ABSTRACT
Abstract The incidence of non-alcoholic fatty liver (NAFLD) remains high, and many NAFLD patients suffer from severe ischemia-reperfusion injury (IRI). Currently, no practical approach can be used to treat IRI. Puerarin plays a vital role in treating multiple diseases, such as NAFLD, stroke, diabetes, and high blood pressure. However, its role in the IRI of the fatty liver is still unclear. We aimed to explore whether puerarin could protect the fatty liver from IRI. C57BL/6J mice were fed with a high‐fat diet (HFD) followed by ischemia reperfusion injury. We showed that hepatic IRI was more severe in the fatty liver compared with the normal liver, and puerarin could significantly protect the fatty liver against IRI and alleviate oxidative stress. The PI3K-AKT signaling pathway was activated during IRI, while liver steatosis decreased the level of activation. Puerarin significantly protected the fatty liver from IRI by reactivating the PI3K-AKT signaling pathway. However, LY294002, a PI3K-AKT inhibitor, attenuated the protective effect of puerarin. In conclusion, puerarin could significantly protect the fatty liver against IRI by activating the PI3K-AKT signaling pathway.
ABSTRACT
Objective To identify and validate co-expressed genes associated with myocardial ischemia/reperfusion injury(MI/RI)and necrotic apoptosis by bioinformatics analysis.Methods Gene expression profile data for MI/RI were obtained by GSE67308 and GSE19875 datasets from the Gene Expression Omnibus(GEO)database.Differential expression analysis was conducted on the GSE67308 dataset to identify differentially expressed genes(DEGs),followed by gene set enrichment analysis and biological pathway analysis.More-over,immune cell infiltration analysis was performed on the GSE67308 dataset.Necrotic apoptosis-related genes were retrieved from the Molecular Signatures Database and the Kyoto Encyclopedia of Genes and Genomes(KEGG).A protein-protein interaction(PPI)network was constructed by overlapping DEGs with these necrotic apoptosis-related genes to identify key genes.Furthermore,the expression pat-terns of these key genes across various cardiac cell types were analyzed using a single-cell sequencing analysis platform,and validation of key gene expression was performed using the GSE19875 dataset.Results A total of 1054 DEGs were identified,comprising 363 upregu-lated and 691 downregulated genes.Gene enrichment analysis revealed that DEGs were primarily associated with processes related to apoptosis,immune responses,and intracellular signaling regulation.Moreover,biological pathway analysis demonstrated that DEGs were predominantly involved in the regulation of signaling pathways such as tumor necrosis factor(TNF)and NF-κB.Immune infiltration anal-ysis indicated a high degree of immune infiltration,particularly with natural killer cells and monocytes,in MI/RI myocardial tissue.PPI network analysis identified Il1b,TNF,Birc3,and Ripk1as crucial genes in the context of necrotic apoptosis.Single-cell sequencing anal-ysis showed the elevated expression of key genes within white blood cells.In comparison to the control group,the MI/RI model group in the GSE19875 dataset exhibited significantly increased expression of Il1b,TNF,Birc3,and Ripk1(P<0.01).Conclusion MI/RI is strongly correlated with the TNF signaling pathway and the NF-κB signaling pathway,both of which play pivotal roles in regulating necrotic apop-tosis.Il1b,TNF,Birc3,and Ripk1emerge as key genes that concurrently regulate both MI/RI and necrotic apoptosis.It is plausible that IL-1b,TNF,Birc3,and Ripk1 may serve as critical regulatory factors in the context of necrotic apoptosis during MI/RI.
ABSTRACT
Acute myocardial infarction(AMI)is a common cardiovascular emergency in clinic.Early reperfusion is a typical and effective method for the treatment of AMI.However,the recovery of blood supply after reperfusion therapy will accelerate the damage of ischemic myocardium and cause myocardial ischemia-reperfusion injury(MI/RI).In recent years,studies have found that TCM has the unique advantages of multi-component,multi-channel and multi-target in the intervention of MI/RI.Janus tyrosine kinase/signal transducer and activator of transcription(JAK/STAT)signaling pathway is closely related to MI/RI,which can reduce MI/RI process by regulating inflammation,oxidative stress,cell proliferation,differentiation and apoptosis.This article reviewed the mechanism of JAK/STAT signaling pathway in MI/RI and the research of TCM targeting this pathway,in order to provide references for the prevention and treatment of MI/RI and further drug development.
