Hypoxic postconditioning protects myocardium by regulating autophagy in aging cardiomyocytes through piRNA-005854 / 中国组织工程研究

BACKGROUND:Ischemic postconditioning is one of the effective ways to reduce ischemia-reperfusion injury and has been more and more widely used in clinical practice in recent years,but its specific molecular mechanism has yet to be studied. OBJECTIVE:To investigate the role and mechanism of piRNA-005854 in the aging cardiomyocytes caused by hypoxic postconditioning. METHODS:In vitro,cardiomyocytes were administered 8 mg/mL D-galactose for 9 days to induce their aging.β-Galactosidase staining was used to observe the aging of cardiomyocytes.Senescent cells were treated with hypoxia/reoxygenation and hypoxic postconditioning.ELISA was utilized to detect changes in myocardial injury markers creatine kinase isoenzyme MB and lactate dehydrogenase levels.Western blot assay was applied to detect the expression changes of autophagy-related proteins LC3II,p62,ULK1 and phosphorylated ULK1 in aging cardiomyocytes.qRT-PCR was employed to determine the expression level of piRNA-005854.piRNA-005854 inhibitor and piRNA-005854 mimics were transferred into aging cardiomyocytes and followed with hypoxic postconditioning.Western blot assay was used to examine the expression of LC3II,p62,ULK1 and phosphorylated ULK1. RESULTS AND CONCLUSION:(1)D-galactose induced obvious senescence of cardiomyocytes 9 days later.(2)Compared with the normoxia group,creatine kinase isoenzyme MB and lactate dehydrogenase levels increased in the hypoxia/reoxygenation group(P<0.01);LC3 II/I expression was increased;p62 expression was decreased;ULK1 phosphorylation level was increased,and piRNA-005854 expression was increased(P<0.01).(3)Compared with the hypoxia/reoxygenation group,creatine kinase isoenzyme MB and lactate dehydrogenase levels significantly reduced in the hypoxic postconditioning group(P<0.01);LC3 II/I expression significantly decreased(P<0.05);p62 expression increased(P<0.01);ULK1 phosphorylation level decreased(P<0.05),and piRNA-005854 expression decreased(P<0.01).(4)After transfection of piRNA-005854 inhibitor,LC3II/I expression was decreased(P<0.01);the expression of p62 was increased significantly(P<0.05);the phosphorylation level of ULK1 was decreased significantly(P<0.01).After transfection of piRNA-005854 mimics,LC3II/I expression was increased significantly;the expression of p62 was decreased,and the phosphorylation level of ULK1 was increased significantly(P<0.01).(5)The results show that piRNA-005854-mediated reduction of ULK1-dependent autophagy level is a possible mechanism that hypoxic postconditioning exerts its protective effect on aging cardiomyocytes.

Vascular endothelial growth factor 165/bone morphogenetic protein improves osteoblast injury under hypoxic and reoxygenated conditions / 中国组织工程研究

BACKGROUND:It has been found that vascular endothelial growth factor 165 and bone morphogenetic proteins interact with each other during hypoxia-reoxygenation and are involved in the repair process of osteoblast injury by regulating the activation of intracellular signaling pathways. OBJECTIVE:To further investigate the relationship between vascular endothelial growth factor 165/bone morphogenetic protein and hypoxic-reoxygenated osteoblast injury. METHODS:Osteoblasts were selected and the hypoxic-reoxygenated injury model was established.Vascular endothelial growth factor 165 and bone morphogenetic protein expressions at mRNA and protein levels were detected by real-time PCR and western blot before and after modeling.After modeling,osteoblasts were given different concentrations of vascular endothelial growth factor 165 and bone morphogenetic protein 2(10,20,40 ng/mL).Cell proliferation was detected by cell counting kit-8 method and apoptosis was detected by DAPI at 12,24,36,48,and 72 hours after treatment. RESULTS AND CONCLUSION:Compared with before modeling,the mRNA and protein expressions of vascular endothelial growth factor 165 and bone morphogenetic protein 2 in osteoblasts after modeling were significantly decreased(P＜0.05).The proliferation rate of osteoblasts was significantly increased with the increase of vascular endothelial growth factor 165 concentration(P＜0.05),while the apoptosis rate of osteoblasts decreased significantly with the increase of vascular endothelial growth factor 165 concentration(P＜0.05).The proliferation rate of osteoblast was significantly increased with the increase of bone morphogenetic protein 2 concentration(P＜0.05),while the apoptosis rate of osteoblast decreased significantly with the increase of bone morphogenetic protein 2 concentration(P＜0.05).To conclude,vascular endothelial growth factor 165 and bone morphogenetic protein are lowly expressed in hypoxic-reoxygenated osteoblast injury,and treatment with vascular endothelial growth factor 165 and bone morphogenetic protein can reduce the injury of hypoxic-reoxygenated osteoblast in a concentration-dependent manner,suggesting that vascular endothelial growth factor 165 and bone morphogenetic protein have a significant protective effect against the injury of hypoxic-reoxygenated osteoblasts.

