BACKGROUND:Whether coiled-coil-helix-coiled-coil-helix domain-containing 2(CHCHD2)can regulate the neuroprotective role of PINK1/Parkin-mediated mitochondrial autophagy in Parkinson's disease remains unknown.OBJECTIVE:To explore the role and mechanisms of CHCHD2 in the 6-hydroxydopamine-induced Parkinson's disease cell model in mediating the PINK1/Parkin signaling pathway and its involvement in mitochondrial autophagy.METHODS:Utilizing recombinant plasmid transfection technology to overexpress or knockdown CHCHD2,SH-SY5Y cells were constructed with a Parkinson's disease model using 6-hydroxydopamine,and divided into control group,model group,overexpression negative control+6-hydroxydopamine group,knockdown negative control+6-hydroxydopamine group,overexpression CHCHD2+6-hydroxydopamine group,and knockdown CHCHD2+6-hydroxydopamine group.Western blot assay and RT-qPCR were used to detect CHCHD2 expression.Western blot assay was utilized to detect the protein expression of LC3Ⅰ/Ⅱ,p62,MFN1,COXIV,DRP1,PINK1,Parkin,TIM23,Bax,Bcl-2,and cleaved-caspase 3.CCK-8 assay,JC-1,and reactive oxygen species assay kits were used to measure cell viability,mitochondrial membrane potential,and reactive oxygen levels.Monodansylcadaverine staining was used to observe cell autophagy.Transmission electron microscopy was used to observe autophagolysosomes.RESULTS AND CONCLUSION:(1)Compared with the control group,cell activity,mitochondrial membrane potential,and the protein expression levels of CHCHD2,PINK1,and Parkin were decreased,and the levels of reactive oxygen species,apoptosis,and LC3Ⅰ/Ⅱ and p62 proteins were increased(P＜0.05),and the presence of autophagic lysosomes was observed in the model group.(2)Compared with the model group,overexpression of CHCHD2 could reduce the level of cellular reactive oxygen species,increase the mitochondrial membrane potential,and the expression levels of PINK1,Parkin,and MFN1 proteins,and observed an increase in mitochondrial autolysosomes,and the knockdown of CHCHD2 had the opposite effect with the increase of COXIV,TIM23 and p-DRP1 protein expression(P＜0.05).(3)Compared with the model group,overexpression of CHCHD2 reduced apoptosis,up-regulated Bcl-2,and down-regulated the expression of Bax and cleaved-caspase3 proteins,while knockdown of CHCHD2 had the opposite effect(P＜0.05).(4)The results suggest that CHCHD2 can play a neuroprotective role by promoting PINK1/Parkin-mediated mitochondrial autophagy,improving mitochondrial function,and alleviating apoptosis in 6-hydroxydopamine-induced Parkinson's disease cell models.