ABSTRACT
Objective:To investigate the myocardial protective effect of extracorporeal cardiac shock wave therapy (CSWT) combined with sulfur hexafluoride microbubble post-conditioning on myocardial ischemia-reperfusion injury (MI/RI) in rats, and to provide theoretical support for clinical treatment of MI/RI.Methods:A total of 32 male SD rats were randomly divided into 4 groups: sham operation group (Sham group), MI/RI group (IR group), CSWT group (IR+ SW group), and CSWT combined with sulfur hexafluoride microbubble treatment group (IR+ SW+ MB group), with 8 rats in each group. Therapeutic intervention was performed in IR+ SW group and IR+ SW+ MB group on the 1st, 3rd and 5th day after modeling. The left ventricular end-diastolic diameter (LVEDD), left ventricular end-systolic diameter (LVESD), left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS) of the rats were measured by echocardiography before and after treatment. On the 7th day, myocardial fibrosis was assessed by Masson staining, and cardiomyocyte apoptosis was observed by TUNEL staining. The myocardial apoptotic proteins Bcl-2, BAX, Cleaved-Caspase-3 and Cleaved-Caspase-9 in the infarct boundary area were detected by Western blot. The differences of the above indexes among different groups were compared.Results:①There was no significant change in heart rhythm and heart rate among the groups before and after treatment, and there was no significant difference in heart rate ( P>0.05). ②The echocardiographic results after treatment showed that, compared with IR group, LVEDD and LVESD in IR+ SW group and IR+ SW+ MB group decreased in turn, while LVEF and LVFS increased in turn with significant differences between each two groups (all P<0.05). ③Compared with IR group, the degrees of myocardial fibrosis and apoptosis in IR+ SW group and IR+ SW+ MB group were alleviated in turn, and the relief in IR+ SW+ MB group was the most obvious. Quantitative analysis showed that compared with IR group, the proportions of cardiomyocyte apoptosis in IR+ SW group and IR+ SW+ MB group decreased in turn, and there were significant differences between each two groups (all P<0.05). ④The results of Western blot detection showed that compared with IR group, the levels of Bcl-2 in IR+ SW group and IR+ SW+ MB group increased in turn, while the levels of BAX and the activation level of Caspase-3 and Caspase-9 protein decreased in turn. These differences were all statistically significant between each two groups (all P<0.05) except for the activation level of Caspase-3 protein between IR+ SW group and IR+ SW+ MB group ( P>0.05). Conclusions:CSWT combined with sulfur hexafluoride microbubble therapy can improve left ventricular remodeling and left ventricular systolic function by inhibiting cardiomyocyte apoptosis.
ABSTRACT
@#Objective To explore the effect of microRNA-138(miR-138)on injury of ischemia/reperfusion(I/R)induced human renal tubular epithelium(HK-2)cells through neutrophil gelatinase-associated lipocalin(NGAL).Methods HK-2 cells were used to construct I/R model cells,and transfected with miR-138 mimic,miR-138 inhibitor,NGAL,NGAL + miR-138 mimic plasmids,respectively.qRT-PCR determined the expression of miR-138 or NGAL mRNA in different cells to identify the transfection results.Cell counting kit-8(CCK-8)method and flow cytometry were used to detected the activities and apoptosis of cells.ELISA and western blot were used to determine the effects of miR-138 mimic or miR-138 inhibitor on levels of interleukin(IL)-6,IL-1β,tumor necrosis factor(TNF-α)and protein expression of toll like receptor 4(TLR4),nuclear factor kappa-B(NF-κB),inhibitor of NF-κB(IκBα),pho-IκBα(p-IκBα),NGAL of cells.Results miR-138 mRNA expression and cell activity were decreased,while apoptosis increased in I/R cells(P<0.01).Plasmid transfected well,miR-138 mimic increased activity while decreased apoptosis and NGAL mRNA expression of I/R cell.miR-138 inhibitor or NGAL mimic inhibited activity and increased apoptosis and NGAL mRNA expression of I/R cell.The negative effects of NGAL mimic on I/R cell were reversed by miR-138 mimic.miR-138 inhibitor increased levels of IL-6,IL-1β,TNF-α of I/R cell,and increased TLR4,NF-κB,p-IκBα,NGAL protein expression and decreased IκBα protein expression(P<0.05).While miR-138 mimic decreased levels of IL-6,IL-1β,TNF-α of I/R cell,and decreased TLR4,NF-κB,p-IκBα,NGAL protein expression and increased IκBα protein expression(P<0.05).Conclusion miR-138 reduced apoptosis and inflammation factor levels to play a protective role on I/R induced HK-2 cells may through regulating NGAL and TLR4/NF-κB pathway.
