Caspase-3 Protein Expression Research Articles

Studies on the proliferation inhibition effects of TUA from Actinidia chinensis Radix on lung cancer xenografts in nude mice and its preliminary mechanism

Objective: To investigate inhibitory effect of TUA (2β, 3β, 23-trihydroxy-urs-12-en-28-oic acid) isolated from Actinidia chinensis Radix on the lung cancer xenografts in nude mice and explore its preliminary mechanism. Methods: NCI-H460 cells were implanted into nude mice and the transplantation tumor block from nude mice of more than 2 generations was inoculated to the right armpits of BALB/c mice with dissecting needle to establish a lung cancer xenograft model. When the transplanted volume was about 50 mm3, the mice were randomly divided into 6 groups: (1) model group; (2) 10 mg·kg–1 cisplatin group; (3) 10 mg·kg–1 PDTC group; (4) TUA high dose group (30 mg·kg–1); (5) TUA middle dose group (12 mg·kg–1); (6) TUA low dose group (6 mg·kg–1). Administration approach was intratumoral injection. The effects of each group on the weight of transplanted tumor animals, the volume and weight of tumor were continuously observed for 14 days. Tumor volume growth curve was drawn and tumor inhibitory rate and index were calculated; HE staining was used to observe nude mice tumor tissue pathological changes. The effects of TUA on NF-κB signaling pathway related proteins were detected by immunohistochemistry and Western blot. Results: In vivo experiments showed that the transplanted tumors in nude mice became smaller compared with the models. With the increase of TUA dose, the tumor tissue became smaller and smaller, especially in high TUA dose (30 mg·kg–1). It had the similar size with the NF-κB inhibitor PDTC (10 mg·kg–1) group. HE dyeing observation results confirmed the degree of tumor necrosis and fission in TUA treated tumor tissues obviously decreased. Immunohistochemical results showed that comparing the TUA treatment group with the model group, p65 expression in tumor tissues was reduced, and expression of IκBα increased. Western blot results also showed that the NF-κB related p65 protein expression levels decreased, at the same time IκBα protein expression level increased; the apoptosis related proteins Survivin protein expression was depressed, Caspase-3 protein expression was promoted. Conclusion: TUA significantly inhibits the growth of lung transplantation tumor and its mechanism. It may be related to the decreasing the expression of p65, Survivin and increasing the expression of IκBα, Caspase-3 in tumor tissues.

Inhibition of autophagy enhances apoptosis induced by Ce6-photodynamic therapy in human colon cancer cells

ObjectiveTo evaluate the therapeutic effect of Chlorin e6 photodynamic therapy (Ce6-PDT) in human colorectal cancer cells and investigate the role of autophagy in Ce6-PDT. MethodsSW480 cells underwent Ce6-PDT with and without pretreatment with the autophagy inhibitor 3-methyladenine (3MA). Cell viability was assessed using 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. Apoptosis was evaluated using an Annexin V assay, using a rhodamine 123 (RH123) assay to evaluate mitochondrial membrane potential (MMP), and by measuring Caspase-3 and Bcl-2 protein expression using western blotting. Autophagy was evaluated by directly visualizing acridine orange-stained acidic vesicular organelles (AVOs) using fluorescent microscopy and by measuring LC3Ⅰ/Ⅱand Atg5 expression using western blotting. ResultsCe6-PDT decreased SW480 viability in a dose-dependent manner. Ce6-PDT induced apoptosis in SW480 cells via the mitochondrial apoptosis pathway as indicated by decreased mitochondrial membrane potential, increased Annexin V staining, and increased Caspase-3 expression. Ce6-PDT was also shown to induce autophagy as demonstrated by increased acridine-orange stained AVOs as well as increased expression of the autophagy-associated proteins Atg5. Inhibition of autophagy with 3MA potentiated SW480 cell response to Ce6-PDT and increased the rate of apoptosis in the treated cells. ConclusionsCe6-PDT induces autophagy and apoptosis of SW480 cells in a dose-dependent manner. Inhibition of autophagy increases the apoptosis induced by Ce6-PDT. Modulation of autophagy may be a potential therapeutic target for colon cancer cells treated with Ce6-PDT.

Vitamin D decreases expression of NLRP1 and NLRP3 inflammasomes in placental explants from women with preeclampsia cultured with hydrogen peroxide

This study aimed to evaluate the immunomodulatory effect of vitamin D (VD) on the NLRP1 and NLRP3 inflammasomes in placental explants from preeclamptic (PE) and normotensive (NT) pregnant women. Placental explants from eight PE and eight NT pregnant women were cultured with or without hydrogen peroxide (H2O2), VD or H2O2 + VD. Gene and protein expression of NLRP1, NLRP3, HMGB1, caspase-1, IL-1β, TNF-α and IL-18 were determined by qPCR and Western blotting/ELISA. Compared to NT pregnant women, the endogenous gene expression of NLRP1, NLRP3, HMGB1, IL-1β, TNF-α and IL-18 was significantly higher in explants from PE and became decreased after VD treatment. Similarly, VD decreased the protein expression of NLRP1, NLRP3, caspase-1, HMGB1, IL-1β, TNF-α and IL-18 in PE. Placental explants from NT cultured with H2O2 showed increased gene and protein expression of NLRP1, NLRP3, caspase-1, IL-1β, TNF-α and HMGB1, while H2O2 was also able to increase TNF-α and caspase-1 gene expression in PE. Treatment with H2O2 + VD decreased gene/protein expression of NLRP1, NLRP3, caspase-1, HMGB1, IL-1β, TNF-α and IL-18 in PE and NT explants with H2O2. NLRP1 and NLRP3 are upregulated in the PE. VD may play an immunomodulatory role in the placental inflammation and downregulates oxidative stress induced in vitro by H2O2.