BACKGROUND:Whether coiled-coil-helix-coiled-coil-helix domain-containing 2(CHCHD2)can regulate the neuroprotective role of PINK1/Parkin-mediated mitochondrial autophagy in Parkinson's disease remains unknown.OBJECTIVE:To explore the role and mechanisms of CHCHD2 in the 6-hydroxydopamine-induced Parkinson's disease cell model in mediating the PINK1/Parkin signaling pathway and its involvement in mitochondrial autophagy.METHODS:Utilizing recombinant plasmid transfection technology to overexpress or knockdown CHCHD2,SH-SY5Y cells were constructed with a Parkinson's disease model using 6-hydroxydopamine,and divided into control group,model group,overexpression negative control+6-hydroxydopamine group,knockdown negative control+6-hydroxydopamine group,overexpression CHCHD2+6-hydroxydopamine group,and knockdown CHCHD2+6-hydroxydopamine group.Western blot assay and RT-qPCR were used to detect CHCHD2 expression.Western blot assay was utilized to detect the protein expression of LC3Ⅰ/Ⅱ,p62,MFN1,COXIV,DRP1,PINK1,Parkin,TIM23,Bax,Bcl-2,and cleaved-caspase 3.CCK-8 assay,JC-1,and reactive oxygen species assay kits were used to measure cell viability,mitochondrial membrane potential,and reactive oxygen levels.Monodansylcadaverine staining was used to observe cell autophagy.Transmission electron microscopy was used to observe autophagolysosomes.RESULTS AND CONCLUSION:(1)Compared with the control group,cell activity,mitochondrial membrane potential,and the protein expression levels of CHCHD2,PINK1,and Parkin were decreased,and the levels of reactive oxygen species,apoptosis,and LC3Ⅰ/Ⅱ and p62 proteins were increased(P＜0.05),and the presence of autophagic lysosomes was observed in the model group.(2)Compared with the model group,overexpression of CHCHD2 could reduce the level of cellular reactive oxygen species,increase the mitochondrial membrane potential,and the expression levels of PINK1,Parkin,and MFN1 proteins,and observed an increase in mitochondrial autolysosomes,and the knockdown of CHCHD2 had the opposite effect with the increase of COXIV,TIM23 and p-DRP1 protein expression(P＜0.05).(3)Compared with the model group,overexpression of CHCHD2 reduced apoptosis,up-regulated Bcl-2,and down-regulated the expression of Bax and cleaved-caspase3 proteins,while knockdown of CHCHD2 had the opposite effect(P＜0.05).(4)The results suggest that CHCHD2 can play a neuroprotective role by promoting PINK1/Parkin-mediated mitochondrial autophagy,improving mitochondrial function,and alleviating apoptosis in 6-hydroxydopamine-induced Parkinson's disease cell models.

BACKGROUND:Studies have found that nicotine can activate the dopamine system,slowing the progression of Parkinson's disease,but the specific mechanism is still unclear.Research on the neuroprotective mechanism of nicotine in animal models of Parkinson's disease is lacking. OBJECTIVE:To investigate the neuroprotective effect of nicotine on rotenone-induced Parkinson's disease in mice. METHODS:Twenty-eight C57BL/6 mice were randomly divided into vehicle group,rotenone group,autophagy agonist group and nicotine group,with seven mice in each group.Dopaminergic nerve damage was induced by rotenone in C57BL/6 mice,and the autophagy agonist(rapamycin)or nicotine was given before modeling.The spatial exploration function of the mice was observed by open field test.Western blot and Q-PCR were used to detect the expression of α-synuclein,autophagy related factors Beclin-1 and P62,and apoptosis-related factors Bax,Bcl-2 and Cleaved-caspase3 in the nigra of each group.The deposition of mitochondria,autophagosomes and lipofuscin in nigra cells were observed by transmission electron microscopy.The survival of neurons was observed by Nissl staining.The expression of tyrosine hydroxylase was observed by immunofluorescence and immunohistochemical staining. RESULTS AND CONCLUSION:The open field test showed that the distance,average speed and time of movement were reduced in the rotenone group compared with the solvent group.Compared with the rotenone group,the exercise distance,average speed and exercise time of mice were increased in the nicotine group and autophagy agonist group(P＜0.05).The results of immunofluorescence and immunohistochemistry showed that the mean fluorescence intensity and mean absorbance value of tyrosine hydroxylase in the rotenone group decreased compared with that in the solvent group.Compared with the rotenone group,the mean fluorescence intensity and mean absorbance value of tyrosine hydroxylase were increased in the nicotine group and autophagy agonist group.Western blot and Q-PCR results showed that compared with the solvent group,the expressions of α-synuclein and P62 in the rotenone group were increased,while Beclin-1 expression was decreased(P＜0.05);compared with the rotenone group,the expression of α-synuclein and P62 decreased in the nicotine group and autophagy agonist group,and the expression of Beclin-1 increased(P＜0.05).Compared with the solvent group,the expressions of Bax and Cleaved caspase3 were increased and Bcl-2 expression was decreased in the rotenone group(P＜0.05);compared with the rothenone group,the expressions of Bax and Cleaved-caspase3 were decreased and the expression of Bcl-2 was increased in the nicotine and autophagy agonist groups(P＜0.05).To conclude,nicotine may have a dopaminergic neuroprotective effect on rotenone-induced Parkinson's disease mouse models by improving autophagy dysfunction and reducing apoptosis.