ABSTRACT
ObjectiveAcute myocardial infarction (AMI) is a highly prevalent and deadly disease globally, with its incidence continuing to rise in recent years. Timely reperfusion therapy is crucial for improving the prognosis of AMI patients. However, myocardial reperfusion can lead to irreversible myocardial ischemia/reperfusion (MI/R) injury, which is associated with adverse cardiovascular outcomes following AMI. Studies have shown that microRNAs (miRNAs) are abnormally expressed during MI/R injury and play an important role in the fate of cardiomyocytes. Effective preventive and therapeutic strategies against MI/R injury remain lacking in clinical practice, necessitating elucidation of the molecular mechanisms underlying MI/R onset and progression. This study investigated the role of microRNA-878 (miR-878) in the regulation of mitochondria-mediated apoptosis in MI/R injury. MethodsThe H9c2 cells were flushed with a gas mixture containing 1% O2, 5% CO2 and 94% N2 for 3 h. Then the cells were incubated in complete culture medium under 5% CO2 and 95% air for 6 h to mimic in vivo hypoxia/reoxygenation (H/R) injury. Cell viability were detected by CCK-8 assay. The concentrations of lactate dehydrogenase (LDH) were then measured.The level of apoptosis was analyzed by flow cytometry. The morphology of mitochondria was analyzed by immunofluorescence and laser confocal microscopy. The levels of mitochondrial reactive oxygen species (mtROS) were detected by immunofluorescence. Dual luciferase reporter gene assay was used to study the binding site of miR-878 and Pim1. RNA immunoprecipitation (RIP) assay was used to verify the binding relationship between miR-878 and Pim1. The gene expression levels were detected by real-time fluorescent quantitative PCR (RT-qPCR) and Western blot. ResultsThe study found that compared with the control group, the expression of miR-878 in H/R-treated H9c2 cells was significantly increased ((1.00±0.25) vs (9.70±2.63), P<0.01). In H/R-induced cells, transfection of miR-878 inhibitor significantly increased cell viability ((46.67±3.00) vs (74.62±4.08), P<0.000 1), and decreased LDH release ((358.58±41.71) vs (179.09±15.59), P<0.000 1) and cell apoptosis rate ((43.41±0.72) vs (27.42±4.48), P<0.01). At the same time, downregulation of miR-878 expression significantly inhibited DRP1-mediated mitochondrial overdivision and mtROS production ((6.60±0.57) vs (4.32±0.91), P<0.000 1). The mechanism study showed that miR-878 could target and bind Pim1 and inhibit the expression level of Pim1 ((1.00±0.13) vs (0.38±0.03), P<0.01). Rescue experiments confirmed that down-regulation of Pim1 expression significantly reversed the anti-injury effect of miR-878 inhibitor in H9c2 cells (P<0.01), promoted mitochondrial overdivision and mtROS production ((1.00±0.12) vs (2.41±0.12), P<0.01), and decreased the expression level of p-DRP1 ((1.00±0.15) vs (0.59±0.06), P<0.05). ConclusionThe present study demonstrates that miR-878 expression is upregulated in H9c2 cardiomyocytes subjected to H/R injury. Inhibition of miR-878 expression alleviates H/R-induced cardiomyocyte damage. Notably, downregulation of miR-878 significantly inhibits DRP1-mediated mitochondrial fission and mitigates mtROS production. Mechanistically, miR-878 targets and binds to the 3'-UTR of the Pim1 gene, thereby suppressing Pim1 protein expression. Collectively, these findings suggest that under H/R conditions, miR-878 promotes excessive mitochondrial fragmentation through DRP1 activation by targeting Pim1, ultimately contributing to cardiomyocyte injury. Modulation of the miR-878/Pim1 axis may represent a potential therapeutic strategy for mitigating MI/R-induced cardiac damage.
ABSTRACT
Objective To evaluate the effect of spliced X-box binding protein 1 (XBP1s) on hypoxia/reoxygenation (H/R) injury of mouse renal tubular epithelial cells and unravel underlying mechanism. Methods Mouse renal tubular epithelial cells were divided into adenovirus negative control group (Ad-shNC group), targeted silencing XBP1s adenovirus group (Ad-shXBP1s group), Ad-shNC+H/R group and Ad-shXBP1s+H/R group. The apoptosis level, mitochondrial reactive oxygen activity, mitochondrial membrane potential and mitochondrial calcium ion level were detected in each group. Chromatin immunocoprecipitation followed by sequencing (ChIP-seq) was employed to analyze the binding sites of XBP1s in regulating the inositol 1,4,5-trisphosphate receptor (ITPR) family. The expression levels of XBP1s and ITPR family messenger RNA (mRNA) and protein were determined in each group. Results Compared with the Ad-shNC group, the apoptosis level was higher, mitochondrial reactive oxygen species level was increased, mitochondrial membrane potential was decreased and mitochondrial calcium ion level was elevated in the Ad-shNC+H/R group. Compared with the Ad-shNC+H/R group, the apoptosis level was lower, mitochondrial reactive oxygen species level was decreased, mitochondrial membrane potential was elevated, and mitochondrial calcium ion level was decreased in the Ad-shXBP1s+H/R group (all P<0.05). Compared with the Ad-shNC group, relative expression levels of XBP1s, ITPR1, ITPR2 and ITPR3 mRNAs and proteins were down-regulated in the Ad-shXBP1s group (all P<0.05). Compared with the Ad-shNC group, relative expression levels of XBP1s, ITPR1, ITPR2 and ITPR3 proteins were up-regulated in the Ad-shNC+H/R group. Compared with the Ad-shNC+H/R group, relative expression levels of XBP1s, ITPR1, ITPR2 and ITPR3 were down-regulated in the Ad-shXBP1s+H/R group (all P<0.05). ChIP-seq results showed that XBP1s could bind to the promoter and exon of ITPR1, the exon of ITPR2, and the exon of ITPR3. Conclusions XBP1s may affect mitochondria-associated endoplasmic reticulum membrane structure and function by directly regulating ITPR transcription and translation. Down-regulating XBP1s may inhibit ITPR expression and mitigate mitochondrial damage.