Enhanced Anticancer Response of Curcumin- and Piperine-Loaded Lignin-g-p (NIPAM-co-DMAEMA) Gold Nanogels against U-251 MG Glioblastoma Multiforme.

Glioblastoma multiforme (GBM) is the most aggressive and commonly diagnosed brain cancer and is highly resistant to routine chemotherapeutic drugs. The present study involves the synthesis of Lignin-g-p (NIPAM-co-DMAEMA) gold nanogel, loaded with curcumin and piperine, to treat GBM. The ongoing study has the application potential to (1) overcome the limitations of drugs biodistribution, (2) enhance the toxicity of anticancer drugs against GBM, and (3) identify the drugs uptake pathway. Atom transfer radical polymerization was used to synthesize the Lignin-g-PNIPAM network, crosslinked with the gold nanoparticles (GNPs) to self-assemble into nanogels. The size distribution and morphological analysis confirmed that the drug-loaded gold nanogels are spherical and exist in the size of 180 nm. The single and combinatorial toxicity effects of curcumin- and piperine-loaded Lignin-g-p (NIPAM-co-DMAEMA) gold nanogels were studied against U-251 MG GBM cells. A cytotoxicity analysis displayed anticancer properties. IC50 of curcumin- and piperine-loaded gold nanogels were recorded at 30 μM and 35 μM, respectively. Immunostaining and Western blot analysis confirmed the protein expression of caspase-3 and cleaved caspase-3 in cells treated with drug-loaded nanogels. Kinetic drug release revealed 86% release of hybrid curcumin–piperine from gold nanogel after 250 min at pH 4. Atomic absorption spectroscopic analysis confirmed that the drug-loaded nanogels have better internalization or association with the cancer cells than the GNPs or nano-gels alone. Morphological studies further confirmed that the curcumin and piperine nanogels penetrate the cells via endocytic pathways and induce caspase-3-related apoptosis. The experimental evidence shows the enhanced properties of combinatorial curcumin–piperine gold nanogels (IC50: 21 μM) to overcome the limitations of conventional chemotherapeutic treatments of glioma cells.

Effects of Sidaxue, a Miao ethnomedicine recipe, on apoptosis and pyrolysis of human fibroblast-like synovial cells in rheumatoid arthritis

To investigate the effects of Sidaxue (SX), a recipe in Miao ethnomedicine, on apoptosis and pyrolysis of human fibroblast-like synovial cells in rheumatoid arthritis (RA-FLS). The target proteins related with RA and those involved in cell apoptosis and pyroptosis were searched in different online databases, and Venny software was used to obtain apoptosis- and pyroptosis-related proteins in RA. RA-apoptosis-pyroptosis protein interaction (PPI) network was constructed to identity the key target proteins related with apoptosis and pyroptosis in RA. Autodock vina software was used to perform molecular docking to verify the binding ability of the main active ingredients in SX with the apoptosis- and pyroptosis-related proteins. In the cell experiment, MH7A cells were treated with 5 mg/L TGT (positive control) or 5, 10, 20, and 40 mg/L SX, and the changes in cell migration and invasion abilities and expressions of apoptosis- and pyroptosis-related proteins were examined using wound healing assay, Transwell assay, ELISA and Western blotting. We identified 9 RA-related apoptotic target proteins, 15 RA-related pyroptosis target proteins, and 4 overlapping target proteins related with RA, apoptosis and pyroptosis. The main active ingredients in SX had a high affinity with the target proteins including TNF-α, Fas, and Bax. In MH7A cells, SX treatment concentration-dependently increased the cell inhibition rate at 24, 48 and 72 h (P < 0.05), and significantly lowered the cell migration ability at 6, 12 and 24 h (P < 0.05); treatment with 20 and 40 mg/L SX for 24 h obviously suppressed MH7A cell invasion (P < 0.05). SX treatment (20 and 40 mg/L) and TGT treatment both significantly lowered the expression levels of TNF-α, IL-1β, and IL-18 in the cells (P < 0.05). The Bax/Bcl-2 ratio and Fas and FasL expressions were increased significantly in cells treated with 20 and 40 mg/L SX (P < 0.05), and caspase-1 expression was decreased significantly in cells treated with 5 and 40 mg/L SX (P < 0.05). SX can induce apoptosis and pyroptosison in RA-FLSs possibly by down-regulating the expressions of TNF-α, IL-1β and IL-18, up-regulating the expressions of Bax, Fas, and FasL, and inhibiting Bcl-2 and caspase-1 protein expressions.