Regulatory effect of retinoid X receptor on oxidative stress response in-duced by hypoxia/reoxygenation in rat type Ⅱ alveolar epithelial cells / 中国病理生理杂志

AIM:To investigate the regulatory role of retinoid X receptor(RXR)in oxidative stress response of rat type Ⅱ alveolar epithelial cells(AECII)induced by hypoxia/reoxygenation(HR).METHODS:The AECII were di-vided into control(C)group,HR group,HR+solvent dimethyl sulfoxide(DMSO)group(HD group),HR+RXR agonist 9-cis-retinoic acid(9-RA)group(RA group),and HR+RXR antagonist HX531 group(HX group).Cell Counting Kit-8(CCK-8)method was used to measure the cell viability.Immunofluorescence staining was used to detect the expression of surfactant protein A(SP-A)and RXRα in AECII.Kits were detected to the levels of superoxide dismutase(SOD)and malondialdehyde(MDA)in cells.Transmission electron microscopy was used to observe the ultrastructural changes of the cells.Western blot was used to detect the protein level of nuclear factor E2-related factor 2(Nrf2).RT-PCR was used to detect the expression level of Nrf2 mRNA.RESULTS:Compared with C group,the cell viability and SOD activity in HR,HD,RA and HX groups were decreased significantly(P<0.05),the MDA content were increased significantly(P<0.05),the Nrf2 mRNA and protein expression levels were decreased significantly(P<0.05 or P<0.01),and the immuno-fluorescence expression of RXRα was significantly increased(P<0.01).Compared with HR and HX groups,the cells in RA group showed significantly increased cell viability(P<0.05),increased SOD activity(P<0.05),decreased MDA con-tent(P<0.05),increased Nrf2 mRNA and protein expression levels(P<0.01),and significantly increased immunofluo-rescence expression of RXRα(P<0.01).CONCLUSION:Hypoxia/reoxygenation can aggravate the oxidative stress re-sponse of rat AECII,and RXR agonist intervention can alleviate HR-induced rat AECII injury by inhibiting oxidative stress.

Rat cardiac fibroblasts inhibit gap junction between cardiomyocytes through increasing matrix metalloproteinase 2 activity / 中国病理生理杂志

AIM:To investigate the effect of conditioned medium from hypoxia/reoxygenation(H/R)-treated rat cardiac fibroblasts(CFs)on gap junction between cardiomyocytes and determine whether its mechanism is related to matrix metalloproteinase 2(MMP2)activity.METHODS:(1)H9c2 cells were randomly divided into five groups:con-trol group,normal group,ARP-100 group,H/R group,and H/R+ARP-100 group.Scrape loading/dye transfer assay was used to assess the gap junction function.Western blot was used to detect the expression and phosphorylation levels of Cx43.Gelatin zymography assay was performed to measure MMP2 activity.(2)SD rats were randomly divided into control group,ARP-100 group,ischemia-reperfusion(I/R)group,and I/R+ARP-100 group,with 8 rats in each group.Micro-electrode array technology was used to record the type and duration of arrhythmia.Immunohistochemistry experiment was performed to assess expression levels and distribution of Cx43 in myocardial tissues.RESULTS:Compared with the con-trol group,the H/R group showed decreased protein expression of Cx43(P＜0.01),narrowed distance of lucifer yellow dif-fusion(P＜0.01),and increased MMP2 activity(P＜0.01).ARP-100 attenuated H/R-induced gap junction dysfunction(P＜0.05).The arrhythmia score was also reduced after perfusion with ARP-100(P＜0.01).CONCLUSION:H/R-treated rat CFs can inhibit gap junction between cardiomyocytes,and its mechanism may involve increased MMP2 activity.

Neoflavonoids from Dalbergia cochinchinensis and their anti-hypoxia/reoxygenation injury activities on H9c2 myocardial cells / 中成药

AIM To study the neoflavonoids from Dalbergia cochinchinensis Pierre ex Laness and their anti-hypoxia/reoxygenation injury activities on H9c2 myocardial cells.METHODS The 70%ethanol extract from D.cochinchinensis was isolated and purified by silica gel,Sephadex LH-20 and reverse-preparative HPLC,then the structures of obtained compounds were identified by physicochemical properties and spectral data.The CCK-8 method was used to detect their activities on H9c2 cells and protective effects on hypoxia-reoxygenation injury of H9c2 cells,and their structure-activity relationship was analyzed.RESULTS Twelve compounds were isolated and identified as latifolin(1),5-O-methyllatifolin(2),mimosifoliol(3),5-O-methydalbergiphenol(4),dalbergiphenol(5),cearoin(6),2,4-dihydroxy-5-methoxy-benzophenone(7),2-hydroxy-4,5-dimethoxybenzophenone(8),melannoin(9),2,2′,5-trihydroxy-4-methoxybenzophenone(10),dalbergin(11),4-methoxydalbergione(12).The dalbergiphenols and dalbergins had little toxicity to H9c2 cells,and dalbergiphenols had strong activity against hypoxia-reoxygenation injury of H9c2 cells.CONCLUSION Compound 8 is a new natural product.Compounds 4,9 are isolated from this plant for the first time.Dalbergiphenols may be the main neoflavonoids against hypoxia-reoxygenation injury of H9c2 cells.

Effect of Salvianolic Acid B Combined with Puerarin on Pyroptosis of SH-SY5Y Cells Injured by Oxygen-glucose Deprivation/Reoxygenation / 中国实验方剂学杂志

