Tumor necrosis factor-α inhibitors for the treatment of four cases of hidradenitis suppurativa / 中华皮肤科杂志

Four male cases of hidradenitis suppurativa (Hurley stage Ⅱ/Ⅲ) aged 20 - 45 years were collected from Department of Dermatology, Xijing Hospital, the Fourth Military Medical University from August 2017 to December 2019. All the patients presented with sinuses, abscesses and scars on the buttocks, axillary and inguinal regions, and showed a poor response to previous treatment with antibiotics, glucocorticoids, retinoids, traditional Chinese medicine, etc. The patients were treated with intravenous drips of infliximab at a dose of 5 mg/kg at weeks 0, 2 and 6, followed by an every-9-week treatment regimen, or with subcutaneous injection of adalimumab at a dose of 80 mg at weeks 0 and 2, followed by an every-3-week regimen at a dose of 40 mg. Two patients experienced infusion reactions after intravenous drips of infliximab, and then were switched to adalimumab. Three of these patients achieved hidradenitis suppurativa clinical response, whereas 1 showed no response.

Role and related mechanism of Mst-1 on regulating hypoxic reoxygenation induced autophagy and apoptosis in cardiomyocytes of mouse / 中华心血管病杂志

Objective: To explore the role and related mechanism of mammalian sterile 20-like kinase 1(Mst-1)in regulating hypoxia reoxygenation (HR) induced myocardial cell autophagy and apoptosis. Methods: Enzyme digestion method combined with differential adherent method was used to culture neonatal mouse myocardial cells. HR model was established by hypoxia for 24 hours and reoxygenation for 6 hours. The experimental groups including control group (normal cultured cardiomyocytes), Mst-1 empty virus group (cardiomyocytes transfected with recombinant lentiviral empty vector for 48 hours), Mst-1 knockdown group (recombinant lentivirus carrying Mst-1small interfering RNA (siRNA) was transfected into cardiomyocytes for 48 hours), Mst-1 overexpression group (cardiomyocytes were transfected with recombinant lentivirus carrying Mst-1 gene for 48 hours), HR group (cardiomyocytes exposed to HR), Mst-1 knockdown+HR group (HR model of cardiomyocyte was established 48 hours after transfection with recombinant lentivirus carrying Mst-1siRNA) and Mst-1 overexpression+HR group (HR model of cardiomyocyte was established 48 hours after transfection with recombinant lentivirus carrying Mst-1 gene). Real-time fluorescence quantitative RCR (qPCR) and Western blot were used to detect the relative expression of Mst-1 mRNA and protein in the cells, immunofluorescence staining was used to detect cardiomyocyte troponin T (cTnT), and autophagosomes and autophagy enzyme changes. TUNEL method was used to detect myocardial cell apoptosis, Western blot was adopted to detect autophagy-related protein microtubule-related protein 1 light chain 3 (LC3) Ⅱ/LC3 Ⅰ, P62 and apoptosis-related protein cleaved-caspase 9, pro-caspase 9, cleaved-caspase-3, pro-caspase-3, and myeloid leukemia 1 (MCL-1) expression. MCL-1 inhibitor A1210477 was used to validate the signaling pathway of Mst-1 on regulating cardiomyocyte apoptosis and autophagy. Results: Immunofluorescence detection revealed that the cultured cells expressed cardiomyocyte-specific marker cTnT. The expression of Mst-1 in cardiomyocytes increased in HR model. Lentiviral transfection could effectively inhibit or overexpress Mst-1 in treated cells. The levels of autophagosomes and autophagolysosomes in cardiomyocytes undergoing HR and in Mst-1 overexpression+HR group were lower than those of control group, while autophagosomes and autophagolysosomes in cardiomyocytes of Mst-1 knockdown+HR group was significantly higher than in the HR group (all P<0.05). The TUNEL results showed that the proportion of TUNEL positive cells was significantly increased in the HR group and Mst-1 overexpression+HR group than in the control group, while the proportion of TUNEL positive cells was significantly decreased in the Mst-1 knockdown group+HR group as compared to the HR group (all P<0.05). Western blot results showed that the LC3 Ⅱ/LC3 Ⅰ levels were significantly lower, while the expression levels of P62, cleaved-caspase-9 and cleaved-caspase-3 were significantly higher in the HR group and Mst-1 overexpression+HR group than in control group (all P<0.05). The LC3 Ⅱ/LC3 Ⅰ value was significantly higher, and the expression levels of P62, cleaved-caspase-9 and cleaved-caspase-3 were significantly lower in the Mst-1 knockdown+HR group than in the HR group (P both<0.05). The expression level of P-MCL-1 protein was significantly lower in cardiomyocytes of HR and Mst-1 overexpression+HR group than in control group, and the expression level of P-MCL-1 protein was higher in Mst-1 knockdown+HR group than in HR group (P both<0.05). The recovery experiment showed that inhibiting MCL-1 in cells can block the regulatory effect of Mst-1 siRNA on cell autophagy and apoptosis. Conclusion: Inhibiting Mst-1 expression in cardiomyocytes can promote the autophagy of cardiomyocytes induced by hypoxic reoxygenation and reduce the apoptosis of cardiomyocytes via activating McL-1.