Procyanidin protects human retinal pigment epithelial cells from high glucose by inhibiting autophagy.

The damage of hyperglycemia to the retinal pigment epithelial (RPE) cells is a critical event in diabetic retinopathy (DR). Procyanidin (PC), a kind of polyphenol compounds, has shown to be effective in preventing and treating diabetes as well as its complications, in which autophagy disorder is involved in the pathological mechanism. However, it remains unclear whether PC can play a protective role in DR by regulating the autophagy of RPE. Here, the effect of PC on RPE under high glucose conditions and the role of autophagy were investigated. The cell viability of ARPE-19, a human RPE cell line, was detected by cell counting kit-8 (CCK-8) and the apoptosis rate was measured by flow cytometry. The protein expressions of apoptosis markers, including Bax, Bcl-2, and Caspase-3, as well as autophagy markers including LC3, p62, p53, and mTOR were detected by Western blotting. Autophagic flux in ARPE-19 cells was detected by transfection with Ad-mCherry-GFP-LC3B. Under high glucose conditions, the viability of ARPE-19 was decreased and the apoptosis rate increased, the protein expressions of Bax, Caspase-3, LC3-II/LC3-I, and p-p53 were all increased and the expressions of Bcl-2, p62, and p-mTOR decreased, and autophagic flux was increased compared with that of the controls. Treatment with PC weakened all these changes caused by high glucose. When rapamycin (RPM), an autophagy agonist was added, the cell viability of ARPE-19 by PC treatment was decreased while the apoptosis was increased. Our findings indicate that through the p53/mTOR autophagy pathway, PC may protect RPE cells from high glucose-induced injury.

Downregulation of phosphoglycerate mutase 5 improves microglial inflammasome activation after traumatic brain injury

Traumatic brain injury (TBI) is considered as the most common cause of disability and death, and therefore an effective intervention of cascade pathology of secondary brain injury promptly can be a potential therapeutic direction for TBI prognosis. Further study of the physiological mechanism of TBI is urgent and important. Phosphoglycerate mutase 5 (Pgam5), a mitochondrial protein, mediate mitochondrial homeostasis, cellular senescence, and necroptosis. This study evaluated the effects of Pgam5 on neurological deficits and neuroinflammation of controlled cortical impact-induced TBI mouse model in vivo and LPS + ATP-induced microglia model in vitro. Pgam5 was overexpressed post-TBI. Pgam5 depletion reduced pyroptosis-related molecules and improved microglia activation, neuron damage, tissue lesion, and neurological dysfunctions in TBI mice. RNA-seq analysis and molecular biology experiments demonstrated that Pgam5 might regulate inflammatory responses by affecting the post-translational modification and protein expression of related genes, including Nlrp3, caspase1, Gsdmd, and Il-1β. In microglia, Pgam5-sh abrogated LPS + ATP-induced Il-1β secretion through Asc oligomerization-mediated caspase-1 activation, which was independent of Rip3. The data demonstrate the critical role Pgam5 plays in nerve injury in the progression of TBI, which regulates Asc polymerization and subsequently caspase1 activation, and thus reveals a fundamental mechanism linking microglial inflammasome activation to Asc/caspase1-generated Il-1β-mediated neuroinflammation. Thus, our data indicate Pgam5 worsens physiological and neurological outcomes post-TBI, which may be a potential therapeutic target to improve neuroinflammation after TBI.

Extracorporeal Cardiac Shock Waves Therapy Improves the Function of Endothelial Progenitor Cells After Hypoxia Injury via Activating PI3K/Akt/eNOS Signal Pathway.

Background: Extracorporeal cardiac shock waves (ECSW) have great potential in the treatment of coronary heart disease. Endothelial progenitor cells (EPCs) are a class of pluripotent progenitor cells derived from bone marrow or peripheral blood, which have the capacity to migrate to ischemic myocardium and differentiate into mature endothelial cells and play an important role in neovascularization and endothelial repair. In this study, we investigated whether ECSW therapy can improve EPCs dysfunction and apoptosis induced by hypoxia and explored the underlying mechanisms.Methods: EPCs were separated from ApoE gene knockout rat bone marrow and identified using flow cytometry and fluorescence staining. EPCs were used to produce in vitro hypoxia-injury models which were then divided into six groups: Control, Hypoxia, Hypoxia + ECSW, Hypoxia + LY294002 + ECSW, Hypoxia + MK-2206 + ECSW, and Hypoxia + L-NAME + ECSW. EPCs from the Control, Hypoxia, and Hypoxia + ECSW groups were used in mRNA sequencing reactions. mRNA and protein expression levels were analyzed using qRT-PCR and western blot analysis, respectively. Proliferation, apoptosis, adhesion, migration, and angiogenesis were measured using CCK-8, flow cytometry, gelatin, transwell, and tube formation, respectively. Nitric oxide (NO) levels were measured using an NO assay kit.Results: Kyoto encyclopedia of genes and genomes (KEGG) enrichment analysis showed that differentially expressed genes were enriched in cancer signaling, PI3K-Akt signaling, and Rap1 signaling pathways. We selected differentially expressed genes in the PI3K-Akt signaling pathway and verified them using a series of experiments. The results showed that ECSW therapy (500 shots at 0.09 mJ/mm2) significantly improved proliferation, adhesion, migration, and tube formation abilities of EPCs following hypoxic injury, accompanied by upregulation of p-PI3K, p-Akt, p-eNOS, Bcl-2 protein and NO, PI3K, and Akt mRNA expression, and downregulation of Bax and Caspase3 protein expression. All these effects of ECSW were eliminated using inhibitors specific to PI3K (LY294002), Akt (MK-2206), and eNOS (L-NAME).Conclusion: ECSW exerted a strong repaired effect on EPCs suffering inhibited hypoxia injury by inhibiting cell apoptosis and promoting angiogenesis, mainly through activating the PI3K/Akt/eNOS signaling pathway, which provide new evidence for ECSW therapy in CHD.

