Caspase-3 Protein Expression Research Articles

Myrislignan Induces Apoptosis in Gastric Cancer Cell Line Through PI3K/AKT Signaling Pathway

To investigate the effect of myrislignan (MYR) on the apoptosis of gastric cancer cell line and its relationship with phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) signaling pathway. The gastric cells (SGC-7901) were treated with MYR at different concentrations, i.e., 0, 25, 50, 100, and 200 μmol/L, for 48 h and 72 h and the effect of MYR on the proliferation of SGC-7901 cells was measured by CCK-8 assay. Then, SGC-7901 cells were treated with different concentrations of MYR at 50, 100, and 200 μmol/L for 48 h. Meanwhile, a normal control group and a dimethyl sulfoxide (DMSO) solvent control group (0.1% DMSO) were established. Flow cytometry was used to determine the apoptosis rate of SGC-7901 cells. The protein expression levels of PI3K, AKT, Bcl-2-associated X protein (BAX), cysteine-dependent aspartate-specifc protease-3 (Caspase-3), and Caspase-9 were determined by Western blot. Then, PI3K activator (20 μmol/mL) was used to treat SGC-7901 cells for 48 h in 4 groups, the control group, 0.1% DMSO group, MYR group, and MYR+PI3K activator group, and the effect on MYR's induction of apoptosis and regulation of the protein expression levels of PI3K, AKT, BAX, Caspase-3, and Caspase-9 in SGC-7901 cells. Compared with the control group, MYR at 50, 100 and 200 μmol/L inhibited the proliferation of gastric cancer cells, increased the apoptosis rate, down-regulated the protein expression levels of PI3K and AKT, and up-regulated the protein expression levels of BAX, Caspase-3, and Caspase-9 in a dose-dependent manner ( P<0.05). However, PI3K activator attenuated MYR-induced apoptosis in gastric cancer cells and MYR's regulation of PI3K, AKT, BAX, Caspase-3, and Caspase-9 protein expression ( P<0.05). MYR induces the expression of BAX, Caspase-3, and Caspase-9 proteins by inhibiting the PI3K/AKT signaling pathway, thereby promoting the apoptosis of gastric cancer cells.

Pharmacokinetic Characteristics of Bisdemethoxycurcumin and Its Mechanism of Reversing Cardiomyocyte Apoptosis

To investigate the pharmacokinetic characteristics of bisdemethoxycurcumin in vivo and the reversal effect of phosphatidylinositol 3-kinase/protein kinase B/caspase-9 signaling pathway on thapsigargininduced cardiomyocyte apoptosis in neonatal rat. Demethoxycurcumin and bisdemethoxycurcumin were intragastrically administered to adult Sprague–Dawley rats. Plasma was collected and the drug concentration was measured by high performance liquid chromatography. The pharmacokinetic parameters of the drug were calculated by Drug and Statistics 2.1.1 pharmacokinetic software. After that, primary cardiomyocytes from Sprague–Dawley neonatal rats were isolated and cultured, and they were divided into three groups; control (conventional culture without any drug treatment), model (endoplasmic reticulum stress model obtained by treatment with 1 mg/ml thapsigargin for 24 h), demethoxy curcumin group (treated with 1 μmol/l thapsigargin and 10 μmol/l demethoxy curcumin for 24 h at the same time) and bisdemethoxycurcumin group (treated with 1 μmol/l thapsigargin and 10 μmol/l bisdemethoxycurcumin for 24 h at the same time). Cell survival activity was measured by cell counting kit-8 assay. Western blot was used to detect the expression of intracellular apoptotic factors Bcl-2-associated X, B-cell lymphoma protein 2 and B-cell lymphoma protein 2/Bcl-2-associated X protein and the expression levels of phosphatidylinositol 3-kinase/protein kinase B/caspase-9 signaling pathway molecules phosphatidylinositol 3-kinase, protein kinase B, caspase-3 and caspase-9 protein. The results showed that the main pharmacokinetic parameters area under the timeconcentration curve 0-12 h, mean residency time 0-12 h, maximum plasma concentration, time to maximum plasma concentration of demethoxycurcumin and bisdemethoxycurcumin were (1.35±0.36) μg/ml•h and (1.01±0.14) μg/ml•h, (4.05±0.53) h and (3.43±0.21) h, (0.46±0.09) μg/ml and (0.45±0.11) μg/ml, (0.14±0.00) h and (0.20±0.00) h, respectively. In contrast with the model group, the survival activity of demethoxycurcumin and bisdemethoxycurcumin groups was significantly increased, the apoptosis rate was significantly decreased; the lactate dehydrogenase, malondialdehyde content and reactive oxygen species level were decreased, the superoxide dismutase activity was increased, the Bcl-2-associated X, caspase-3 and caspase-9 protein expression was decreased and the B-cell lymphoma protein 2/Bcl-2-associated X, phosphatidylinositol 3-kinase and protein kinase B protein expression was increased (p<0.05). However, bisdemethoxycurcumin was slightly superior to demethoxycurcumin in improving the above indicators, but the difference was not statistically meaningful (p>0.05). In summary, demethoxy curcumin and bisdemethoxycurcumin can mediate phosphatidylinositol 3-kinase/protein kinase B/caspase-9 signaling pathway to reduce thapsigargin-induced endoplasmic reticulum stress injury in cardiomyocytes, which in turn plays a protective role in cardiomyocytes.