Intranasal Administration of Oxytocin Attenuates Stress Responses Following Chronic Complicated Stress in Rats

BACKGROUND/AIMS: Gastrointestinal (GI) symptoms may develop when we fail to adapt to various stressors of our daily life. Central oxytocin (OXT) can counteract the biological actions of corticotropin-releasing factor (CRF), and in turn attenuates stress responses. Administration (intracerebroventricular) of OXT significantly antagonized the inhibitory effects of chronic complicated stress (CCS) on GI dysmotility in rats. However, intracerebroventricular administration is an invasive pathway. Intranasal administration can rapidly deliver peptides to the brain avoiding stress response. The effects of intranasal OXT on hypothalamus-pituitary-adrenal axis and GI motility in CCS conditions have not been investigated. METHODS: A CCS rat model was set up, OXT 5, 10, or 20 μg were intranasal administered, 30 minutes prior to stress loading. Central CRF and OXT expression levels were analyzed, serum corticosterone and OXT concentrations were measured, and gastric and colonic motor functions were evaluated by gastric emptying, fecal pellet output, and motility recording system. RESULTS: Rats in CCS condition showed significantly increased CRF expression and corticosterone concentration, which resulted in delayed gastric emptying and increased fecal pellet output, attenuated gastric motility and enhanced colonic motility were also recorded. OXT 10 μg or 20 μg significantly reduced CRF mRNA expression and the corticosterone concentration, OXT 20 μg also helped to restore GI motor dysfunction induced by CCS. CONCLUSION: Intranasal administration of OXT has an anxiolytic effect and attenuates the hypothalamus-pituitary-adrenal axis in response to CCS, and gave effects which helped to restore GI dysmotility, and might be a new approach for the treatment of stress-induced GI motility disorders.

Effect of remote medical information platform on efficiency of chest pain diagnosis and treatment and on a clinical decision analysis in chest pain center / 中华老年医学杂志

Objective To investigate the effect of remote medical information platform on efficiency of chest pain diagnosis and treatment and on clinical decision analyses in chest pain center.Methods A total of 537 chest pain patients who met the inclusion and exclusion criteria were consecutively enrolled and divided into two groups.The group without the chest pain platform(before setting up the platform)was 251 cases,and the group with chest pain platform(after setting up the platform)was 286 cases.The constituent ratio of acute coronary syndrome (ACS),the numbers of cases of both emergency thrombolysis and emergency percutaneous coronary intervention(PCI),the mean transfer treatment time,the first time medical contact to balloon catheter technique(FMC-to-B) and the door-to-balloon(D-to-B) time were compared between the two groups.The important multivariate factors affecting the D-to-B time were analyzed.Results The group with versus without chest pain platform showed the statistically significant improvements in the parameters as follows:(1)getting long range treatment (249 cases or 87.1％ vs.92 cases or 36.7 ％,x2 =146.56,P ＜0.05),(2) receiving thrombolysis(64 cases or 22.4％ vs.15 cases or 6.0％,x2 =28.61,P＜0.05),(3)average transfer treatment time(TTT) (176.3 ± 86.1 min vs.360.7 ± 107.4 min,t =11.53,P ＜0.05),(4)FMC-to-B(203.8±65.9 min vs.583.4±125.1 min,t =8.41,P＜0.05)and (5)D-to-B time(86.5±30.6 min vs.148.2 ± 41.7 min,t =4.49,P ＜ 0.05).Especially,patients after setting up the chest pain platform reached the standard of D-to-B time less than 90 min.According to whether reaching the standard of D-to-B time or not,clinical decision-making model analysis showed that the average Gini coefficient achieving the millennium development goal(MDG) was highest in the hospital referral,followed by the average transfer treatment time and emergency thrombolysis.Conclusions Reducing average transfer treatment time,improving the efficiency of hospital referral,and refining the remote terminal information platform for chest pain diagnosis and treatment are important for chest pain center by analyzing clinical data of chest pain patients.

Impact and related mechanism on the improvement of hyperglycemia-induced pyroptosis in H9c2 cells by mircoRNA-214 / 中华心血管病杂志