ABSTRACT
CD47 is a transmembrane protein widely expressed on cell surface, which is considered as a key molecule for immune escape. With an increasing number of related studies, the role of CD47 and its ligands in immunomodulatory effects has been gradually understood. Recent studies have investigated the role of CD47 in ischemia-reperfusion injury of allogenetic kidney transplantation, rejection and xenotransplantation. Nevertheless, the specific role and the key mechanism remain elusive. In this article, the structure and function of CD47, common CD47 ligands, the relationship between CD47 and kidney transplantation, and the application of CD47 in kidney transplantation were reviewed, the latest research progress of CD47 in kidney transplantation was summarized, and the limitations of current research and subsequent research direction were analyzed, aiming to provide reference for subsequent application of CD47 in allogeneic and kidney xenotransplantation.
ABSTRACT
Purpose: To investigate the protective effect of breviscapine on myocardial ischemia-reperfusion injury (MIRI) in diabetes rats. Methods: Forty rats were divided into control, diabetes, MIRI of diabetes, and treatment groups. The MIRI of diabetes model was established in the latter two groups. Then, the treatment group was treated with 100 mg/kg breviscapine by intraperitoneal injection for 14 consecutive days. Results: After treatment, compared with MIRI of diabetes group, in treatment group the serum fasting blood glucose, fasting insulin, homeostasis model assessment of insulin resistance, and glycosylated hemoglobin levels decreased, the serum total cholesterol, triacylglycerol, and low-density lipoprotein cholesterol levels decreased, the serum high-density lipoprotein cholesterol level increased, the heart rate decreased, the mean arterial pressure, left ventricular ejection fraction, and fractional shortening increased, the serum cardiac troponin I, and creatine kinase-MB levels decreased, the myocardial tumor necrosis factor α and interleukin-6 levels decreased, the myocardial superoxide dismutase level increased, and the myocardial malondialdehyde level decreased (all P < 0.05). Conclusions: For treating MIRI of diabetes in rats, the breviscapine can reduce the blood glucose and lipid levels, improve the cardiac function, reduce the myocardial injury, and decrease the inflammatory response and oxidative stress, thus exerting the alleviating effect.
Subject(s)
Animals , Rats , Myocardial Reperfusion Injury , Oxidative Stress , Diabetes Mellitus , Inflammation , IschemiaABSTRACT
Abstract Background: Cuproptosis is known to regulate diverse physiological functions in many diseases, but its role in regulating Myocardial Ischemia-Reperfusion Injury (MI/RI) remains unclear. Methods: For this purpose, the MI/RI microarray datasets GSE61592 were downloaded from the Gene Expression Omnibus (GEO) database, and the Differently Expressed Genes (DEGs) in MI/RI were identified using R software. Moreover, the MI/RI mice model was established to confirm further the diagnostic value of Pyruvate Dehydrogenase B (Pdhb), Dihydrolipoamide S-acetyltransferase (Dlat), and Pyruvate dehydrogenase E1 subunit alpha 1 (Pdhα1). Results: The analysis of microarray datasets GSE61592 revealed that 798 genes were upregulated and 768 were downregulated in the myocardial tissue of the ischemia-reperfusion injury mice. Furthermore, Dlat, Pdhb, Pdhα1, and cuproptosis-related genes belonged to the downregulated genes. The receiver operating characteristics curve analysis results indicated that the Dlat, Pdhb, and Pdhα1 levels were downregulated in MI/RI and were found to be potential biomarkers for MI/RI diagnosis and prognosis. Similarly, analysis of Dlat, Pdhb, and Pdhα1 levels in the MI/RI mice revealed Pdhb being the key diagnostic marker. Conclusions: This study demonstrated the prognostic value of cuproptosis-related genes (Dlat, Pdhb, and Pdhα1), especially Pdhb, MI/RI, providing new insight into the MI/RI treatment.
ABSTRACT
AIM To explore the effects of Buyang Huanwu Decoction on mitochondrial oxidative damage and PKCε-Nampt pathway in rats following cerebral ischemia reperfusion(I/R).METHODS The rats were randomly divided into the sham operation group,the model group,Buyang Huanwu Decoction group(14.3 g/kg)and edaravone group(3 mg/kg).Except those of the sham operation group,SD rats of other groups were induced into models of brain I/R injury by MCAO method,followed by corresponding drug administration 24 hours after operation.After 7 days of administration,the rats had their neurological deficit evaluated by neurological function scoring;thier expression of neuron marker MAP-2 detected by immunofluorescence staining;their neuron damage observed and the oxidative damage evaluated through assessment of their ROS levels and MDA and SOD activities;their changes of mitochondrial membrane potential detected by fluorescent probe JC-1;their ratio of NAD+/NADH detected using modified enzyme circulation method;their expressions of PKCε,p-PKCε and Nampt proteins detected with Western blot;and their positive expressions of p-PKCε and Nampt proteins detected with immunohistochemistry method.RESULTS Compared with the model group,Buyang Huanwu Decoction group shared decreased cerebral infarction volume and neurological function score(P<0.05);increased cerebral fluorescence intensity of MAP-2(P<0.05);reduced neuronal damage,decreased cerebral levels of ROS and MDA(P<0.05);increased SOD activity,mitochondrial membrane potential and NAD+/NADH ratio(P<0.05);and increased protein expressions of p-PKCε and Nampt(P<0.05).CONCLUSION Buyang Huanwu Decoction can improve mitochondrial function and reduce brain I/R injury in rats by activating their PKCε-Nampt signaling pathway.