Objective:To explore the risk factors for death within 1 year after hip fracture surgery in the elderly.Methods:A case control study was made on the clinical data of 551 elderly patients with hip fracture treated in Second Affiliated Hospital of Soochow University from January 2019 to December 2020, including 182 males and 369 females; aged 65-100 years [80(73,86)years]. Joint replacement, cannulated screw fixation or proximal femoral nail fixation were performed. The patients were divided into survival group ( n=494) and death group ( n=57) based on the death within 1 year after surgery recorded at postoperative telephone follow-up. The gender, age, hypertension, diabetes, cardiovascular diseases, chronic respiratory diseases, neurological diseases, chronic renal failure, anemia on admission, fracture types, American anesthesiologist Association (ASA) classification, operative methods, preoperative waiting time, duration of operation and perioperative blood transfusion were recorded in two groups. Univariate Cox regression was used to analyze the correlation between the above indexes and death within 1 year after surgery. All indexes with P<0.2 in the univariate analysis were included in multivariate Cox regression analysis to clarify the independent risk factors for death within 1 year after surgery. Results:Univariate Cox regression analysis showed that death within 1 year after surgery correlated with gender, age chronic respiratory diseases, chronic renal failure and anemia on admission (all P<0.01), but not with hypertension, diabetes, cardiovascular diseases, neurological diseases, fracture types, ASA classification, operative methods, preoperative waiting time, duration of operation or perioperative blood transfusion (all P>0.05). Multivariate Cox regression analysis showed that male ( HR=2.08, 95% CI 1.20, 3.61, P<0.01), age ≥ 80 years ( HR=2.22, 95% CI 1.15, 4.28, P<0.05), chronic respiratory diseases ( HR=2.54, 95% CI 1.19, 5.40, P<0.05), chronic renal failure ( HR=4.57, 95% CI 1.27, 16.44, P<0.05), anemia on admission ( HR=2.82, 95% CI 1.38, 5.76, P<0.01) were significantly associated with death within 1 year after surgery. Conclusion:Male age≥ 80 years, chronic respiratory disease, chronic renal failure and anemia on admission are independent risk factors for death within 1 year after hip fracture surgery in the elderly.

Objective: To investigate the guiding significance of Daping orthopedics operative risk scoring system for senile patient (DORSSSP) for stratified treatment of elderly patients with severe hip fractures. Methods: A retrospectively case-control study was performed for data of 440 elderly patients with hip fracture admitted to Second Affiliated Hospital of Soochow University from January 2014 to January 2018, including 130 male and 310 female patients aged 60-98 years [(79.3±6.3)years]. According to the DORSSSP scoring system, the patients were divided into low risk group (Group A, n=208), medium risk group (Group B, n=157) and high risk group without SICU transfer after operation (Group C, n=23) and high risk group with SICU transfer after operation (Group D, n=52). The risk prediction results of each group were recorded and compared with the actual complications and mortality. Results: (1)According to the prediction of DORSSSP, the number of postoperative complications in Groups A, B, C and D were 52, 60, 14 and 31, respectively, while the number of actual complications after operation was 45, 55, 13 and 16. There was significant difference between the predicted value and the actual value of postoperative complications in Group D (P<0.01), which was not found in other three groups (P>0.05). The incidence of postoperative complications in Group D was lower than that in Group C (P<0.05). (2) According to the prediction of DORSSSP, the number of postoperative death in Groups A, B, C and D were 0, three, two and four, respectively, while the number of actual death after operation was 0, one, two and one, respectively. The predicted value and the actual value of death were significantly different in Group D (P<0.05), but were not in other three groups (P>0.05). The incidence of postoperative death in Group D was lower than that in Group C (P>0.05). Conclusions There is a good correlation between DORSSSP score and postoperative complications and mortality. Based on DORSSSP score for stratified treatment, the interventional treatment of elderly patients with severe hip fracture after operation into SICU can better reduce the incidence of complications.