Phenotypic and Functional Transformation in Smooth Muscle Cells Derived from a Superficial Thrombophlebitis-affected Vein Wall

Superficial thrombophlebitis (ST) is a frequent pathology, but its exact incidence remains to be determined. This study tested the hypothesis whether relationships exist among smooth muscle cells (SMCs) derived from ST, varicose great saphenous veins (VGSVs), and normal great saphenous veins (GSVs). Forty-one samples of ST, VGSVs, and GSVs were collected. SMCs were isolated and cultured. Proliferation, migration, adhesion, and senescence in SMCs from the three vein walls were compared by various methods. Bax, Bcl-2, caspase-3, matrix metalloproteinase-2 (MMP-2), MMP-9, tissue inhibitor of metalloproteinase-1 (TIMP-1), and TIMP-2 messenger RNA (mRNA) and protein expressions were detected by fluorescence quantitative PCR and Western blot. An obvious decrease in cytoskeletal filaments was observed in thrombophlebitic vascular smooth muscle cells (TVSMCs). The quantity of proliferation, migration, adhesion, and senescence in TVSMCs was significantly higher than in varicose vascular smooth muscle cells and normal vascular smooth muscle cells (NVSMCs) (all P < 0.05). Bax and caspase-3 mRNA and protein expression were decreased, while Bcl-2 mRNA and protein expression were increased in the TVSMCs compared with the varicose vascular smooth muscle cells and the NVSMCs (all P < 0.05). MMP-2, MMP-9, TIMP-1, and TIMP-2 mRNA and protein expression were significantly increased in the TVSMCs compared with the VVGSVs and the NVSMCs (all P < 0.05). SMCs derived from ST are more dedifferentiated and demonstrate increased cell proliferation, migration, adhesion, and senescence, as well as obviously decreased cytoskeletal filaments. These results suggest that the phenotypic and functional differences could be related to the presence of atrophic and hypertrophic vein segments during the disease course among SMCs derived from ST, VGSVs, and GSVs.

Mafenide derivatives inhibit neuroinflammation in Alzheimer's disease by regulating pyroptosis.

The main mechanism of pyroptosis is Caspase‐1–mediated GSDMD cleavage, and GSDMD is also the executive protein of pyroptosis. Our previous study has shown that mafenide can inhibit pyroptosis by inhibiting the GSDMD‐Asp275 site to suppress cleavage. In this study, sulfonamide was used as the parent nucleus structure to synthesize sulfa‐4 and sulfa‐20. Screening of drug activity in the pyroptosis model of BV2 and iBMDM cell lines revealed the efficacy of five compounds were superior to mafenide, which exerted a better inhibitory effect on the occurrence of pyroptosis. For in vivo assay, Sulfa‐4 and Sulfa‐22 were intervened in the neuroinflammation APP/PS1 mice. As a result, the administration of Sulfa‐4 and Sulfa‐22 could significantly inhibit the activation of microglia, decrease the expression of inflammatory factors in the central nervous system and simultaneously suppress the production of p30‐GSDMD as well as the expression of upstream NLRP3 inflammasome and Caspase‐1 protein. Immunoprecipitation and Biotin‐labelled assay confirmed the targeted binding relationship of Sulfa‐4 and Sulfa‐22 with GSDMD protein in the iBMDM model in vitro. In this study, we investigated a new type inhibitor of GSDMD cleavage, which exerted a good inhibitory effect on pyroptosis and provided new references for the development of inflammatory drugs in the future.

Increase in NO causes osteoarthritis and chondrocyte apoptosis and chondrocyte ERK plays a protective role in the process.