Objective@#To investigate whether microRNA(miR)-214 can improve hyperglycemia induced pyroptosis in H9c2 cells through targeting caspase-1.@*Methods@#H9c2 cells of rats those in good growth condition were selected and incubated into the T25 culture bottle after digestion and passage. Cells were cultured in an incubator at 37 ℃ with 5%CO2, repeat passage was made after cell density reached about 80%, The 5th to 8th generations of cells were selected for the subsequent experiments. To observe the effect of overexpression of miR-214 on pyroptosis and caspase-1 expression in H9c2 cells induced by hyperglycemia, the cells were divided into 4 groups: Control group(H9c2 cells cultured normally), Hyperglycemia group (HG group, 50 mmol/L glucose was used to intervene H9c2 cells for 24 hours), miR-214 mimics+hyperglycosis group (mimics+HG group, H9c2 cells were transfected with miR-214 mimics for 24 hours and then treated with 50 mmol/L hyperglycosis for 24 hours), miR-214 mimic-negative control+hyperglycaemic group(MNC+HG group, H9c2 cells were transfected with miR-214 mimic-negative control for 24 hours and then treated with 50 mmol/L hyperglycaemic for 24 hours). In order to further verify the anti-pyroptosis effect of miR-214 was mediated by targeted inhibition on caspase-1, cells overexpressing caspase-1 were used in the rescue experiment. The cells overexpressing caspase-1 were divided into 4 groups: Hyperglycemia group (HG group, 50 mmol/L glucose was used to intervene H9c2 cells for 24 hours), miR-214 mimics+hyperglycosis group (mimics+HG group, H9c2 cells were transfected with miR-214 mimics for 24 hours and then treated with 50 mmol/L hyperglycosis for 24 hours), miR-214 mimics+hyperglycosis+recombinant adenovirus (Ad-caspase-1-EGFP) group with caspase-1 gene and EGFP green fluorescent protein expression (mimics+HG+Ad-caspase-1-EGFP group, H9c2 cells were transfected with caspase-1-green fluorescent protein-carrying adenovirus for 48 hours, followed by transfection of miR-214 mimics for 24 hours, and then treated with 50 mmol/L hyperglycaemia for 24 hours), miR-214 mimics+HG+Ad-EGFP empty virus group (mimics+HG+Ad-EGFP group, H9c2 cells were transfected with empty adenovirus containing green fluorescent protein for 48 hours, followed by transfection with miR-214 mimics for 24 hours, and then treated with 50 mmol/L hyperglycosis for 24 hours). The mRNA expression levels of miRNA-214 and caspase-1 in cells were detected by real-time quantitative PCR. The expression and localization of caspase-1 protein were detected by immunofluorescence assay. Western blot was used to detect protein expression levels of procaspase-1, cleaved caspase-1, NLRP3 and ACS with β-actin as internal reference. The secretion of IL-1β and IL-18 in cell culture medium was detected by ELISA. The correlation between miR-214 and caspase-1 was detected by double luciferase reporter gene.@*Results@#(1) The mRNA expression levels of miR-214 and caspase-1 in each group: the mRNA expressions of miR-214 in HG group and MNC+HG group were significantly lower than that in control group(P<0.05). The mRNA expression of miR-214 in mimics+HG group was significantly higher than that in control group (P<0.05). The mRNA expression levels of caspase-1 in HG group and MNC+HG group were significantly higher than that in control group(P<0.05). The mRNA expression level of caspase-1 in mimics+HG group was lower than that in control group(P<0.05). (2) The expression of caspase-1 in each group: the green fluorescence intensity in the control group was weak, which was strong in the HG group and MNC+HG group. The green fluorescence expression was weaker in mimics+HG group than in HG group. (3) ASC and NLRP3 protein expression levels in each group: ASC and NLRP3 protein expression levels in HG group and MNC+HG group were higher than those in control group(P<0.05). ASC and NLRP3 protein expression levels were significantly lower in mimics+HG group than in mimics+HG group (P<0.05). (4) The secretion of IL-1β and IL-18 in the cell culture medium of each group: the content of IL-1β and IL-18 in the cell culture medium of HG group and MNC+HG group was significantly higher than that of control group (P<0.05). The content of IL-1β and IL-18 in the cell culture medium of mimics+HG group was significantly lower than that of the HG group (P<0.05). (5) Correlation between miR-214 and caspase-1: miR-214 specifically binds to caspase-1 3 ′UTR. Meanwhile, Western blot results showed that cleaved caspase-1 protein expression levels were significantly higher in both HG group and MNC+HG group than in control group (P<0.05). The levels of cleaved caspase-1 were significantly lower in mimics+HG group than in HG group (P<0.05). There was no significant difference in procaspase-1 expression among groups (P>0.05). (6) The expression levels of procaspase-1, cleaved caspase-1, ASC and NLRP3 in each group in rescue experiment: there was no significant difference in the expression of procaspase-1 in each group (P>0.05). Cleaved caspase-1, ASC and NLRP3 protein expressions were significantly lower in mimics+HG group than in HG group (P<0.05). However, cleaved caspase-1, ASC and NLRP3 protein expressions were significantly higher in mimics+HG+ Ad-caspase-1-EGFP group than in mimics+HG group (P<0.05). (7) The expression of IL-1β and IL-18 in rescue experiment: the secretions of IL-1β and IL-18 in the cell culture medium of the mimics+HG group were significantly lower than that of HG group (P<0.05), which were significantly higher in mimics+HG+Ad-caspase-1-EGFP group than in mimics+HG group (P<0.05).@*Conclusion@#miR-214 can improve the hyperglycemia induced pyroptosis in H9c2 cells by targeted inhibition of the caspase-1.