ABSTRACT
AIM To investigate the effects of Zhuangyao Shuanglu Tongnao Formula on neuronal apoptosis in rats with cerebral ischemia-reperfusion injury based on the study of oxidative stress and inflammatory response.METHODS The rats were randomly divided into the sham operation group,the model group,the edaravone group(3.0 mg/kg),the low,medium and high dose groups(9.0,18.0,36.0 g/kg)of Zhuangyao Shuanglu Tongnao Formula,with 18 rats in each group.The middle cerebral artery occlusion/reperfusion was conducted by thread embolism method to simulate cerebral ischemia reperfusion injury in rats followed by 6 days corresponding drugs administration.Subsequently,the rats had their neurological function deficit scored by Zeal Longa scoring method;their sizes of cerebral infarction areas measured by TTC staining;their pathological damage and apoptosis of neurons in hippocampal CA1 area of ischemic penumbra of the brain tissue detected by HE staining and TUNEL staining;their SOD activity and levels of GSH,MDA,IL-6,IL-1β,TNF-α in brain tissue detected by kits;and their protein expressions of Bax,Bcl-2,caspase-3,cleaved-capase-3,TLR4,NF-κB p65,Nrf2,HO-1 in rat brain tissue determined by Western blot.RESULTS Compared with the model group,the groups intervened with edaravone,medium and high dose of Zhuangyao Shuanglu Tongnao Formula displayed improvements in the scores of nerve function defects,the rate of cerebral infarction,the rate of neuronal apoptosis,the levels of IL-6,IL-1β,TNF-α and MDA in the ischemic penumbra of brain tissues,the protein expressions of Bax and TLR4,the ratio of cleaved-capase-3/caspase-3 and p-NF-κB p65/NF-κB p65(P＜0.05),the levels of GSH,the activity of SOD and the protein expressions of Bcl-2,Nrf2 and HO-1(P＜0.05).CONCLUSION Being an inhibitor of oxidative stress and inflammatory response,Zhuangyao Shuanglu Tongnao Formula can alleviate brain injury in rats with cerebral ischemia reperfusion injury through the inhibition of neuronal apoptosis and improvement of neural function mediated by the inhibition of TLR4/NF-κB signal pathway and activation of Nrf2/HO-1 signal pathway.
ABSTRACT
The nucleotide-binding oligomerization domain-like receptor protein 3(NLRP3)inflammasome is an inflammatory protein complex, and can participate into the inflammatory response. Upon activation, these inflammasomes can lead to Caspase-1 activation, thereby inducing a cascade of inflammatory factor activation and further cell pyroptosis. Excessive activation of inflammasomes will induce the overexpression of inflammatory factors, persistently triggering immune dysregulation and inflammatory chain reactions, even causing severe damage. The recent studies have confirmed a close association between retinal diseases, such as diabetic retinopathy(DR), retinal ischemia-reperfusion injury(RIRI), and proliferative vitreoretinopathy(PVR)with immune dysregulation and inflammatory responses, which is serving as crucial factors in the progression of retinal diseases. This article reviews the NLRP3 inflammasome signaling pathway and its role in the occurrence and development of retinal diseases, in order to provide new ideas for the pathogenesis and prevention of retinal diseases.