Nitric oxide (NO) and reactive oxygen species (ROS) play an important role in the pathology of human osteoarthritis (OA). Ankylosing spondylitis (AS) and atypical OA have similar clinical manifestations and often require differential diagnosis. The mechanism is however not totally clear yet. This study aims to investigate the effects of excessive NO-ROS in OA patients and the effects of extracellular signal-regulated kinases (ERK) pathway in NO-induced apoptosis of chondrocytes during OA progress. Serum samples from OA or AS as pathological control patients and healthy controls were collected for NO and related chemical measurements. The rabbit articular chondrocytes were cultured in vitro, and NO was applied by Sodium Nitroprusside (SNP) in culture medium to mimic OA condition in patients. The level of SNP-evoked chondrocyte apoptosis with or without PD98059 (ERK-specific inhibitor) was evaluated by TUNEL assay, Annexin V flow cytometry and Western blotting. The activity and mRNA expression of caspase-3 in chondrocytes were measured by assay kits and RT-PCR. The levels of NO and malondialdehyde (MDA) in serum were significantly higher in OA patients, while only MDA was significantly higher in AS patients. However, the level of superoxide dismutase (SOD) was lower in both OA and AS patients. SNP induced chondrocyte apoptosis was enhanced by PD98059 with increased protein expression and functional activity of caspase-3. The increase in nitric oxide occurs specifically in OA patients. ERK pathway may play a protective role on the NO-induced chondrocyte apoptosis, and inhibition of ERK pathway enhances the NO-induced apoptosis.

Melatonin Suppresses Apoptosis of Nucleus Pulposus Cells through Inhibiting Autophagy via the PI3K/Akt Pathway in a High-Glucose Culture.

Diabetes mellitus- (DM-) associated hyperglycemia promotes apoptosis of disc nucleus pulposus (NP) cells, which is a contributor to intervertebral disc degeneration (IDD). Melatonin is able to protect against cell apoptosis. However, its effects on apoptosis of NP cell in a high-glucose culture remain unclear. The purpose of the present study was to investigate the effects and molecular mechanism of melatonin on NP cell apoptosis in a high-glucose culture. NP cells were cultured in the baseline medium supplemented with a high-glucose concentration (0.2 M) for 3 days. The control cells were only cultured in the baseline medium. Additionally, the pharmaceutical inhibitor LY294002 was added along with the culture medium to investigate the possible role of the PI3K/Akt pathway. Apoptosis, autophagy, and activity of the PI3K/Akt pathway of NP cells among these groups were evaluated. Compared with the control NP cells, high glucose significantly increased cell apoptosis ratio and caspase-3/caspase-9 activity and decreased mRNA expression of Bcl-2, whereas it increased mRNA or protein expression of Bax, caspase-3, cleaved caspase-3, cleaved PARP, and autophagy-related molecules (Atg3, Atg5, Beclin-1, and LC3-II) and decreased protein expression of p-Akt compared with the control cells. Additionally, melatonin partly inhibited the effects of high glucose on those parameters of cell apoptosis, autophagy, and activation of PI3K/Akt. In conclusion, melatonin attenuates apoptosis of NP cells through inhibiting the excessive autophagy via the PI3K/Akt pathway in a high-glucose culture. This study provides new theoretical basis of the protective effects of melatonin against disc degeneration in a DM patient.

Protective Mechanism of Trimetazidine in Myocardial Cells in Myocardial Infarction Rats through ERK Signaling Pathway.

Objective To study the protective effect of trimetazidine on myocardial cells in rats with myocardial infarction and explore its effect on ERK signaling pathway. Methods 40 SD rats were randomly divided into the sham operation group, model group, low-dose group, and high-dose group (intra-abdominal injection of trimetazidine 5 mg/kg and 10 mg/kg, respectively), construction of rat myocardial infarction model by coronary artery left anterior descending artery ligation. 7 days after surgery, the survival rate and cardiac function of each group of rats were recorded. The myocardial infarct size was detected by TTC staining. The apoptosis level of rat cardiomyocytes was detected by TUNEL staining. The content of ROS in rat cardiomyocytes was detected by DCFH-DA. Western-blot was used to detection of Caspase-3, Bcl-2/Bax, and ERK signaling pathway-related proteins in myocardial tissue. Results Compared with the model group, the survival rate of the rats in the low-dose group and the high-dose group was significantly increased (P < 0.01), the cardiac function was significantly improved (P < 0.01), the myocardial infarct size was significantly decreased (P < 0.01), the level of apoptosis was significantly decreased (P < 0.01), the content of ROS in cardiomyocytes was significantly decreased (P < 0.01), the protein expression of Caspase-3 and NF-κB in cardiomyocytes was significantly decreased (P < 0.01), and the expression of Bcl-2/Bax and p-ERK were significantly increased (P < 0.01). Conclusion Trimetazidine can activate ERK signaling pathway in cardiomyocytes of rats with myocardial infarction, increase the expression of p-ERK, decrease the content of ROS in cardiomyocytes, decrease the expression of apoptotic proteins, reduce myocardial infarct size, improve cardiac function, and increase myocardial function.