Experiment of effects of HMGB-1 combined with MSCs transplantation on cardiac function in rats with acute myocardial infarction / 重庆医学

Objective To investigate the effects and mechanisms of HMGB-1 combined with MSCs transplantation on the heart function in rat with acute myocardial infarction.Methods A total of 144 male SD rats were divided into the healthy control group,model control group,MSCs transplantation group,HMGB-1 injection group,HMGB-1 injection+ MSCs transplantation group,HMGB-1 BoxA injection+MSCs transplantation group.On the 28th day after surgery,the heart function,myocardial pathological section,myocardial infarction area and new vessel density in infarction area were detected.Andthe level of related serum cytokines were measured on the 3rd,7th and 28th days after surgery.Results On the 28th day after surgery,left ventricular end diastolic dimension (LVDd) and left ventricular iiaternal diameter at end-systole (LVDs) in the HMGB-1 injection+ MSCs transplantation group were significantly decreased and the fractional shortening (FS) and ejection fraction (EF) value were significantly increased compared with the other five groups (P＜0.05);the infarction area in the HMGB-1 injection+MSCs transplantation group was significantly decreased and the new vessels number in the infarction area was significantly increased compared with the other model groups (P＜0.05).On the 3rd and 7th days after surgery,serum TLR4 and VEGF levels in the HMGB-1 injection+MSCs transplantation group were the highest (P＜0.05).On the 7th and 28th days after surgery,the levels of serum IL-6,NF-κB and TNF-α in the HMGB-1 injection+MSCs transplantation group were the lowest among all groups (P＜0,05).Conclusion HMGB-1 injection combined with MSCs transplantation treatment can effectively improve the prognosis of myocardial infarction.

Effect of miR-21 over-expression on proliferation, migration and diffe-rentiation of c-Kit+ cardiac stem cells / 中国病理生理杂志

AIM:To explore the effect of microRNA (miR)-21 on proliferation, migration and differentiation abilities of c-Kit+ cardiac stem cells (CSCs). METHODS:c-Kit+ CSCs were cultured and selected by the methods of en-zyme digestion and magnetic bead separation. miR-21 mimics (50 nmol/L) and mimics negative control ( MNC) were transfected into c-Kit+CSCs with Lipofectamine?2000. The cells was divided into 3 groups:control group:c-Kit+ CSCs without any pretreatment; MNC group:the cells were transfected with MNC for 48 h; mimics group:the cells were trans-fected with miR-21 mimics for 48 h. qPCR was used to assess the expression of miR-21 in each group. CCK-8 and EdU as-says were used to determine the cell proliferation. qPCR and immunofluorescence were used to detect the differentiation in each group. Scratch assay was adopted to explore the migration ability of the cells. RESULTS:The expression of c-Kit in the c-Kit+ CSCs were 90.8%, with 0.6% of CD45 and 0.5% of CD34. A significant increase in miR-21 expression was observed when the cells were transfected with miR-21 mimics for 48 h ( P<0.05). CCK-8 and EdU assays showed that miR-21 significantly increased cell proliferation as compared with MNC group and control group (P<0.05). No difference in the expression of Nkx2.5, CD31 and α-SMA at mRNA and protein levels was observed, and no difference of the migra-tion ability in 3 groups of the c-Kit+ CSCs was found. CONCLUSION:Over-expression of miR-21 significantly promotes the proliferation of c-Kit+ CSCs, without any effect on the cell migration and differentiation.

Optical coherence tomographic analysis of in-stent neoatherosclerosis in lesions with restenosis after drug-eluting stent implantation / 中华心血管病杂志

Objective@#To explore the imaging characteristics and related influencing factors of in-stent neoatherosclerosis (ISNA) in patients with restenosis after drug-eluting stent(DES) implantation with optical coherence tomography(OCT).@*Methods@#A total of 25 cases of coronary heart disease patients(DES placement time ≥8 months) with coronary artery angiography showing DES in-stent restenosis (ISR) in Zunyi medical college affiliated hospital from July 2013 to December 2015 were included in this study and patient's data were retrospectively analyzed.In these patients with ISR, OCT images were acquired before percutaneous coronary intervention. Patients were divided into the ISNA group (12 patients and 12 lesions) and non-ISNA group(13 patients and 13 lesions) according to the result of OCT. ISNA on OCT was defined as neointima formation with the presence of lipids or calcification.@*Results@#(1) The incidence of chronic kidney disease and increased low-density lipoprotein cholesterol level in ISNA group were significant higher than that in non-ISNA group(all P<0.05). The stent implantation time in ISNA group was longer than that in the non-ISNA group(53.0(14.0, 81.0) months vs. 15.0(8.5, 32.5) months, P<0.01). In addition, clinical manifestation of acute coronary syndrome was present in 8 out of 12 patientsin ISNA group, and stable angina pectoris was found in 10 out of 13 casesin non-ISNA group(P<0.01). (2) Quantitative analysis of OCT showed that the lumen area was less in ISNA group than in non-ISNA group((3.45±1.82)mm2 vs. (4.17±1.68)mm2, P<0.01), and neointimal area(3.89(2.26, 5.52)mm2 vs. 2.96(1.99, 4.22)mm2, P<0.01), neointimal load (53.15(40.18, 67.30)% vs. 41.54(32.08, 56.91)%, P<0.01), neointimal thickness(0.98(0.63, 1.36)μm vs. 0.72(0.51, 1.03)μm, P<0.01) were higher in ISNA group than in non-ISNA group.(3)Qualitative analysis of OCT showed that the prevalence of homogeneous intima was less in the ISNA group than in the non-ISNA group ((41.42±22.56)% vs.(72.06±18.68)%, P<0.05), on the contrary, the heterogeneous intima was more common in the ISNA group ((58.57±22.56)% vs. (27.94±18.68)%, P<0.05). There was no significant difference between two groups in the peri-stentmicrovessels (9/12 vs. 5/13,P>0.05), and prevalence of intraintimalmicrovessels was higher in the ISNA group than in non-ISNA group (7/12 vs. 2/13, P<0.05). In addition, thin cap fibrous plaque(7/12 vs. 0, P<0.01), disrupted intima with visible cavity (7/12 vs. 1/13, P<0.05),andintraluminal red thrombus(7/12 vs. 1/13, P<0.05) were significantly higher in ISNA group than in non-ISNA group.@*Conclusions@#Results of OCT show that ISNA occurs frequently in patients with ISR after DES implantation. The stent implantation time, incidence of chronic kidney disease and higher low-density lipoprotein cholesterol level are associated with the formation of ISNA in these patients.