ABSTRACT
ObjectiveTo investigate the effect and mechanism of Taohong Siwutang （TSD） on myocardial ischemia reperfusion injury （MIRI） in ovariectomized （OVX） female mice. MethodAfter the OVX model of female mice was established， the estradiol （E2） level was detected by enzyme-linked immunosorbent assay （ELISA） to verify the model. Sixty OVX mice were randomly divided into six groups： Sham operation group， model group， low， medium， and high dose groups （9， 18， 36 g·kg-1） of TSD， and nuclear factor E2-related factor 2 （Nrf2） inhibitor group， with 10 mice in each group. The MIRI model was verified by a laser speckle flowmeter. The pathological changes in myocardial tissue were detected by 2，3， 5-triphenyltetrazolium chloride （TTC） and hematoxylin-eosin （HE） staining. Serum creatine kinase isoenzyme （CK-MB）， cardiac troponin Ⅰ （CTnⅠ）， malondialdehyde （MDA）， superoxide dismutase （SOD）， interleukin-6 （IL-6）， and interleukin-10 （IL-10） levels were detected by ELISA. The expression of Nrf2 and heme oxygenase-1 （HO-1） were observed by immunofluorescence staining. The expressions of Nrf2， HO-1， apoptotic B-cell lymphomato-2 （Bcl-2）， Bcl-2 associated X protein （Bax）， inflammatory cytokines interleukin-18 （IL-18）， and interleukin-1β （IL-1β） were detected by Western blot. ResultCompared with the sham operation group， the serum levels of CK-MB， CTnⅠ， MDA， and IL-6 in the model group were increased （P<0.01）， and the levels of SOD and IL-10 were decreased （P<0.01）. The damage scores of TTC and HE staining in myocardial tissue were increased （P<0.01）. The expressions of Nrf2 and HO-1 in myocardial tissue by immunofluorescence were decreased （P<0.01）， and the protein expressions of Nrf2， HO-1， and Bcl-2 in myocardial tissue were decreased. The protein expressions of Bax， IL-18， and IL-1β were increased （P<0.01）. Compared with the model group， serum levels of CK-MB， CTnⅠ， MDA， and IL-6 of TSD groups were significantly decreased （P<0.05， P<0.01）， and SOD and IL-10 were significantly increased （P<0.05， P<0.01）. TTC staining and HE staining damage scores of myocardial tissue were significantly decreased （P<0.01）. The expressions of Nrf2 and HO-1 in myocardial tissue by immunofluorescence were increased （P<0.01）. The protein expressions of Nrf2， HO-1， and Bcl-2 were significantly increased （P<0.05， P<0.01）， and those of Bax， IL-18， and IL-1β were significantly decreased （P<0.05， P<0.01）. The effect of the high dose group of TSD was the most significant. The serum levels of CK-MB， CTnⅠ， MDA， and IL-6 in the Nrf2 inhibitor group were significantly increased （P<0.05， P<0.01）， and the levels of SOD and IL-10 were significantly decreased （P<0.05， P<0.01）. The damage scores of TTC and HE staining in myocardial tissue were significantly increased （P<0.01）. The expressions of Nrf2 and HO-1 in myocardial tissue by immunofluorescence were significantly decreased （P<0.01）. The protein expressions of Nrf2， HO-1， and Bcl-2 in myocardial tissue were significantly decreased， and those of Bax， IL-18， and IL-1β were significantly increased （P<0.01）. ConclusionTSD can alleviate MIRI in OVX mice， reduce oxidative stress response and the release of inflammatory factors， and inhibit apoptosis， playing a protective role in OVX mice with MIRI， which may be related to the activation of Nrf2/HO-1 signaling pathway.
ABSTRACT
【Objective】 To explore the role of ZFP36 in cardiomyocyte injury and autophagy induced by hypoxia/reoxygenation (H/R) so as to clarify its molecular regulatory mechanism. 【Methods】 H9C2 rat cardiomyocytes were infected with ZFP36 overexpressing lentivirus (OE-ZFP36) or its negative control lentivirus (OE-ZFP36 NC) to construct stable cell lines, respectively. Transfection of ATG4D overexpression plasmid (OE-ATG4D) improved the expression of ATG4D. Hypoxia/reoxygenation (H/R) induced myocardial cell injury. H9C2 cells were mainly divided into control group, H/R group, OE-ZFP36 NC+H/R group, OE-ZFP36+H/R group, OE-ATG4D NC+H/R group, OE-ATG4D+H/R group, OE-ZFP36+OE-ATG4D NC+H/R group, and OE-ZFP36+OE-ATG4D+H/R group. The protein expressions of ATG4D, Beclin1, LC3 and ZFP36 in H9C2 cells were detected by Western blotting. The mRNA levels of ZFP36 and ATG4D in H9C2 cells were detected by Real-time fluorescence quantitative PCR (qPCR). The viability of H9C2 cells was detected by CCK-8 assay. The levels of interleukin (IL-6) and tumor necrosis factor (TNF-α) in H9C2 cells were detected by enzyme-linked immunosorbent assay (ELISA). Reactive oxygen species (ROS) in H9C2 cells were detected by DCFH-DA method. SOD detection kit was used to detect the SOD level in H9C2 cells. The apoptosis of H9C2 cells was detected by flow cytometry. LC3 autophagosomes in H9C2 cells were detected by cellular immunofluorescence. Dual-luciferase reporter gene assay was used to detect the binding of ZFP36 and ATG4D mRNA in H9C2 cells. 【Results】 Compared with control group, H/R group showed decreased cell viability, increased IL-6 and TNF-α levels, increased ROS levels and decreased SOD levels, increased cell apoptosis. Up-regulated ATG4D and Beclin1 protein expression, increased LC3Ⅱ/LC3Ⅰ ratio, as well as upregulated ZFP36 expression were found in H/R group (all P<0.05). Compared with OE-ZFP36 NC+H/R group, elevated cell viability, decreased IL-6 and TNF-α levels, decreased ROS levels and increased SOD levels, reduced cell apoptosis (P<0.05), and downregulated ATG4D and Beclin1 protein expression, decreased LC3Ⅱ/LC3Ⅰ ratio were shown in OE-ZFP36+H/R group (all P<0.05). Compared with infection with OE-ZFP36 NC lentivirus, infection with OE-ZFP36 lentivirus decreased the luciferase activity of ATG4D 3′-UTR reporter gene, decreased the stability of ATG4D mRNA, and downregulated the H/R-induced ATG4D mRNA expression (all P<0.05). Compared with OE-ATG4D NC+H/R group, OE-ATG4D+H/R group had upregulated ATG4D mRNA and protein expression, decreased cell viability, increased IL-6 and TNF-α levels, increased ROS levels, decreased SOD levels and elevated cell apoptosis (all P<0.05). Compared with OE-ZFP36+OE-ATG4D NC+H/R group, OE-ZFP36+OE-ATG4D+H/R group had decreased cell viability, increased IL-6 and TNF-α levels, increased ROS levels, decreased SOD levels and elevated cell apoptosis (all P<0.05). 【Conclusion】 The expression of ZFP36 is upregulated in H/R-induced cardiomyocyte injury. The overexpression of ZFP36 inhibits H/R-induced cardiomyocyte injury and autophagy by regulating ATG4D, thus resisting cardiomyocyte H/R injury. It proves that ZFP36 is an important regulatory molecule against MI/RI.