Verbascoside inhibits paraquate-induced pulmonary toxicity via modulating oxidative stress, inflammation, apoptosis and DNA damage in A549 cell

Paraquat (PQ), one of the most frequently used herbicides, can cause serious health problems in an exposed individual. In the present study, we investigated the protective effect of verbascoside (VB), a phenylpropanoid glycoside from lemon verbena, against PQ-induced A549 cell injury with a particular focus on the possible molecular pathways involved. A549 cells were exposed to PQ (300 µM) and different concentrations of VB (12.5, 25, and 50 µM). Cell viability, ROS content, the level of antioxidant enzymes (SOD, CAT and GPx) and inflammatory markers (IL-6 and TNF-α), as well as 8-OHdG, were detected using MTT assay and an ELISA kit. Western blotting and qRT-PCR were performed to measure the levels of caspase3 and NF-κB mRNA and protein expression. Exposure of cells to PQ caused viability loss and ROS increase. PQ also increased the levels of IL-6, TNF-α and 8-OHdG and decreased the antioxidant enzymes content. PQ treatment resulted in cell death by increasing the gene and protein expression level of caspase 3 and NF-κB. Treatment with VB notably increased cell survival, antioxidant enzymes activity, which concomitantly attenuated ROS, NF-κB and inflammatory mediator production. VB also inhibited apoptosis expression markers. These results indicated that VB could protect A549 cells against PQ induced cell injury by attenuation of ROS and inflammatory marker production and modulation of antioxidant enzymes. VB efficiently suppressed increased NF-κB and caspase-3 activity and formation of 8-OHdG and ultimately improved cell viability. Therefore, VB may be useful in the development of a new therapy for PQ-induced pulmonary toxicity.

Shenqi Lixin Decoction improves cardiac function in rats with adriamycin-induced heart failure through modulation of PGC-1α and mitochondrial apoptosis pathway.

BackgroundHeart failure (HF) is a complex clinical syndrome and a serious manifestation or late stage of various heart diseases. This study aimed to explore the protective effects and underlying mechanisms of Shenqi Lixin Decoction (SQLXD) in HF.MethodsA HF rat model was induced by intraperitoneal injection of adriamycin (3 mg/kg in the first 3 weeks, 2 mg/kg in the next 3 weeks, once a week, subcutaneous injection, 6 weeks cumulative dose is 15 mg/kg). After 4 weeks of intragastric administration of SQLXD (9.975, 19.95, 39.90 g/kg, once a day, gavage), the indexes of cardiac function were measured by cardiac color Doppler ultrasound, the cardiac muscle structure and pathological changes were observed by transmission electron microscope, hematoxylin-eosin (HE) staining and Masson. The plasma N-terminal B-type natriuretic peptide (NT-proBNP) level and myocardial tissue adenosine triphosphate (ATP) content were detected by ELISA. FITC detected the cardiomyocyte apoptosis rate (CMAR) labeled Annexin V/PI. Expression of B cell lymphoma factor 2 (Bcl-2), Bcl-2 associated X (Bax), cysteine protease-3 (Caspase-3), and peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) mRNA in myocardial tissue were detected by real-time PCR (RT-PCR). The expression of Bcl-2, Bax, Caspase-3 and P53 protein in myocardial tissue were detected by Western blot.ResultsCompared to the normal group, left ventricular end systolic diameter (LVSD), left ventricular end diastolic diameter (LVDD), CMAR and the expression of P53 protein, mRNA and protein of Bax and Caspase-3 were significantly increased in model group, while left ventricular ejection fraction (LVEF), left ventricular fractional shortening (LVFS), stroke volume (SV) and the expression of Bcl-2 protein, mRNA of PGC-1α and Bcl-2 were significantly reduced. Compared to the model group, LVSD, LVDD, CMAR and the expressions of P53 protein, mRNA and protein of Bax and Caspase-3 in the medium and high dose SQLXD groups and the control group were significantly decreased, while LVEF, LVFS, SV and the expression of Bcl-2 protein, mRNA of PGC-1α and Bcl-2 were obviously increased. Pathological findings by transmission electron microscope, Masson, and HE staining all revealed protective effects of SQLXD on heart.ConclusionsSQLXD can effectively protect HF rats’ hearts. The potential mechanism may be related to the modulation of the expression of PGC-1α and the mitochondrial apoptosis pathway.

Mechanism of microRNA-1 regulating H9c2 cardiomyocyte apoptosis after hypoxia/reoxygenation