Objective To evaluate the role of serine-threonine kinase (Akt)/glycogen synthase kinase-3 beta (GSK-3β) signaling pathway in isoflurane preconditioning-induced inhibition of mitochondrial permeability transition pore protein (mPTP) opening during myocardial ischemia-reperfusion (I/R) in rats.Methods Ninety-six male Sprague-Dawley rats,aged 3-4 months,weighing 200-250 g,were randomly divided into 4 groups (n =24 each) using a random number table:control group (group C);I/R group;isoflurane preconditioning group (group IPC);Akt inhibitor MK-2206 group (group MK).Myocardial I/R was induced by occlusion of the anterior descending branch of the left coronary artery for 30 min followed by 2 h of reperfusion.In group IPC,1.5％ isoflurane was inhaled for 30 min followed by 45 min washout,and then the model of myocardial I/R injury was established.In group MK,MK-2206 300 μg/kg (in dimethyl sulfoxide) was injected intraperitoneally at 30 min before isoflurane inhalation.At 2 h of reperfusion,8 rats were selected and sacrificed,and the hearts were removed for determination of myocardial infarct size.At 2 h of reperfusion,8 rats were selected,and blood samples were collected from the right internal jugular vein for determination of serum cardiac troponin Ⅰ (cTnI) concentrations.The rats were then sacrificed,and myocardial specimens were obtained for determination of the expression of phosphorylated GSK-3β (p-GSK-3β) in cytoplasm and mitochondria (by Western blot) and co-expression of p-GSK-3β with adenine nucleotide translocator (ANT),voltage-dependent anion channel or cyclophilin D in myocardial tissues (using co-immunoprecipitation).At 2 h of reperfusion,8 rats were selected and sacrificed,myocardial cells were obtained,and the opening time of mPTP was determined with a laser scanning confocal microscope.Results Compared with group C,the myocardial infarct size and serum cTnI concentrations were significantly increased,and the expression of p-GSK-3β in cytoplasm and mitochondria was up-regulated in I/R and IPC groups,the co-expression of p-GSK-3β with ANT was significantly down-regulated,and the opening time of mPTP was shortened in group I/R,and the co-expression of p-GSK-3β with ANT was significantly up-regulated,and the opening time of mPTP was prolonged in group IPC (P＜0.05).Compared with group I/R,the myocardial infarct size and serum cTnI concentrations were significantly decreased,the expression of p-GSK-3β in cytoplasm and mitochondria was up-regulated,the co-expression of p-GSK-3β with ANT was significantly up-regulated,and the opening time of mPTP was prolonged in group IPC,and the opening time of mPTP was significantly prolonged (P＜0.05),and no significant change was found in the other parameters in group MK (P＞0.05).Compared with group IPC,the myocardial infarct size and serum cTnI concentrations were significantly increased,the expression of p-GSK-3β in cytoplasm and mitochondria was up-regulated,the co-expression of p-GSK-3β with ANT was significantly down-regulated,and the opening time of mPTP was shortened in group MK (P＜0.05).No co-expression of p-GSK-3β with voltage-dependent anion channel or cyclophilin D was found in myocardial tissues.Conclusion The mechanism by which isoflurane preconditioning inhibits mPTP opening during myocardial ischemia-reperfusion is partially related to activation of Akt/GSK-3β signaling pathway in rats.

Research based learning (RBL) is a brand new teaching mode implicated by Shanghai Jiaotong University School of Medicine specifically designed for clinical undergraduates.The nature of RBL is about exploring unknown knowledge and designing and executing comprehensive experiments.RBL aims at creating an open,active student participation,and close student-mentor interaction teaching mode.IBL includes literature study,research aim selection,experiment design and implementation,statistical analysis,results and conclusions,final report writing and defense of the report.Our experience indicates that RBL can substantially improve students' scientific logic and critical thinking and experimental skills,and develop the spirit of a team-player.This scientific training enables students to receive more comprehensive scientific training,facilitate their subsequent clinical research and practice,and assist them to participate in more international scientific exchanges.We propose that the RBL mode should be adopted for clinical student education in other universities.