ABSTRACT
AIM: To investigate the protective effect and mechanism of Danlou tablet on retinal ischemia-reperfusion injury(RIRI)in mice.METHODS: A total of 40 ApoE-/- mice were fed with high fat diet for 6 wk, and the RIRI model was established by anterior chamber infusion of pressurized saline. The mice were divided into control group(normal saline for 8 wk), RIRI model group(normal saline for 8 wk), and low-, medium-, and high-dose Danlou tablets groups [1, 2, and 4 g/(kg·d), respectively, for 8 wk]. The morphological changes of retina were observed by hematoxylin-eosin(HE)staining, retinal cell apoptosis was detected by terminal-deoxynucleoitidyl transferase mediated Nick-End Labeling(TUNEL)staining. The Western-blot assay was used to detect the expression of retinal tissue sample Kelch-like ech-associated protein 1(Keap1), nuclear factor E2 related factor 2(Nrf2), heme oxygenase 1(HO-1), and superoxide dismutase(Sod2)proteins.RESULTS: Compared with the control group, the mouse retina was atrophic with thinning thickness and increasing cell apoptosis, down-regulation of Sod2 protein expression, and up-regulation of Keap1 protein expression in the RIRI model group(all P<0.01). Compared with the RIRI model group, the retinal thickness increased in the medium- and high-dose of Danlou tablets groups(all P<0.01), and the cell apoptosis of retina decreased in the low-, medium- and high-dose of Danlou tablets groups(all P<0.05). There were no significant differences in the expression of Keap1 and HO-1 proteins of mouse retina tissue in the low-dose of Danlou tablets group(P>0.05). The expression of Sod2, Nrf2 and HO-1 proteins up regulated, and the expression of Keap1 protein down regulated in the medium- and high-dose of Danlou tablets groups(all P<0.05).CONCLUSION: Danlou tablet can alleviate RIRI-induced atrophy and thinning of retina and retinal cell apoptosis by regulating Keap1-Nrf2/HO-1 signal pathway and reducing oxidative stress.
ABSTRACT
OBJECTIVE To explore the protective effect and mechanism of Longshengzhi capsules on cerebral ischemia- reperfusion injury in rats. METHODS The model of middle cerebral artery occlusion （MCAO） was established by using the improved thread occlusion method. The experiment was divided into six groups： sham surgery group （only separating blood vessels without inserting thread plugs， given the same volume of normal saline）， model group （modeling， given the same volume of normal saline）， nimodipine group （positive control， modeling， dose of 20 mg/kg）， and low-dose， medium-dose， and high-dose groups of Longshengzhi capsules （modeling， doses of 0.72， 1.44 and 2.88 g/kg， respectively）， with 10 mice in each group. Each group was given corresponding medication solution/normal saline by gavage， once a day， for 7 consecutive days. One hour after the last administration， the Zea Longa scoring method was used to score the neurological deficits in each group of rats， and the ABC enzyme-linked immunosorbent assay was used to detect the serum levels of tumor necrosis factor-α （TNF-α） and interleukin-6 （IL-6） in rats； TTC staining was used to observe the volume of cerebral infarction in rats and calculate the cerebral infarction volume ratio. Hematoxylin eosin staining was used to observe the pathological changes in the brain tissue of rats. Immunohistochemical staining was used to detect the positive expression of NLRP3 protein in the brain tissue of rats. Real-time fluorescence quantitative PCR was used to detect mRNA relative expressions of Toll-like receptor 4 （TLR4） and nuclear factor-κB （NF-κB） in the brain tissue of rats. Western blot assay was adopted to detect the relative expressions of TLR4， NLRP3 and phosphorylated NF-κB （p-NF-κB） protein in the brain tissue of rats and its intracellular NF-κB protein. RESULTS Compared with the sham surgery group， the neural dysfunction score， serum levels of TNF-α and IL-6， cerebral infarction volume ratio， relative expression levels of NF-κB and TLR4 mRNA， as well as protein relative expressions of TLR4， NLRP3 and p-NF-κB in the brain tissue， and relative protein expression of intracellular NF-κB were increased significantly in the model group （P＜0.01）； the enlarged gap and significant edema were observed in cortical nerve cells of brain tissue in rats， with a large amount of inflammatory cell infiltration； the positive expression of NLRP3 protein in brain tissue of rats obviously increased. Compared with the model group， the levels of the above indicators in the medium-dose and high-dose groups of Longshengzhi capsules， as well as the Nimodipine group， were reversed to varying degrees， and most differences were statistically significant （P＜0.05 or P＜0.01）； the pathological morphology observation showed a significant improvement， and the positive expression of NLRP3 protein in the brain tissue of rats was obviously reduced. CONCLUSIONS Longshengzhi capsules may inhibit TLR4/NF-κB/NLRP3 signaling pathway and neuroinflammatory response， thereby achieving a protective effect against cerebral ischemia-reperfusion injury in rats.