Valuation Traceability of Reference Extract of Total Lactones from Ginkgo Leaf for Quantitative Analysis / 医药导报

Objective To prepare the phmaceutical reference materials of total lactones extract from ginkgo leaf for quantitative analysis.Methods A HPLC determination method was developed to investigate the uniformity and stability of the reference extract of ginkgo leaf total lactones using with bilobalide, ginkgolide A, ginkgolide B and ginkgolide C as the indexes.Three laboratories participated in the collaborative calibration test.Results The four components in reference extract had good uniformity and stability with RSD less than 2.0%;the marked values of the four components had been determined through statistical data analysis which provided by assigned traceability values.The values of bilobalide, ginkgolide A, ginkgolide B, and ginkgolide C were 39.54%, 29.03%, 15.96% and 11.69%, respectively.Conclusion The reference extract of ginkgo leaf total lactones can be prepared for quality control in future quantitative analysis.

Proportion of regulatory T cells and expression of interleukin-17A and-27 in the peripheral blood of patients with chronic spontaneous urticaria and their significance / 中华皮肤科杂志

Objective To explore the role of peripheral blood CD4+CD25+CD127low Treg cells,interleukin-17A (IL-17A) and IL-27 in the pathogenesis of chronic spontaneous urticaria (CSU).Methods Peripheral blood samples were obtained from 37 patients with CSU (CSU group)and 40 healthy controls (control group).Flow cytometry was performed to determine the percentage of CD4+CD25+CD127low Treg cells in the peripheral blood,and enzyme-linked immunosorbent assay (ELISA) to detect the serum levels of IL-17A and IL-27.Results The percentage of CD4+CD25+CD127low Treg cells in the peripheral blood (5.99％ ± 2.72％ vs.9.07％ ± 3.44 ％,t =4.325,P ＜ 0.01) and the serum level of IL-27 (20.54 ± 7.65 ng/L vs.26.63 ± 9.72 ng/L,t =3.039,P =0.003) were both significantly lower in the CSU group than in the control group.However,there was no significant difference in the serum level of IL-17A between the 2 groups (P =0.529).Among the patients with CSU,the level of IL-17A was negatively correlated with the percentage of CD4+CD25+CD127low Treg cells (r =-0.359,P =0.029),while the urticaria activity score (UAS) was uncorrelated with the levels of IL-17A and IL-27 as well as the percentage of CD4+CD25+ CD127low Treg cells (r =-0.076,-0.083,-0.053 respectively,all P ＞ 0.05).Conclusion There may be Th17/Treg imbalance in the peripheral blood of patients with CSU,and IL-27 may be involved in the occurrence of CSU.

Applications of Reference Extract from Ginkgo Leaf Total Lactones for Quantitative Assay of Ginkgo Folium / 医药导报

Objective To explore the feasibility of using the quantitative reference extract of ginkgo leaf total lactones instead of single component reference for the quantitative assay of Ginkgo Folium.Methods HPLC-ELSD method was performed by using a Diamonsil C18 column (250 mm×4.6 mm,5 μm) with methanol-water as the mobile phase at the gradient elution mode.Flow rate was 1.0 mL·min-1.The parameters of ELSD detector were as follows,the drifit tube temperature was 105 ℃,and the flow rate of nitrogen(N2) was 3 L·min-1.Results The linear ranges of ginkgolide A,ginkgolide B,ginkgolide C,and bilobalide were 0.735-5.879 μg (r=0.999 6),0.404-6.060 μg (r=0.999 6),0.296-4.439 μg (r=0.999 6),and 1.001-6.006 μg (r=0.999 7),respectively.The recoveries and RSD of the four components were 95.6% (4.0%),97.3% (4.5%),99.3% (5.0%),and 100.4% (2.1%),respectively.Conclusion The quantitative reference extract of ginkgo leaf total lactones can be used as the substitute for the determination of terpene lactones.