ObjectiveTo investigate the effect and mechanism of salvianolic acid B combined with puerarin in protecting the SH-SY5Y cells from the damage by oxygen-glucose deprivation/reoxygenation （OGD/R） based on pyroptosis. MethodSH-SY5Y cells were used to establish the model of OGD/R， and cells were classified into the control， OGD/R， 10 μmol·L-1 salvianolic acid B， 100 μmol·L-1 puerarin， 10 μmol·L-1 salvianolic acid B + 100 μmol·L-1 puerarin， and 10 μmol·L-1 NOD-like receptor protein 3 （NLRP3） inhibitor MCC950 groups. Except the control group， other groups were rapidly reoxygenated for 12 h after 6 h OGD for modeling. The cell survival rate was determined by the methyl thiazolyl tetrazolium （MTT） assay. An optical microscope was used to observe the cell morphology. A spectrophotometer was used to determine the content of lactic dehydrogenase （LDH） in culture supernatant. Cell damage was measured by Hoechst/PI staining. The mRNA levels of NLRP3， cysteinyl aspartate specific proteinase-1 （Caspase-1）， gasdermin D （GSDMD）， apoptosis-associated speck-like protein （ASC）， and interleukin-1β （IL-1β） were determined by real-time fluorescence quantitative polymerase chain reaction （Real-time PCR）. The protein activation of Caspase-1 and NLRP3 was detected by immunofluorescence. Western blot was employed to determine the protein levels of IL-1β， ASC， NLRP3， Caspase-1， and cleaved Caspase-1. ResultCompared with the control group， the OGD/R group showed decreased cell survival rate （P<0.01）， damaged cell morphology， increased leakage rate of LDH （P<0.01）， up-regulated mRNA levels of NLRP3， Caspase-1， GSDMD， ASC， and IL-1β （P<0.01）， and up-regulated protein levels of IL-1β， ASC， NLRP3， Caspase-1， and cleaved Caspase-1 （P<0.01）. Compared with the OGD/R group， salvianolic acid B， puerarin， and salvianolic acid B combined with puerarin improved cell survival rate （P<0.01）， and the combined treatment group outperformed salvianolic acid B and puerarin used alone （P<0.01）. Salvianolic acid B combined with puerarin and MCC950 both improved cell morphology， reduced the leakage of LDH （P<0.01）， alleviated cell damage， and down-regulated the mRNA levels of NLRP3， Caspase-1， GSDMD， ASC， and IL-1β （P<0.05， P<0.01） and also the protein levels of IL-1β， ASC， NLRP3， Caspase-1， and cleaved Caspase-1 （P<0.05， P<0.01）. ConclusionThe results indicated that salvianolic acid B combined with puerarin can alleviate the OGD/R-induced damage of SH-SY5Y cells by inhibiting pyroptosis.

Effect of exogenous hydrogen sulfide on hypoxia/reoxygenation injury in glomerular mesangial cells and its mechanism / 中国药理学通报

Aim To explore the effect of exogenous hydrogen sulfide ( H2 S ) on hypoxia/reoxygenation ( H/R) injury in glomerular mesangial cells and elucidate its relevant mechanism. Methods H/R induced mouse mesangial cell line ( SV40MES13 ) to establish cell damage model. Cell viability was detected by cell proliferation kit ( CCK8 ), the content of H

Methyltransferase WTAP aggravates hypoxia/reoxygenation-induced myocardial cell injury by regulating the expression of activator of transcription 4 / 中华危重病急救医学

Objective:To investigate the regulatory role of Wilms tumor 1-associating protein (WTAP) in hypoxia/reoxygenation (H/R)-induced cardiomyocyte injury and its molecular mechanism.Methods:① Experiment Ⅰ: H9C2 cardiomyocytes were divided into blank control group and H/R model group. H/R was used to induce myocardial ischemia/reperfusion (I/R) injury model in H9C2 cells. The blank control group was not treated. N6-methyladenosine (m6A) RNA methylation assay kit was used to detect the level of m6A. Real-time fluorescent quantitative polymerase chain reaction (RT-qPCR) and Western blotting were used to detect the mRNA and protein expression levels of methyltransferases [WTAP, methyltransferase-like proteins (METTL3, METTL14)], respectively. ② Experiment Ⅱ: H9C2 cardiomyocytes were divided into blank control group, H/R+sh-NC group, and H/R+sh-WTAP group. sh-WTAP was transfected to knock down the expression of WTAP in H/R+sh-WTAP group, and the model establishment method in the other groups was the same as experimentⅠ. At 48 hours after transfection, the apoptosis rate of cells was detected by flow cytometry. The protein expressions of WTAP, activated caspase-3, activated poly (ADP-ribose) polymerase (PARP), activating transcription factor 4 (ATF4), proline-rich receptor-like protein kinase (PERK), phosphorylated PERK (p-PERK) and CCAAT/enhancer-binding protein homologous protein (CHOP) were detected by Western blotting. The positive expression of ATF4 was observed by immunofluorescence staining. ③ Experiment Ⅲ: H9C2 cardiomyocytes were divided into blank control group, H/R+sh-NC group, H/R+sh-WTAP group and H/R+sh-WTAP+ATF4 group. The overexpression plasmid ATF4 was transfected into H9C2 cardiomyocytes, and the modeling method of the other groups were modeled the same as experimentⅡ. The apoptosis rate was detected by flow cytometry. Western blotting was used to detect the protein expressions of ATF4, CHOP, activated caspase-3 and activated PARP.Results:① ExperimentⅠ: the methylation level of m6A in the H/R group was significantly higher than that in the blank control group. RT-qPCR results showed that the gene expressions of METTL3, METTL14 and WTAP in the H/R model group were significantly higher than those in the blank control group, and WTAP was the most significantly up-regulated. Western blotting results showed the same trend. These results suggested that the expression level of methyltransferase WTAP is significantly up-regulated in H/R-induced cardiomyocytes. ②Experiment Ⅱ: the apoptosis level in H/R+sh-WTAP group was significantly lower than that in H/R+sh-NC group [(14.16±1.58)% vs. (24.51±2.38)%, P < 0.05]. Western blotting results showed that the protein expressions of WTAP, activated caspase-3, activated PARP, p-PERK, ATF4 and CHOP in the H/R+sh-WTAP group were significantly lower than those in the H/R+sh-NC group. Fluorescence microscopy results showed that the ATF4 positive signal in the H/R+sh-WTAP group was significantly weaker than that in the H/R+sh-NC group [(19.36±1.81)% vs. (32.83±2.69)%, P < 0.01]. The above results suggested that knockdown of WTAP could inhibit H/R-induced cardiomyocyte apoptosis and endoplasmic reticulum stress. ③ Experiment Ⅲ: the apoptosis level of H/R+sh-WTAP+ATF4 group was significantly higher than that of H/R+sh-WTAP group [(26.61±2.76)% vs. (17.14±0.87)%, P < 0.05]. Western blotting results showed that the protein expressions of ATF4, CHOP, activated caspase-3 and activated PARP in the H/R+sh-WTAP+ATF4 group were significantly higher than those in the H/R+sh-WTAP group. These results suggested that overexpression of ATF4 reversed the inhibitory effect of sh-WTAP on endoplasmic reticulum stress and apoptosis in H/R-induced cardiomyocytes. Conclusion:Methyltransferase WTAP could regulate ATF4 expression, mediate cell apoptosis and endoplasmic reticulum stress, and promote H/R-induced myocardial cell injury.