ABSTRACT
Aim To investigate the regulatory effect of geraniol on Nrf2/HO-1 signaling pathway after cerebral ischemia-reperfusion(I/R)in rats. Methods In this experiment,all the male SD rats were randomly divided into nine groups receiving the following treatments:sham operation(sham); sham operation+200 mg·kg-1 geraniol; I/R; I/R+50 mg·kg-1 geraniol; I/R+100 mg/kg geraniol; I/R+200 mg·kg-1 geraniol; edaravone; I/R+ brusatol(Nrf2 inhibitor); I/R+200mg·kg-1 geraniol+brusatol. All rats received intraperitoneal injection of geraniol for five consecutive days before MCAO and again after MCAO. During the construction of cerebral I/R injury models,the blood vessels were isolated without any suture in the sham operation and the sham operation +200 mg·kg-1 geraniol groups while the blood vessels with suture in other groups. The damage of neurological function was evaluated by the modified rating scale for neurological function. The TTC,HE and Tunel staining methods were used to determine the cerebral infarction volume,the damage of the ischemic cortex and the apoptosis of cortical cells,respectively. The oxidative stress-related parameters then were measured. The protein expressions of Nrf2 and HO-1 were detected by immunohistochemical staining and the target protein expressions of the injured cortex were detected by Western blot. Results Compared with the model group,it was found that the geraniol treatment significantly repaired neural injury,reduced cerebral infarction volume,cerebral cortex damage and cell apoptosis. Meanwhile,geraniol intervention could significantly increase the expression of Nrf2/HO-1 protein in the right-sided cortex and reduce oxidative stress level. Conclusion Geraniol can attenuate cerebral injury induced by ischemia-reperfusion in rats via activating Nrf2/HO-1 signaling pathway.
ABSTRACT
Myocardial ischemia-reperfusion injury （MIRI） is a serious complication of revascularization in patients with myocardial infarction. The nuclear factor erythroid 2-related factor 2 （Nrf2）/heme oxygenase-1 （HO-1） signaling pathway plays an important role in the pathological process of MIRI. Currently，research has found that traditional Chinese medicine has a good effect on myocardial injury caused by ischemia-reperfusion. Based on the Nrf2/HO-1 signaling pathway，this article summarizes the action mechanism of traditional Chinese medicine formulas and monomers in intervening with MIRI. It is found that traditional Chinese medicine formulas （Yixin formula，Wenyang tongmai formula，Dingxin formula Ⅰ），monomers such as terpenoids （ginkgolides， astragaloside Ⅳ，ginsenosides），phenols （brazilin，hematoxylin A，resveratrol） and quinones （aloe，emodin） can alleviate MIRI by activating the Nrf2/HO-1 signaling pathway，inhibiting oxidative stress and inflammatory reactions，etc.
ABSTRACT
Ischemia-reperfusion injury (IRI) is an extremely complicated pathophysiological process, which may occur during the process of myocardial infarction, stroke, organ transplantation and temporary interruption of blood flow during surgery, etc. As key molecules of immune system, macrophages play a vital role in the pathogenesis of IRI. M1 macrophages are pro-inflammatory cells and participate in the elimination of pathogens. M2 macrophages exert anti-inflammatory effect and participate in tissue repair and remodeling and extracellular matrix remodeling. The balance between macrophage phenotypes is of significance for the outcome and treatment of IRI. This article reviewed the role of macrophages in IRI, including the balance between M1/M2 macrophage phenotype, the mechanism of infiltration and recruitment into different ischemic tissues. In addition, the potential therapeutic strategies of targeting macrophages during IRI were also discussed, aiming to provide reference for alleviating IRI and promoting tissue repair.