To investigate the changes of cardiomyocyte apoptosis after hypoxia/reoxygenation (H/R) regulated by microRNA-1 (miR-1). Cardiomyocyte strain H9c2 derived from rat embryonic heart tissue were cultured in vitro. The cells in logarithmic growth phase were divided into blank control group, H/R group, miR-1 mimics+H/R group, miR-1 inhibitor antisense oligonucleotide (ASO)+H/R group and microRNA negative control fragment (miRNA NC)+H/R group. The low sugar DMEM medium containing low concentration of fetal bovine serum (FBS) was used as the medium under anoxic condition. After being cultured in a closed anaerobic incubator at 37 centigrade (95% N2 and 5% CO2) for 12 hours, the cells were cultured with the fresh high sugar DMEM medium containing 5% FBS in a closed incubator at 37 centigrade for reproducing cardiomyocyte H/R model. The blank control group was cultured in high glucose DMEM medium containing 10% FBS in 37 centigrade and 5% CO2 incubator. In miR-1 mimics+H/R group, miR-1 ASO+H/R group and miRNA NC+H/R group, the corresponding transfectants were mixed in high glucose DMEM medium and transfected into cells before H/R model was established, and the final concentration was 50 nmol/L. The blank control group and H/R group were added with DMEM medium at the same time. After the establishment of the model, the expression level of miR-1 was detected by real-time fluorescence quantitative polymerase chain reaction (qPCR). The expression levels of apoptosis-related proteins caspase-9, Bcl-2 and Bax were detected by Western blotting, and cardiomyocyte apoptosis was detected by flow cytometry. Compared with the blank control group, the expression levels of miR-1, caspase-9 and Bax protein and the apoptosis rate of cardiomyocytes were significantly increased, while the expression level of Bcl-2 was significantly decreased, which indicated that the expression of miR-1 and the level of apoptosis were increased in H/R group. Compared with H/R group, the expressions of miR-1, caspase-9 and Bax and the apoptosis rate of cardiomyocytes in miR-1 mimics+H/R group were further increased [miR-1 (2-ΔΔCt): 11.59±1.48 vs. 2.57±0.38, caspase-9 protein (caspase-9/β-actin): 2.59±0.12 vs. 1.56±0.20, Bax protein (Bax/β-actin): 4.09±0.38 vs. 1.97±0.13, apoptosis rate: (25.23±0.87)% vs. (17.86±0.73)%, all P < 0.01], while the expression of Bcl-2 was decreased (Bcl-2/β-actin: 0.37±0.02 vs. 0.49±0.03, P < 0.01). The expressions of miR-1, caspase-9 and Bax and the apoptosis rate were significantly decreased in miR-1 ASO+H/R group [miR-1 (2-ΔΔCt): 1.16±0.06 vs. 2.57±0.38, caspase-9 protein (caspase-9/β-actin): 1.05±0.24 vs. 1.56±0.20, Bax protein (Bax/β-actin): 0.93±0.11 vs. 1.97±0.13, apoptosis rate: (11.19±0.85)% vs. (17.86±0.73)%, all P < 0.05], while the expression of Bcl-2 was increased (Bcl-2/β-actin: 0.84±0.17 vs. 0.49±0.03, P < 0.05). There was no significant difference in miR-1 expression, caspase-9, Bax and Bcl-2 protein expressions, and apoptosis rate between H/R+miRNA NC group and H/R group. The expression of miR-1 and level of apoptosis were increased in H/R cells, and miR-1 could aggravate cardiomyocyte apoptosis.

Effects of miR-26a-5p and PTEN on Myocardium of Myocardial Ischemia-reperfusion Mice

This study aimed to investigate the effects of miR-26a-5p and PTEN on myocardium of myocardial ischemia-reperfusion mice. Twelve C57/B6 male mice were randomly selected and divided into control group and mouse model group with 6 mice in each group, in which no surgical modeling was normally performed. Mice were killed 2 hours after operation to collect myocardial tissue. H9C2 was transfected with miR-26a-5p-mimic and blank vector to overexpress miR-26a-5p, and then normal group, hypoxia/reoxygenation group (cell model group), blank group and overexpression group were established respectively. Protein expressions of PTEN, Caspase-3, Caspase-9, P13K and p-Akt in tissues and cells of mice were detected. Expressions of miR-26a-5p and PTEN in myocardial tissue and cells of mice were detected. Apoptosis was detected. The relationship between miR-26a-5p and PTEN was determined. Expressions of miR-26a-5p in tissues of mouse model group were lower than those of normal group, while PTEN expressions were opposite (p&lt;0.05). Expressions of miR-126 in overexpression group were increased compared with those of cell model group and blank group, and decreased compared with those of control group (P&lt;0.05). Expressions of PTEN in overexpression group were lower than those in cell model group and blank group, and higher than those in control group (p&lt;0.05). Compared with control group, miR-26a-5p was decreased and PTEN was increased in cell model group (P&lt;0.05). Compared with blank group and cell model group, expressions of PTEN, Caspase-3 and Caspase-9 proteins in overexpression group were significantly decreased, while expressions of P13K and p-Akt proteins were significantly increased (p&lt;0.05). Apoptosis rate in overexpression group was significantly lower than that in cell model group and blank group (p&lt;0.05). Dual-luciferase reporter proved that there was a targeted regulatory relationship between miR-26a-5p and PTEN. In conclusion, up-regulation of miR-26a-5p regulates PTEN/PI3K/p-Akt signaling pathway and reduces myocardial cell apoptosis, thus improving ischemia-reperfusion injury.

MSCs enhances the protective effects of valsartan on attenuating the doxorubicin-induced myocardial injury via AngII/NOX/ROS/MAPK signaling pathway.