Effect and related mechanism of miRNA-21 on hydrogen peroxide-induced C-kit+ cardiac stem cells apoptosis / 中华心血管病杂志

Objective@#To explored the effect and related mechanism of miRNA-21 on hydrogen peroxide-induced C-kit+ cardiac stem cells apoptosis.@*Methods@#C-kit+ cardiac stem cells were isolated from SD rats by the methods of enzyme digestion and magnetic bead. Cells were divided into the following experimental groups: (1) negative control mimics (NCM)group: cells were transfected with negative control miRNA-21 mimics for 48 hours; (2)mimics group: cells were transfected with miRNA-21 mimics for 48 hours; (3) NCM+ H2O2 group: negative control miRNA-21 mimics were transfected into cells for 48 hours and then treated with 100 μmol H2O2 for 2 hours; (4)mimics+ H2O2 group: miRNA-21 mimics were transfected into cells for 48 hours and then treated with 100 μmol H2O2 for 2 hours. mRNA of miRNA-21 was detected by RT-PCR. The apoptosis rate of C-kit+ cardiac stem cells was determined using the annexin V-FITC/PI staining assay. Western blot was employed to measure the expression of apoptosis related proteins(Caspase-3, Bax, and Bcl-2).@*Results@#(1) Compared with the NCM group, the mRNA expression level of miRNA-21 was significantly up-regulated in mimics group, while obviously down-regulated in NCM+ H2O2 group(all P<0.05). Compared with the mimics group, the mRNA expression levels of miRNA-21 in mimics+ H2O2 group was significantly downregulated (P<0.05), but remarkably upregulated compared with the NCM+ H2O2 group(P<0.05). (2) Flow cytometry results indicated that the early apoptosis rates were similar between the NCM group and mimics group ((4.57±3.45)% vs. (5.13±3.21)%, P>0.05). Compared with the NCM group, the early apoptosis rates were remarkably increased ((79.07±5.75)% vs.(4.57±3.45)%, P<0.05) in NCM+ H2O2 group. Compared with the mimics group, the early apoptosis was significantly up-regulated in the mimics+ H2O2 group ((30.27±1.36)% vs.(5.13±3.21)%, P<0.05), which were further down-regulated in mimics+ H2O2 group compared with the NCM+ H2O2 group ((30.27±1.36)% vs.(79.07±5.75)%, P<0.05). (3) Western blot results showed similar protein expression of Caspase-3, Bax and Bcl-2 between NCM group and mimics group(all P>0.05). Compared with the NCM group, the Caspase-3 and Bax protein expression was significantly increased in NCM+ H2O2 group (all P<0.05), but the protein expression level of Bcl-2 was similar between the 2 groups(P>0.05). The Caspase-3 and Bax protein expression was markedly decreased, while Bcl-2 apparently increased in the mimics+ H2O2 group compared with the NCM+ H2O2 group(all P<0.05).@*Conclusion@#Overexpression of miRNA-21 protects the C-kit+ cardiac stem cells from apoptosis caused by oxidative stress through downregulating proapoptotic and upregulating the antiapoptotic proteins.

Observation for the Effect of Vacuum Sealing Drainage in Treating the Patients With Pacemaker Pocket Infection / 中国循环杂志

Objective: Pocket infection in patients after total removal of implanted pacemaker has many problems for their electronic system; our research intends to explore the feasibility of conservatively treating such infection and retain the electronic system. Methods: A total of 4 patients with pacemaker pocket infection in our hospital from 2015-01 to 2016-02 were studied. Thorough debridement and disinfection were conducted in infected pockets and devices, meanwhile vacuum sealing drainage was applied. Electrode wire was kept and intravenous antibiotics were given for (7-10) days after the operation in all patients. Results: The average time of infection occurred at 14.75 months after operation with the type of isolated pacemaker pocket infection. Pocket vacuum suction drainage was performed, with the mean of 7.25 (5-10) months follow-up observation, infection was disappeared and the patients had good wound healing. Conclusion: With thorough debridement of infected pocket, rational treatment of residual electronic system and vacuum sealing drainage, the infection might be effectively controlled for complete recovery without lead extraction in relevant patients.

Transfection of calcitonin gene-related peptide mediated by lentivirus vec-tor in vitro and its effects on cardiac stem cell viability / 中国病理生理杂志

AIM:To investigate the effect of calcitonin gene-related peptide (CGRP)transfection into c-kitpos cardiac stem cells (c-kit+CSCs) on the cell viability.METHODS: Under the sterile condition, the auricles of SD rats were taken out , and then c-kit +CSCs were collected through enzyme digestion and immunomagnetic bead separation (MACS).The cells were identified by flow cytometry .c-kit +CSCs were transfected with enhanced green fluorescent pro-tein CGRP lentiviral vector ( Lv-EGFP-CGRP) or enhanced green fluorescent protein lentiviral vector ( Lv-EGFP).The cells were randomly divided into Lv-EGFP-CGRP-CSCs group , Lv-EGFP-CSCs group and CSCs group .The transfection was observed under the fluorescence microscope .The transfection efficiency was detected by flow cytometry .The CGRP protein secretion in the cell culture supernatants was detected by ELISA .The viability of c-kit+CSCs transfected with Lv-EGFP-CGRP or Lv-EGFP was measured by CCK-8 assay.RESULTS:c-kit+CSCs were isolated and cultured successfully .The expression positive rate of c-kit was 91.0%and the expression positive rates of CD 45 and CD34 were 4.5% and 4.0%, respectively.After transfected with lentivirus for 48 h, the stable fluorescence in c-kit +CSCs was observed under fluores-cence microscope .The transfection efficiency were 80%when MOI was 20.The level of CGRP was significantly increased in Lv-ECFP-CGRP-CSCs group compared with Lv-EGFP-CSCs group and CSCs group (P<0.05).Meanwhile, transfection with lentiviral vector in each group did not affect the viability of c-kit+CSCs.CONCLUSION:Transfection of Lv-EGFP-CGRP into c-kit+CSCs was successful .The secretion of CGRP was found in the transfected c-kit+CSCs and the viability was not changed after transfection .CGRP-modified c-kit+CSCs may play a role in treating myocardial infarction .