Study on Protective Effect and Mechanism of Danshen Tongluo Jiedu Decoction Medicated Serum for Regulating MALAT1 in Hypoxia/reoxygenation Rats CMECs / 中国中医药信息杂志

Objective To investigate the protective effect and mechanism of Danshen Tongluo Jiedu Decoction medicated serum for hypoxia/reoxygenation rat myocardial microvascular endothelial cells(CMECs)by regulating MALAT1.Methods Rats CMECs cells were cultured in vitro to establish a model of hypoxia/reoxygenation damaged cells,and were transfected overexpressing/silencing blank MALAT1 slow virus,cells were divided into overexpressed blank + TCM group,overexpressed MALAT1 + TCM group,overexpressed MALAT1 group,silenced blank + TCM group,silenced MALAT1 group,and silenced MALAT1 + TCM group.They were cultured with corresponding serum separately.Beclin-1 protein expression was detected by immunofluorescence method,and SRPK1,SRSF1,VEGF and Bax protein expressions were detected by Western blot,MALAT1,SRPK1 and SRSF1 mRNA expressions were detected by RT-PCR.Results Compared with the overexpressed blank + TCM group,Beclin-1 protein expression increased in the overexpressed MALAT1 + TCM group,the protein expressions of SRPK1,SRSF1 and Bax significantly increased(P<0.05,P<0.01),VEGF protein expression significantly decreased(P<0.01),while MALAT1,SRPK1 and SRSF1 mRNA expressions significantly increased(P<0.05,P<0.01).Compared with the overexpressed MALAT1 group,the protein expression of Beclin-1 in overexpressed MALAT1 + TCM group decreased,the expressions of SRPK1,SRSF1 and Bax protein significantly decreased(P<0.01,P<0.05),the expression of VEGF protein significantly increased(P<0.01),the mRNA expressions of MALAT1,SRPK1 and SRSF1 significantly decreased(P<0.05).Compared with the silenced blank + TCM group,the protein expression of Beclin-1 in silenced MALAT1 + TCM group decreased,the expressions of SRPK1,SRSF1 and Bax protein significantly decreased(P<0.01),while the expression of VEGF protein significantly increased(P<0.01),the mRNA expressions of MALAT1,SRPK1 and SRSF1 significantly decreased(P<0.01).Compared with the silenced MALAT1 group,the protein expression of Beclin-1 in silenced MALAT1 + TCM group decreased,the expressions of SRPK1,SRSF1 and Bax protein significantly decreased(P<0.05),the expression of VEGF protein significantly increased(P<0.01),the mRNA expressions of MALAT1,SRPK1 and SRSF1 significantly decreased(P<0.01,P<0.05).Conclusion Upregulation of MALAT1 expression can promote autophagy in hypoxia/reoxygenation model CMECs,while Danshen Tongluo Jiedu Decoction medicated serum can inhibit MALAT1 expression,thus inhibiting autophagy and promoting angiogenesis,and the mechanism may be related to the downregulation of SRPK1 and SRSF1 expressions.

Effect and mechanism of miR-34a-5p targeting Notchl onH/R-induced apoptosis of human cardiomyocytes / 安徽医科大学学报

Objective@#To investigate the improvement of endoplasmic reticulum stress mediated by microRNANotchl)signaling axis on hypoxia/reoxygenation(H/R)human(miR)-34a-5p/transmembrane fusion protein 1 (es were randomly divided into Control group, H/R group, mimiccardiomyocytes. @*Methods@#Human cardiomyocytNC group, mimic group, inhibitor NC group andinhibitor group. Except the Control group, H/R injury model wasof miR-34a-5p and Notchl were detected by quantitative real.established in other groups. The expression levesurvival rate was detected by thiazolyl blue ( MTT), cell apopto-time polymerase chain reaction(qRT-PCR), celtargeting relationship between miR-34a-5p and Notchl was detec-sis rate was detected by flow cytometry, and theexpressions of transcriptional activator 6(ATF6), inositol demandted by dual luciferase gene reporting method. Thesmic reticulum kinase (PERK) and glucose regulatory protein 78 (GRP78)were detected by Western blot. @*Results@#miR-34a-5p targeted Notchl(P<0.05);compared with Con-apoptosis rate and protein expressions of ATF6, IREl, PERK andtrol group, the expression level of miR-34a-5pGRP78 in H/R group increased, while the cellsurvival rate and Notchl mRNA and protein expressions decreased(P<0.05). Compared with H/R group and minic NC group, miR-34a-5p expression, apoptosis rate , and proteinexpressions of ATF6, IRE1, PERK and GRP78in mimic group increased, while cell survival rate and Notchl mRNA and protein expressions decreased (P <0. 05).Compared with H/R group and inhibitor NC group, the exprespressions of ATF6 , IREl , PERK and GRP78 decreased in inhibi-sion of miR-34a-5p, apoptosis rate and protein etor group,while cell survival rate and Notch1 mINA and protein expressions increased ( P <0.05). @*Conclusion@#miR-34a-5p can inhibit the apoptosis of H/R human cardiomyocytes, possibly through the targeted inhibition ofNotchl-mediated endoplasmic reticulum stress.