Objective: To verify if AngII/NOX/ROS/MAPK signaling pathway is involved in Doxorubicin (DOX)-induced myocardial injury and if mesenchymal stem cells (MSCs) could enhance the protective effects of valsartan (Val) on attenuating DOX-induced injury in vitro.Methods: Reactive oxygen species (ROS) formation and the protein expression of AT1R, NOX2, NOX4, caspase-3, caspase-9 and MAPK signaling were assessed in H9c2 cardiomyocytes exposed to DOX for 24 h in the absence or presence of Val, NADPH oxidase inhibitor DPI or knockdown and overexpression of NADPH oxidase subunit: NOX2 and NOX4, co-culture with MSCs, respectively. Finally, MTT assay was used to determine the cell viability of H9c2 cells, MDA-MB-231 breast cancer cells and A549 pulmonary cancer cells under Val, DOX and Val+ DOX treatments.Results: DOX increased ROS formation and upregulated proteins expression of AT1R, NOX2, NOX4, caspase-3, caspase-9 and MAPK signaling including p-p38, p-JNK, p-ERK in H9c2 cells. These effects could be attenuated by Val, DPI, NOX2 siRNA and NOX4 siRNA. Meanwhile, overexpression of NOX2 and NOX4 could significantly increase DOX-induced ROS formation and further upregulate apoptotic protein expressions and protein expressions of MAPK signaling. MSCs on top of Val further enhanced the protective effects of Val on reducing the DOX-induced ROS formation and downregulating the expression of apoptotic proteins and MAPK signaling as compared with Val alone in DOX-treated H9c2 cells. Simultaneous Val and DOX treatment did not affect cell viability of DOX-treated MDA-MB-231 breast cancer cells or A549 pulmonary cancer cells but significantly improved cell viability of DOX-treated H9c2 cardiomyocytes.Conclusions: AT1R/NOX/ROS/MAPK signaling pathway is involved in DOX-induced cardiotoxicity. Val treatment significantly attenuated DOX-induced cardiotoxicity, without affecting the anti-tumor effect of DOX. MSCs enhance the protective effects of Val on reducing the DOX-induced toxicity in H9c2 cells.

Isoformononetin, a dietary isoflavone protects against streptozotocin induced rat model of neuroinflammation through inhibition of NLRP3/ASC/IL-1 axis activation

AimsIsoformononetin (IFN), a methoxyl isoflavone present in most of human dietary supplements. However, being a highly potent antioxidant and anti-inflammatory molecule, its activity against neuronal oxidative stress and neuroinflammation has not been explored till now. The present study was inquested to assess the antioxidant, anti-apoptotic and anti-inflammatory activity of IFN against streptozotocin induced neuroinflammation in different brain regions of rat. Main methodsFour groups of animals were subjected to treatment as control, toxic control (STZ; single intracerebrovascular injection), third group (STZ + IFN; 20 mg/kg p.o.), fourth group (IFN) for 14 days. The different brain regions of rats were evaluated for inflammatory, apoptotic and biochemical antioxidant markers. The brain tissues were further assessed for gene expression, immunohistochemical and western blotting examination for localization of inflammasome cascade expression that plays a pivotal role in neuroinflammation. Key findingsThe modulation in oxidant/antioxidant status after exposure of STZ was significantly balanced after administration of IFN to rats. Further, IFN was also found to be an apoptotic agent as it modulates the apoptotic gene (Bax) and anti-apoptotic gene (BcL2) expression. IFN significantly curtailed the augmented protein expression of NLRP3, NLRP2, ASC, NFκBP65, IL-1β and caspase-1 due to STZ administration in cortex and hippocampus rat brain regions. SignificanceThe aforementioned results proclaim the neuroprotective functioning of IFN against STZ induced inflammation. IFN significantly prevents the neuroinflammation by decreasing the generation of ROS that reduces the activation of NLRP3/ASC/IL-1 axis thereby exerting neuroprotection as evidenced in rat model of STZ induced neuroninflammation.

Neuroprotective Effect of Danggui Shaoyao San via the Mitophagy-Apoptosis Pathway in a Rat Model of Alzheimer's Disease.

Alzheimer's disease (AD) is a serious neurodegenerative disease. While the main pathological characteristic of AD is widely believed to be the accumulation of amyloid-beta (Aβ) in neurons around neurofibrillary plaques, the molecular mechanism of pathological changes is not clear. Traditional Chinese medicine offers many treatments for AD. Among these, Danggui Shaoyao San (DSS) is a classic prescription. In this study, an AD model was established by injecting Aβ 1–42 into the brains of rats, which were then treated with different concentrations of Danggui Shaoyao San (sham operation; model; and Danggui Shaoyao San high-dose, medium-dose, and low-dose intervention groups). The Morris water maze test was used to assess the learning and memory abilities of the animals in each group. Nissl staining was used to detect neurons. Mitophagy was evaluated by transmission electron microscopy and immunofluorescence colocalization. Apoptosis was assessed by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. The expression levels of autophagy- and apoptosis-related proteins were measured by western blot. Compared to the model group, the groups of AD rats administered medium and high doses of Danggui Shaoyao San showed significantly increased learning and memory abilities (P < 0.05), as well as significantly increased autophagosomes in the hippocampus. Moreover, the expression of PTEN-induced kinase 1 (PINK1), Parkin, and microtubule-associated protein light chain 3 (LC3-I/LC3-II) was increased, while that of p62 was significantly decreased (P < 0.05). The neuronal apoptosis rate was also significantly decreased, the Bcl-2/Bax ratio was significantly increased, and the cleaved caspase-3 protein expression was significantly decreased (P < 0.05). Therefore, Danggui Shaoyao San inhibited neuronal apoptosis in AD rats via a mechanism that may be related to the activation of the PINK1-Parkin-mediated mitophagy signaling pathway.