Advances in Research on Stem cell-derived exosomes in Ischemic heart disease / 医学研究生学报

Stem cell therapy provides immense hope for restoring the impaired cardiomyocytes .However, it has been debated on the effectiveness , mechanisms , and survival of the donated cell population in the ischemic myocardial milieu .Protective paracrine effect of stem cells on cardiomyocytes gradually arose great attentions .Exosomes , as typical representative of paracrine effect of stem cells, are 30-100 nm in size and tiny microvesicles released by cells in response to different physiological states , and enriched in pro-teins, messenger RNAs, and miRs characteristic of parental stem cells , represent great potential for treating cardiovascular diseases . Recent studies show that exosomes from different kinds of stem cells can effectively promote cardiac function by means of promoting the donated cell survival , proliferation and angiogenesis in the ischemia heart .The aim of this review is to summarize current research ef-forts on exosomes from different kinds of stem cells , including their potential mechanism to develop a potentially viable therapy for the treatment of impaired cardiomyocytes via promoting cardiomyocytes survival , proliferation and angiogenesis;inhibiting inflammatory re-sponse and cardiac fibrosis after myocardial infarction .

Effect of calcitonin gene-related peptide on myocardin expression and phenotypic switch in vascular smooth muscle cells / 中国病理生理杂志

AIM:Toinvestigatetheeffectsofcalcitoningene-relatedpeptide(CGRP)onmyocardinexpres-sion and phenotypic switch in vascular smooth muscle cells ( VSMCs) .METHODS:VSMCs were obtained by aortic tissue adherent culture and treated with angiotensin Ⅱ( AngⅡ) , AngⅡ+CGRP or AngⅡ+CGRP +CGRP8-37 .The protein expression of myocardin and the phenotypic proteins of the VSMCs was detected by Western blot .RESULTS:The expres-sion of myocardin in cultured VSMCs showed downregulation along with time expansion .The protein level of myocardin was higher at 48 h and 72 h than that at baseline in the cultured VSMCs (P<0.05).However, the myocardin was lower at 48 h and 72 h than that at baseline after treatment with CGRP in cultured VSMCs (P<0.05).Furthermore, at 48 h in cul-tured VSMCs, the myocardin decreased along with α-smooth muscle actin (α-SMA) (P<0.05), and osteopontin (OPN) increased (P<0.05) in AngⅡ group compared with control group .After treatment with CGRP, the levels of myocardin andα-SMA become higher ( P <0.05 ) but OPN was lower ( P <0.05 ) in CGRP group than those in AngⅡ group. CGRP8-37 abrogated CGRP-induced increase in myocardin and α-SMA and decrease in OPN in CGRP 8-37 group compared with CGRP group .CONCLUSION: CGRP may regulate the phenotypic switch of the VSMCs and maintain the cells in contractile phenotype through the upregulation of myocardin protein , which may be accomplished by the combination of CGRP and its receptor .

Capsazepine in rats′lateral ventricles:Effect of different doses on LPS-induced fever / 中国药理学通报

Aim To observe the effects of the transient receptor potential vanilloid receptor 1 ( TRPV1 ) antago-nist Capsazepine on the LPS-induced fever process. Methods Three doses of Capsazepine were respec-tively injected into rats’ lateral ventricles before the in-traperitoneal injection of LPS caused fever. An emitter was implanted inside the rat’ s abdominal cavity to mo-nitor the body temperature changes during the fever process. Then the changes of calcium ion in the hypo-thalamus were detected by fluorescence and the expres-sions of TRPV1 protein were detected by Western blot. Results The higher dosage of Capsazepine was injec-ted, the higher the level of LPS-induced fever was, and the lower the concentration of calcium ion in the hypothalamus as well as the TRPV1 expressions were. Conclusion Capsazepine could increase the level of fever induced by LPS via acting on the rats’ hypothala-mus. This effect might be related to the functions of TRPV1 .