Esketamine alleviates hypoxia/reoxygenation injury of cardiomyocytes by regulating TRPV1 expression and inhibiting intracellular Ca2+ concentration

Abstract Objective This study aimed to investigate the effect of Esketamine (ESK) on the Hypoxia/Reoxygenation (H/R) injury of cardiomyocytes by regulating TRPV1 and inhibiting the concentration of intracellular Ca2+. Methods The H/R injury model of H9c2 cardiomyocytes was established after 4h hypoxia and 6h reoxygenation. H9c2 cells were treated with different concentrations of ESK or TRPV1 agonist capsaicin (10 μM) or TRPV1 inhibitor capsazepine (1 μM). Cell viability was detected by CCK-8 method, and apoptosis by flow cytometry. Intracellular Ca2+ concentration was evaluated by Fluo-4 AM. LDH, MDA, SOD, and GSH-Px were detected with corresponding commercial kits. TRPV1 and p-TRPV1 proteins were detected by Western blot. Results After H/R, H9c2 cell viability decreased, apoptosis increased, intracellular Ca2+ concentration increased, LDH and MDA levels increased, SOD and GSH-Px levels decreased, and p-TRPV1 expression increased. ESK treatment rescued these changes induced by H/R. After up-regulating TRPV1, the protective effect of ESK on H/R injury of H9c2 cells was weakened, while down-regulating TRPV1 could further protect against H/R injury. Conclusion ESK alleviates H/R injury of cardiomyocytes by regulating TRPV1 expression and inhibiting intracellular Ca2+ concentration.

Effect of autophagy regulation on hypoxia/reoxygenation injury of mouse spermatogonia / 重庆医学

Objective To study the effect of autophagy regulation on hypoxia/reoxygenation injury(H/RI)in mouse spermatogonia,and to explore the effect of autophagy on ischemia-reperfusion injury(IRI)in mouse testicular tissue.Methods The mouse spermatogonial cell line GC1 spg was used as the research object to construct the H/RI model.Rapamycin(RAPA)and 3-methyladenine(3-MA)were used as autophagy ago-nists and inhibitors.The control group,the model group,the autophagy agonist intervention group(H/RI+autophagy agonist intervention),and the autophagy inhibitor intervention group(H/RI+autophagy inhibitor intervention)were set up.The cells proliferation ability of each group was detected by methyl thiazol tetrazoli-um(MTT)method.The release level of reactive oxygen species(ROS)and apoptosis level of each group were detected by flow cytometry.The expression levels of autophagy-related gene Beclin1 and apoptosis-relat-ed genes Bcl-2 and Bax mRNA in each group were detected by real-time fluorescence quantitative PCR(qPCR).The expression levels of autophagy-related proteins LC3-Ⅰ,LC3-Ⅱ,Beclin1,p62 and apoptosis-relat-ed proteins Bcl-2,Bax,caspase-3 in each group were detected by Western blot.Results Compared with the control group,the proliferation abiliy,the expression levels of Beclin1 and Bcl-2 mRNA in the model group were significantly decreased(P＜0.01),the relative expression levels of p62 and Bcl-2 proteins were significantly decreased(P＜0.01).The ROS level,apoptosis rate and the mRNA expression level of Bax were significantly increased(P＜0.01),and the protein expresion levels of Beclin1,Bax,caspase-3 and the protein ratio of LC3-Ⅱ/LC3-Ⅰ were significantly increased(P＜0.01).Compared with the model group,the cell proliferation a-bility,the expression levels of Beclin1 and Bcl-2 mRNA in the autophagy agonist intervention group were the protein ratio of significantly increased(P＜0.01),the protein expression levels of Beclin1 and Bcl-2 and the protein ration of LC3-Ⅱ/LC3-Ⅰ were significantly increased(P＜0.01).The ROS level,apoptosis rate and the expression level of Bax mRNA were significantly decreased(P＜0.01),and the protein expression levels of p62,Bax,caspase-3 and the protein ratio of LC3-Ⅱ/LC3-Ⅰ were significantly decreased(P＜0.01).Com-pared with the model group,the cell proliferation ability,the mRNA expression levels of Beclin1 and Bcl-2 in the autophagy inhibitor intervention group were significantly decreased(P＜0.01),the protein expression lev-els of Beclin1 and Bcl-2 protein were significantly decreased(P＜0.01).The ROS level,apoptosis rate and the mRNA expression level of Bax were significantly increased(P＜0.01),and the protein expression levels of p62,Bax,caspase-3 and the protein ratio of LC3-Ⅱ/LC3-Ⅰwere significantly iecreased(P＜0.01).Conclusion Enhanced au-tophagy can inhibit apoptosis of spermatogonia and repair H/RI in mice,which provides a theoretical basis for the treatment of testicular tissue IRI.

Mechanism of Metformin on Oxygen-glucose Deprivation/reoxygenation-induced U251 Cells Injury through Wnt/β-catenin Signaling Pathway / 医学研究杂志