Objective Retrospectively analyze of urate crystal deposition using dual-source CT in asymptomatic hyperuricaemia and symptomatic gout patients.Methods Sixty patients with asymptomatic hyperuricemia (uric acid ≥540 μmol/L) and 48 patients with gout (without any change in the appearance of the limbs and obvious gout nodules) were selected.All patients underwent dual-energy CT imaging.The location number and size were analyzed using CT gout software.Results Eighteen patients with asymptomatic hyperuricemia with urate crystal deposition in limb joints (13 in foot and ankle, 7 in the hand and wrist, 2 in knee), the average size of crystal was (0.12±0.04) cm3.Forty-eight gout patients (28 cases of early gout and 20 cases of late gout and 20 gout cases) had urate crystal deposition (48 in foot and ankle, 39 in hand and wrist, 27 in knee), the average size of crystal was (1.7±0.7) cm3.The number and size of urate crystals deposited in the joints, tendons and ligaments was higher in patients with gout than asymptomatic hyperuricemia (x2=52.076, P＜0.001;t=44.834, P＜0.001).There was no difference between the early gout and late gout in the size of urate crystals (t=0.163, P＞0.05).Conclusion Urate crystals can deposite in asymptomatic hyperuricemia.However, more and larger urate crystals appear in gout patients, there is no difference in size and number of urate crystals between the course duration of gout.

PurposeTo evaluate the value of dual-source CT angiography (DSCTA) in the diagnosis of carotid atherosclerosis, and to explore the relationship between carotid atherosclerosis and diabetes mellitus (DM).Materials and Methods 145 patients with ischemic stroke were divided into DM group (n=80) and non-DM group (n=65). All patients underwent DSCTA. The location and characteristic of carotid atherosclerosis were analyzed using curve planE reconstruction (CPR), maximum intensity projection (MIP), multi-plane reformation (MPR) and volume rendering (VR). The differences between two groups were analyzed.Results Compared with non-DM group, the body mass index and high blood cholesterol were higher in DM group (t=6.197,P<0.05;χ2=8.372,P<0.01). The incidence of carotid atherosclerosis and vulnerable plaques were also higher in DM group than non-DM group (χ2=11.617 and 9.388,P<0.01). There was no significant difference between DM group and non-DM group in the location of carotid atherosclerosis (χ2=0.160, 0.509, 0.419 and 0.016,P>0.05). Carotid atherosclerosis was more common in carotid bifurcation and carotid artery siphon.Conclusion DSCTA can demonstrate the location and characteristics of carotid atherosclerosis in ischemic stroke patients with diabetes mellitus. Diabetes is a risk factor for the formation of carotid atherosclerosis, especially vulnerable plaques.

Objective To investigate the diagnostic value of dual-source CT angiography (DSCTA) for vertebrobasilar atherosclerosis in patients with transient ischemia attack (TIA).Methods 80 TIA patients underwent DSCTA.Common data were collected.Vertebrobasilar plaques and stenosis degree were analyzed by using CT reconstruction.Results 152 plaques were found in 69 patients(86.2％,69/80),59 plaques(38.9％,59/152)in unilateral vertebral artery in 31 patients,83 plaques (54.6％,83/152) inbilateral vertebral artery in 28 cases,10 plaques (6.6％,10/152) in basilar artery in 10 patients,among which 51(33.6％,51/152)plaques were hard,69(45.3％,69/ 152)plaques were soft,32(21.1％,32/152)plaques were mixed.There were 31 cases (44.9％,31/ 69) with mild stenosis,27 cases (39.1％,27/69) with moderate stenosis,7 cases (10.1％,7/69) with severe stenosis,and 4 cases (5.8％,4/69) had vascular occlusion.Conclusions Vertebrobasilar atherosclerosis is a common cause of TIA.DSCTA can be used to analyze the vertebrobasilar plaques and stenosis degree.Soft plaques and severe stenosis promote TIA.