Experimental research on the effects of calcitonin gene related peptide on biological characteristics of bone marrow stem cells / 中华老年医学杂志

Objective To study the effect of the recombinant Lentivirus containing calcitonin gene related peptide (CGRP) gene on cells biological activity and differentiation of rat bone marrow stem cells(MSCs).Methods Rat MSCs were isolated and cultured by granulocytes adherent.MSCs were transfected with Lenti-EGFP CGRP(MSCsCGRP+/+ group),While MSCs were transfected with Lenti-EGFP as control group.Cell transfection rate was detected by flow cytometry,protein secretion in the above-mentioned MSCsCGRP+ + supernatant was detected using ELISA method.Cells surface markers weare detected by flow cytometry and immunohistochemistry.Trypan blue was used to examin the survive rate,β galactosidase staining was used to examin aging of MSCs transfection,and MTT was used to examine cell vitality.Results At first day after transfecting with Lenti-EGFP-CGRP,fluorescence was not observed by fluorescence microscope,but a small amount of CGRP protein was detected by ELISA in MSCsCGRP+/+ group,at 3 days and 4 days after transfecting with MSCs,strong fluorescence was observed by fluorescence microscope (the cell transfection rates were 77.87％ and 79.58％).The CGRP expression was significantly higher in MSCsCGRP+ + group than in control group [(19.53±0.50) pg/ml vs.(3.12±0.00) pg/ml,t=48.964,P＜0.01].At three days after transfection with MSCs,CD29 and CD90 expression were significantly higher,as compared with control group,CD31 expression was increased in MSCsCGRP+ /+ group.Seven days after transfection with MSCs,CD31 expression was significantly increased in MSCsCGRP+ + group,vWF expression was significantly increased in MSCsCGRP+ + group after MSCs were transfected with LentiEGFP CGRP for 14 days,but a SMA expression was decreased in MSCsCGRP+ +group.At 3 days and 7 days after transfection with Lenti-EGFP-CGRP,the proliferation,survive and aging showed no difference in MSCsCGRP+/+group and in control group (the proliferation of cell:t=0.253,0.290the survive of cell t=-0.307,0.690,all P＞0.05).At 14 days after transfection with Lenti-EGFP-CGRP,aging of cell were decreased in MSCsCGRP+ + group as compared with control group (t=2.446,P＜ 0.05).Conclusions After MSCs are transfected with Lenti EGFP-CGRP,biological characteristics of MSCs has no significant effects,there is still proliferation and differentiation activity.Cell secretion of CGRP can promote the endothelial cell differentiation,and inhibit the differentiation to smooth muscle cells.The CGRP modification of MSCs may play a role in the regulation of angiogenesis.

Impact and related mechanism of exogenous receptor activity modifying protein 1 on calcitonin gene-related peptide modified bone marrow mesenchymal stem cells on the migration of vascular smooth muscle cells in vitro / 中华心血管病杂志

<p><b>OBJECTIVE</b>To investigate the impact of calcitonin gene-related peptide (CGRP) modified bone marrow mesenchymal stem cell (MSC) on the migration of vascular smooth muscle cell (VSMC) and related mechanisms.</p><p><b>METHODS</b>The MSC and VSMC were isolated from rats and cultured, CGRP was transfected to MSC with the high expression lentivirus vector, VSMC was transfected with high expression lentivirus vector of receptor activity modifying protein 1 (RAMP1) and the silence expression lentivirus vector of RAMP1. Then MSC was co-cultured with VSMC. Experimental groups were as follows: (1) Ang II group (MSC + VSMC + Ang II); (2) MSC(CGRP+) group (MSC(CGRP+) + VSMC + Ang II); (3) MSC(CGRP+) RAMP1(-) group (MSC(CGRP+) + VSMC(RAMP1-) + Ang II); (4) MSC(CGRP+) RAMP1(+) group (MSC(CGRP+) + VSMC(RAMP1+) + Ang II); (5) RAMP1(+) group (MSC + VSMC(RAMP1+) + Ang II). Transwell assay was applied to detect the migration of smooth muscle cells, Western blot was applied to detect the protein expression of cells in various groups.</p><p><b>RESULTS</b>VSMC migration number was significantly lower in MSC(CGRP+) group compared with Ang II group (50.8 ± 2.6 vs. 71.4 ± 2.3, P < 0.05), but higher than in MSC(CGRP+) RAMP1(+) group (50.8 ± 2.6 vs. 30.4 ± 3.0, P < 0.05). When RAMP1 expression reduced in VSMC, compared with MSC(CGRP+) RAMP1(+) group, VSMC migration increased in the MSC(CGRP+) RAMP1(-) group compared to MSC(CGRP+)RAMP1(+) (69.0 ± 5.6 vs. 30.4 ± 3.0, P < 0.05) and was similar to Ang II group (69.0 ± 5.6 vs. 71.4 ± 2.3, P > 0.05) and RAMP1(+) group (71.6 ± 3.4). According to the result of Western blot, P-P65 protein expression in MSC(CGRP+) group was lower than that in Ang II group (0.475 ± 0.022 vs.0.642 ± 0.035, P < 0.05). P-P65 protein expression in MSC(CGRP+)RAMP1(-) group was higher than that in MSC(CGRP+) RAMP1(+) group (0.670 ± 0.030 vs. 0.373 ± 0.041, P < 0.05), and there was no difference between MSC(CGRP+)RAMP1(-) group and Ang II group (P > 0.05). P-P65 protein expression was similar between RAMP1(+) group (0.643 ± 0.039) and Ang II group (P > 0.05).</p><p><b>CONCLUSIONS</b>CGRP inhibits VSMC migration through RAMP1. NF-κB and RAMP1 play crucial role in the inhibiting effects of CGRP on VSMC migration. Thus, RAMP1-CGRP signaling inhibits VSMC migration through NF-κB signal pathways.</p>