Objective To investigate the mechanism of metformin on oxygen-glucose deprivation/reoxygenation(OGD/R)injury in U251 cells.Methods Human glioma cell line U251 cells were cultured and divided into 6groups:blank control group,model group,metformin medium dose group,metformin high dose group,agonist group(Wnt3a,Wnt/β-catenin signaling pathway agonist),inhibitor group(metformin+XAV939,Wnt/β-catenin signaling pathway inhibitor).Except for the blank control group,the cells in the other groups were subjected to OGD/R for 2h and then reperfusion for 24h to establish the OGD/R model.The animals were treated with met-formin,Wnt3a and XAV939 24h before modeling.Cell viability and toxicity were detected by CCK-8method and lactate dehydrogenase(LDH)assay.ROS formation was detected by DHE staining.Glutathione peroxidase(GSH-Px),superoxide dismutase(SOD)malond-ialdehyde(MDA),interleukin-6(IL-6),inducible nitric oxide synthase(iNOS)and tumor necrosis factor-α(TNF-α)were de-tected by enzyme-linked immunoadsordent assay(ELISA).The protein expression levels of β-catenin,cyclin D1,p-GSK-3β(Ser9)and GSK-3β were detected by Western blot.Results Compared with blank control group,LDH,ROS,MDA,IL-6,iNOS and TNF-α in model group,metformin group,agonist group and inhibitor group were significantly increase.The relative expression lev-els of SOD,GSH-Px,β-catenin,cyclin D1,p-GSK-3β(Ser9)and cell viability were significantly decreased.Compared with mod-el group,the levels of LDH,ROS,MDA,IL-6,iNOS and TNF-α in metformin group and agonist group were significantly decreased,while the relative expression levels of SOD,GSH-Px,β-catenin,cyclin D1,p-GSK-3β(Ser9)and the cell viability were signifi-cantly increased.Compared with metformin group,LDH,ROS,MDA,IL-6,iNOS and TNF-α in metformin group and inhibitor group were significantly increase,the relative expression levels of SOD,GSH-Px,β-catenin,cyclin D1,p-GSK-3 β(Ser9)and the cell viability were significantly decreased.Conclusion Metformin may play a protective role in OGD/R of U251 cells through Wnt/β-cate-nin signaling pathway.

Berberine down-regulates NLRP3 to reduce oxygen-glucose deprivation/reoxygenation injury in PC12 cells / 中风与神经疾病杂志

@#Objective To investigate the mechanism of action of berberine in reducing neuronal pyroptosis by inhibiting the nucleotide-binding oligomerization domain-like receptor protein 3 （NLRP3）/caspase-1 signaling pathway. Methods A PC12 cell model of oxygen-glucose deprivation/reoxygenation （OGD/R） injury was prepared. The control group， OGD/R injury group， and low-and high-dose berberine groups were set up. Cell activity was measured using MTT assay. Cell apoptosis was measured using TUNEL assay. The expression of gasdermin D （GSDMD） was determined using immunofluorescence assay. The levels of interleukin-1β （IL-1β） and IL-18 were measured by enzyme-linked immunosorbent assay. The protein expression levels of NLRP3， GSDMD， and caspase-1 were measured by Western blotting. Results Compared with the OGD/R injury group， cells treated with berberine showed significantly decreased activity， apoptosis， GSDMD fluorescence intensity， and expression levels of IL-1β， IL-18， NLRP3， GSDMD， and caspase-1 （all P<0.01）. Conclusion Berberine can inhibit OGD/R-induced neuronal cell injury， which may be through down-regulating the NLRP3/caspase-1 signaling pathway to inhibit OGD/R-induced cell pyroptosis.

Effect of boschniakia rossica polysaccharides on hypoxia/reoxygenation-induced cardiomyocyte damage by down-regulating miR-302a-3p expression / 中国药理学通报

Aim To explore the effect of boschniakia rossica polysaccharides ( BRPS ) on cardiomyocyte damage induced by hypoxia/reoxygenation (H/R) and its possible mechanism. Methods H/R was used to induce rat cardiomyocyte H9c2 to establish a cell inju¬ry model, and different doses of BRPS were used to treat H9c2 cells. ELISA method was used to detect the level of MDA and the activity of SOD and GSH-Px. Flow cytometry was used to detect the rate of apopto-sis. qRT-PCR was used to detect the expression of miR-302a-3p. anti-miR-NC and anti-miR-302a-3p were respectively transfected into H9c2 cells and then subjected to H/R treatment. miR-NC and miR-302a-3p mimics were respectively transfected into H9c2 cells and treated with 100 mg • L

Research progress on hypoxia/reoxygenation model of nerve cells in vitro / 中国药理学通报

Ischemic stroke is the second leading cause of human death and the third reason of disability. Meanwhile, the incidence is rising year after year worldwide. Ischemic stroke could cause ischemia-reperfusion injury after blood recanalization treat-ment, but the mechanism of ischemia-reperfusion injury is still not very clear, so it is necessary to build a preclinical model with specific characteristics. Up to now, animal experiments have been still complicated, and the culture of brain slices has some limitations. The cell model in vitro has become a simplified and valuable tool widely used by researchers. The paper systematically summarizes the common type of nerve cells, and further analyzes establishment methods and principle, relevant research progress on the in vitro model of ischemia-reperfusion, in order to provide reference for rationally selecting hypoxia and reoxygenation model for basic research on cerebral ischemia and reperfusion and drug screening.

Effect of different hypoxic and hypobaric interventions on blood gas and erythrocyte-related indicators in rats / 浙江大学学报·医学版

