratios (HRs) and their 95%CIs. Results: Among 55 058.50 person-years of the follow-up, the mean follow-up time was 7.67 years, and 898 deaths occurred during the follow-up period. After adjustment, compared with T₁ group, the Cox regression model showed that HRs of T₃ group in SD, CV, ARV and VIM were 1.24 (95%CI: 1.03-1.49), 1.20 (95%CI: 1.01-1.43), 1.28 (95%CI: 1.07-1.55) and 1.20 (95%CI:1.01-1.41), respectively. HRs of per 1 SD higher SD, CV, ARV and VIM were 1.13 (95%CI: 1.06-1.21), 1.08 (95%CI: 1.01-1.15), 1.05 (95%CI: 1.00-1.12) and 1.09 (95%CI: 1.02-1.16) for all-cause mortality, respectively. In the stratified analysis, age, gender, hypoglycemic agent and insulin uses had no effect on the above associations (all P for interaction >0.05). Conclusion: Long-term FPG glycemic variability was positively associated with the risk of all-cause mortality in type 2 diabetes patients.

Humans ; Consensus ; Hypersensitivity ; Desensitization, Immunologic/adverse effects* ; Immunotherapy/adverse effects* ; Injections, Subcutaneous ; Allergens

Abstract: Objective　To construct SARS-CoV-2 receptor binding domain molecular probe for monoclonal memory B cell sorting and obtain RBD specific neutralizing antibodies from peripheral blood mononuclear cells (PBMCs) of COVID-19 convalescents by single-cell sorting. Methods　The SARS-CoV-2 RBD sequence was downloaded from GenBank, and the Avi tag and 6-histidine tags were added at the C-terminal. After codon optimization, it was chemically synthesized, cloned into the pDRVI1.0 vector, expressed after transfection of 293F cells, and biotinylated consequently. RBD-specific B cells were sorted out with this probe1 from the PBMCs of convalescents recovered from COVID-19. After B cells were lysed, the variable regions of heavy chain and light chain were amplified, cloned into the antibody expression vector, and transfected into 293F cells to express the antibody. Then the antibody was purified from the supernatant using protein A column and SARS-CoV-2 pseudovirus was used to test their neutralizing activity. Results　RBD-Avi probe was produced and successfully biotinylated sequentially with an efficiency of 30%-50%. Western blot analysis revealed that the biotinylated probe was recognized by the antibodies purified from COVID-19 convalescent plasma. Using this probe, 7 and 16 RBD-specific memory B cells were successfully isolated from the PBMCs of two convalescent individuals, accounting for 0.24% and 0.17% of the total cell population, respectively. After amplifying the variable regions of antibody heavy and light chains from the lysed B cells, 7 and 12 pairs of antibody heavy-light chains were obtained. A total of 16 antibodies were expressed in the convalescent individuals, and most of the purified antibodies showed neutralizing activity against the pseudovirus, with IC50 values of 6 antibodies below 1 μg/mL. The IC50 values of XJ-A9 and SCF-F1 against the wild-type pseudovirus were 0.07 μg/mL and 0.35 μg/mL, respectively. Conclusion　The SARS-CoV-2 RBD molecular probe constructed in this study has good antigenicity, and the isolated antibodies present neutralizing activity against wild-type SARS-CoV-2 pseudovirus.

Objective:To investigate the association between dietary intake and physical activity category and their combined effects on all-cause and cause-specific mortality risk in patients with type 2 diabetes mellitus (T2DM).Methods:Between December 2013 and December 2021, a prospective cohort study was conducted on 19 863 T2DM patients in Changshu City, Qingjiangpu District (formerly Qinghe District), and Huai'an District, included in the national basic health service management. Information on deaths and underlying causes of death was obtained from the Jiangsu Provincial CDC and Prevention Death Surveillance System. Cox proportional hazards models were used to estimate the intensity of associations between dietary intake, physical activity, and their combined effects with all-cause and cause-specific mortality in patients with T2DM.Results:As of December 31, 2021, the research subjects had been followed up for 150 283 person-years, with a median follow-up time of 8.15 years. During the follow-up period, 3 293 people died, including 1 124 deaths from cardiovascular disease (CVD) and 875 deaths from cancer. Cox regression analysis showed that compared with the population of 0-1 recommended food group, those having more than five recommended food groups had a 19% lower risk of all-cause mortality [hazard ratio ( HR)=0.81, 95% CI: 0.70-0.94] and a 33% lower risk of all-cause mortality ( HR=0.67, 95% CI: 0.52-0.87). Compared with the T2DM population in the physical activity Q1 group, the risk of all-cause mortality, CVD mortality, and cancer mortality among the physical activity Q4 group reduced by 50% ( HR=0.50, 95% CI: 0.45-0.56), 50% ( HR=0.50, 95% CI: 0.41-0.61), and 27% ( HR=0.73, 95% CI: 0.60-0.88), respectively. The combined effect showed that compared with the population in the intake of food categories 0-2 and low physical activity groups, the risk of all-cause, CVD mortality, and cancer mortality in the intake of food categories 4-9 and high physical activity groups reduced by 55% ( HR=0.45, 95% CI: 0.38-0.53), 56% ( HR=0.44, 95% CI: 0.32-0.59), and 40% ( HR=0.60, 95% CI: 0.44-0.82), respectively. Conclusion:Type of dietary intake, physical activity, and their combined effects are associated with a reduced mortality risk in patients with T2DM.

Recent studies have showed that thrombosis is closely related to leucocytes involved in immunity. Interfering with the binding of leukocyte integrin Mac-1 and platelet GPIbα can inhibit thrombosis without affecting physiological coagulation. Mac-1-GPIbα is proposed as a potential safety target for antithrombotic agents. Guanxinning tablet (GXNT) is an oral Chinese patent medicine used for the treatment of angina pectoris, which contains phenolic acid active ingredients, such as salvianolic acids, ferulic acid, chlorogenic acid, caffeic acid, rosmarinic acid, tanshinol, and protocatechualdehyde. Our previous studies demonstrated that GXN exhibited significant antithrombotic effects, and clinical studies suggested that it did not increase bleeding risk. In addition, GXN exerted a significantly regulatory effect on immune inflammation. In the current study, we intended to evaluate the effects of GXN on bleeding events and explore the safety antithrombotic mechanism of GXN based on leukocyte-platelet interaction. First, we established a gastric ulcer model induced by acetic acid in rats and found that GXN not only did not increase the degree of gastrointestinal bleeding when gastric ulcer occurred, but also had a certain promoting effect on the healing of gastric ulcer. Second, in vitroexperiments showed that after pretreatment with GXN and activation by phorbol 12-myristate-13-acetate (PMA), the adhesion and aggregation of leukocytes with human platelets were reduced. It was also found that GXN reduced the expression and activation of Mac-1 in leucocytes, and inhibited platelet activation due to leukocyte engagement via Mac-1. Overall, the results suggest that GXN may be a safe antithrombotic agent, and its low bleeding risk mechanism is probably related to inhibited leukocyte-platelet aggregation and its interaction target Mac-1-GPIbα.
Animals ; Fibrinolytic Agents ; Humans ; Integrins ; Leukocytes ; Macrophage-1 Antigen ; Rats ; Stomach Ulcer ; Tablets ; Thrombosis