OBJECTIVES@#To explore the effects of hypoxic and hypobaric conditions on blood gas and erythrocyte-related indicators in rats.@*METHODS@#SD male rats were exposed to low-pressure hypoxic conditions simulating an altitude of 6500 m in a small or a large experimental cabin. Abdominal aortic blood samples were collected and blood gas indicators, red blood cells (RBCs) count, and hemoglobin (Hb) content were measured. The effects of exposure to different hypoxia times, different hypoxia modes, normal oxygen recovery after hypoxia, and re-hypoxia after hypoxia preconditioning on blood gas indicators, RBCs count and Hb content were investigated.@*RESULTS@#The effect of blood gas indicators was correlated with the length of exposure time of hypoxia and the reoxygenation after leaving the cabin. Hypoxia caused acid-base imbalance and its severity was associated with the duration of hypoxia; hypoxia also led to an increase in RBCs count and Hb content, and the increase was also related to the time exposed to hypoxia. The effects of reoxygenation on acid-base imbalance in rats caged in a small animal cabin were more severe that those in a large experimental cabin. Acetazolamide alleviated the effects of reoxygenation after leaving the cabin. Different hypoxia modes and administration of acetazolamide had little effect on RBCs count and Hb content. Normal oxygen recovery can alleviate the reoxygenation and acid-base imbalance of hypoxic rats after leaving the cabin and improve the increase in red blood cell and hemoglobin content caused by hypoxia. The improvement of hypoxia preconditioning on post hypoxia reoxygenation is not significant, but it can alleviate the acid-base imbalance caused by hypoxia in rats and to some extent improve the increase in red blood cell and hemoglobin content caused by hypoxia.@*CONCLUSIONS@#Due to excessive ventilation and elevated RBCs count and Hb content after hypoxia reoxygenation, oxygen partial pressure and other oxygenation indicators in hypoxic rats are prone to become abnormal, while blood gas acid-base balance indicators are relatively stable, which are more suitable for evaluating the degree of hypoxia injury and related pharmacological effects in rats.

LncRNA SNHG12 regulates miR-138-5p/HIF-1α axis to improve hypoxia/reoxygenation human vascular endothelial cell damage / 中国免疫学杂志

Objective:To study the effect of Long non-coding RNA(LncRNA)small nucleolar RNA host gene 12(SNHG12)regulating miR-138-5p/hypoxia inducible factor-1(HIF-1α)axis on improving the damage of hypoxia/reoxygenation(H/R)human vas-cular endothelial cells.Methods:Human umbilical vein endothelial cells(HUVECs)were cultured in vitro and randomly divided into control group,H/R model group,H/R+LncRNA SNHG12 overexpression group,H/R+miR-138-5p mimics group,H/R+co-transfec-tion group and H/R+co-transfection negative control group,each transfection group was transfected separately,and except for control group,the remaining groups were given hypoxia for 5 hours and then reoxygenated for 1 hour to induce the cell models,and then the cell viability of each group was detected by CCK-8 experiment;the cell apoptosis in each group was detected by flow cytometry experi-ment,and the apoptosis rate of each group was compared;the levels of reactive oxygen species(ROS),lactate dehydrogenase(LDH)and inflammatory factors IL-6,IL-17 and IL-18 in each group were measured by the kit;the expressions of miR-138-5p and HIF-1α mRNA in cells of each group were measured by real-time quantitative PCR(qRT-PCR)experiment;the expressions of apoptotic pro-teins caspase-9,Bcl-2-associated X protein(Bax)and HIF-1α in each group were evaluated by Western blot.Results:Compared with control group,the apoptosis rate,cellular ROS,LDH,IL-6,IL-17 and IL-18 levels,cellular HIF-1α mRNA and protein levels,cellular caspase-9,Bax and HIF-1α protein levels were increased in H/R model group(P<0.05),the cell viability and miR-138-5p level were decreased(P<0.05).Compared with H/R model group and H/R+co-transfection group,the cell viability,cell HIF-1αmRNA and protein levels were increased in H/R+LncRNA SNHG12 overexpression group(P<0.05),the apoptosis rate,cellular ROS,LDH,IL-6,IL-17 and IL-18 levels,cellular caspase-9 and Bax protein levels,and miR-138-5p level were decreased(P<0.05);the cell viability,cellular HIF-1α mRNA and protein levels were decreased in H/R+miR-138-5p mimics group(P<0.05),the apoptosis rate,cellular ROS,LDH,IL-6,IL-17 and IL-18 levels,cellular caspase-9 and Bax protein levels were increased(P<0.05).Com-pared with H/R model group,there was no significant difference in cell index levels between the H/R+co-transfection negative control group and the H/R+co-transfection group(P>0.05).Conclusion:LncRNA SNHG12 can upregulate HIF-1α expression by downregulat-ing miR-138-5p expression,inhibit H/R-induced inflammation and oxidative stress in HUVECs,and reduce cell damage and apoptosis.

Effect and mechanism of lncRNA HCP5 on neurocyte apoptosis induced by oxygen-glucose deprivation/reoxygenation / 中华行为医学与脑科学杂志

Objective:To investigate the effect of long non-coding RNA (lncRNA) human histocompatibility leukocyte antigen complex P5 (HCP5) on the neuronal injury induced by oxygen glucose deprivation/reoxygenation (OGD/R), and to analyze its potential mechanism.Methods:SH-SY5Y cells were divided into control group (CON group, normal medium culture), model group (Model group, OGD/R), interference control group (si-NC group, OGD/R after HCP5 small interfering RNA negative control (si-NC)), HCP5 interference group (si-HCP5 group, OGD/R after HCP5 small interfering RNA (si-HCP5)), HCP5 interference+ inhibitor control group (si-HCP5+ anti-NC group, OGD/R after transfection of si-HCP5, miR-525-5p inhibitor negative control (anti-NC)), HCP5 interference+ miR-525-5p inhibitor group (si-HCP5+ anti-miR-525-5p group, OGD/R after transfection of si-HCP5, miR-525-5p inhibitor). qRT-PCR was used to detect the expression of lncRNA HCP5 and miR-525-5p in cells.The activity of SH-SY5Y cells was detected by MTT.The level of reactive oxygen species (ROS) in the cells was detected by fluorescent probe of dichlorofluorescein diacetate (DCFH-DA). The apoptosis of SH-SY5Y cells was detected by flow cytometry.Western blot was used to detect the expression of BTG2, Bcl-2 related X protein (Bax), B lymphocyte tumor 2 (Bcl-2) and cleaved caspase-3 protein.SPSS 25.0 software was used to analyze the data, and one-way ANOVA was used for comparison between multiple groups, and SNK- q test was used for further comparison between two groups. Results:There were statistically significant differences in lncRNA HCP5, miR-525-5p RNA levels and BTG2 protein expression levels among the 6 groups ( F=28.853, 59.241, 13.731, all P<0.001). Compared with the CON group, the Model group had higher level of lncRNA HCP5, lower level of miR-525-5p, and higher level of BTG2 protein (all P<0.05). Compared with the Model group, the si-HCP5 group had lower level of lncRNA HCP5, higher level of miR-525-5p, and lower level of BTG2 protein (all P<0.05). Compared with the si-HCP5+ anti-NC group, the si-HCP5+ anti-miR-525-5p group had higher level of lncRNA HCP5, lower level of miR-525-5p, and higher level of BTG2 protein (all P<0.05). There were statistically significant differences in cell activity and ROS levels among the six groups of cells ( F=16.180, 59.950, both P<0.001). The cell activity of the Model group was lower than that of the CON group (0.33±0.12, 0.63±0.11) ( P<0.05), and the ROS level was higher than that of the CON group (224.62±23.27, 100.00±0.00) ( P<0.05). The cell activity of the si-HCP5+ anti-miR-525-5p group was lower than that of the si-HCP5+ anti-NC group (0.38±0.08, 0.58±0.08) ( P<0.05), and the ROS level was higher than that of the si-HCP5+ anti-NC group (207.83±19.39, 135.27±14.36) ( P<0.05). There were statistically significant differences in the apoptosis rate and expression levels of apoptotic proteins Bcl-2, Bax, and cleared Caspase-3 among the six groups of cells ( F=27.994, 29.660, 45.000, 52.983, all P<0.001). There were no statistically significant difference in Bax, Bcl-2, cleared Caspase-3 protein levels, and apoptosis rate in SH-SY5Y cells between the Model group and the si-NC group, as well as between the si-HCP5 group and the si-HCP5+ anti-NC group (all P>0.05). Compared with the CON group, the apoptosis rate, levels of Bax and cleared Casase-3 protein in the Model group were significantly upregulated (all P<0.05), while the Bcl-2 protein level was significantly downregulated ( P<0.05). Compared with the Model group and si-NC group, the si-HCP5 group showed significant downregulation of cell apoptosis rate and levels of Bax and cleared Caspase-3 protein (all P<0.05), while the Bcl-2 protein level was upregulated ( P<0.05). Compared with the si-HCP5 group and si-HCP5+ anti-NC group, the si-HCP5+ anti-miR-525-5p group showed significant upregulation of cell apoptosis rate and levels of Bax and cleared Caspase-3 protein levels (all P<0.05), and significant downregulation of Bcl-2 protein levels ( P<0.05). Conclusion:lncRNA HCP5 may inhibit the expression of BTG2 by targeting up-regulation of miR-525-5p, thus leading to apoptosis of nerve cells in OGD/R models.

Effect of miR⁃26a⁃3p targeting Survivin on hypoxia/reoxygenation inj ury of H9c2 cardiomyocyte / 安徽医科大学学报

Objective @# To investigate the effects of miR⁃26a⁃3p on rat myocardial cell ( H9c2) injury induced by hypoxia/reoxygenation (H/R) and its mechanism . @*Methods @# H9c2 cardiomyocytes in logarithmic growth phase were subjected to hypoxia (1% O2 ) for 6 h , and reoxygenated at different times (2 , 4 , 8 , 12 h) to establish H/R model cell . Normoxia group was also set up , and cell proliferation activity was detected by cell counting kit⁃8 (CCK⁃8) . The level of lactic dehydrogenase (LDH) in cell supernatant was determined by colorimetry . The expression levels of miR⁃26a⁃3p and Survivin mRNA were detected by real⁃time fluorescence quantitative PCR (qRT⁃PCR) . The expression level of Survivin protein in the cells was detected by Western blot . H9c2 cells were transfected with miR⁃26a⁃3p inhibitor and negative control inhibitor NC , Survivin gene siRNA interference plasmid ( si⁃Survivin) and negative control si⁃NC , followed by H/R intervention . CCK⁃8 was used to detect cell proliferation in each group . The activity of superoxide dismutase (SOD) and the content of malonaldehyde (MDA) in cell and the level of LDH in supernatant were determined by colorimetry . The apoptosis level of each group was detected by flow cytometry . The protein expression levels of Bcl⁃2 associated X protein ( Bax) , B ⁃cell lymphoma⁃2 ( Bcl⁃2 ) , cleaved caspase⁃3 and Survivin were detected by Western blot . Targeting relationship between miR⁃26a⁃3p and Survivin gene was determined by dual luciferase . @*Results @#Compared with the normoxia group , proliferative activity , mRNA and protein expression levels of Survivin in H9c2 cells gradually decreased with the extension of reoxygen ation time (P < 0. 05) , while LDH and expression level of miR⁃26a⁃3p gradually increased ( P < 0. 05) . Downregulating the expression of miR⁃26a⁃3p increased proliferative activity , SOD activity , and expression level of Bcl⁃2 protein in H9c2 cells exposed to H/R ( P < 0. 05) , while MDA content , LDH release amount , apoptosis rate , expression levels of Bax and cleaved caspase⁃3 protein decreased (P < 0. 05) . Survivin deficiency reversed the protective effect of miR⁃26a⁃3p inhibitor on H9c2 cells induced by H/R . Dual luciferase reporter gene assay confirmed that Survivin was the target gene of miR⁃93 ⁃5p . @*Conclusion @#miR⁃26a⁃3p is highly expressed in cardiomyocyte injury induced by H/R . Inhibition of miR⁃26a⁃3p expression can inhibit H/R⁃induced cardiomyocyte apoptosis and oxidative stress by targeted up⁃regulation of Survivin expression .