Long Non-Coding RNA Metastasis Associated Lung Adenocarcinoma Transcript 1 (LncRNA MALAT1) Shuttling Through Exosome from Bone Marrow Mesenchymal Stem Cell (BMSC) Regulates the Differentiation of Osteoblast in Osteoporosis Rats

This study assesses the effect of LncRNA MALAT1 on osteoblast differentiation in OP rats. The BMSC were transfected with LncRNA MALAT1. The OP rats’ model was established and then assigned into NC set, OP set, BMSC set, BMSCs/LncRNA-NC set and BMSCs/LncRNA MALAT1 set followed by analysis of LncRNA MALAT1, microRNA-34 and cSATB2 expression by RT-PCR, and Bax, Caspase-3 and ALP level in exosome by Western Blot assy. The protein expression of Bax and Caspase-3 in OP set was significantly increased compared with NC set but reduced in BMSCs/LncRNA MALAT1 set without significant differences between BMSC set and BMSC/NC set. There was orderly array of collagenous fiber in lamellar bone of NC set. The quantity of bone lacuna was moderate with orderly array of bone trabecula. There was disorderly array of collagenous fiber in lamellar bone of model set. There were a large number of distribution of osteoblast in the breakdown area with the dissolution of periosteum. The protein level of Bax and Caspase-3 was reduced by LncRNA MALAT1 from BMSC and the level of ALP, microRNA-34 and cSATB2 in the differentiation of osteoblast was increased, thereby promoting the differentiation of osteoblast.

Роль цитокинов и регуляторных молекул клеточного цикла в прогнозировании достижения ответа на терапию у больных хроническим миелолейкозом

Introduction. Despite significant advances in therapy of patients with chronic myeloid leukemia (CML), improved survival rates, development of resistance to tyrosine kinase inhibitors (TKIs) remains an urgent problem. Aim. To study the correlation between the level of expression of regulatory proteins p53, c-myc, ki-67 and caspase-3 on the bone marrow cells and the concentration of certain pro- and anti-inflammatory cytokines in blood serum with the effectiveness of therapy in patients with CML. Materials and methods. Seventy-four CML patients with chronic phase of the disease receiving TKI therapy were examined. In all patients, the concentration of certain cytokines and growth factors (TNF-α, IL-1β, IL-2, IL-4, IL-6, IL-10, IL-17, IL-18, IFN-α, and VEGF-A) was determined in blood serum by enzyme immunoassay and immunocytochemical study of bone marrow smears with monoclonal antibodies against antigens of regulatory molecules ki-67, p53, c-myc, and caspase-3. To determine the role of the biomarkers in predicting the therapy effectiveness, a comparative analysis of their values in groups of patients with a major molecular response (MMR) (n = 50) and without MMR (n = 24) was performed. Results. A comparative analysis of the expression of regulatory molecules on the bone marrow cells and the blood serum concentration of cytokines and growth factors of CML patients, depending on depth of the response to TKI therapy, showed that patients who did not achieve MMR had a significantly higher level of caspase-3 expression and concentration of pro-inflammatory cytokines IL-1β, IL-2, IL-6 and IL-17, as well as growth factor VEGF-A compared with those in patients with MMR. In turn, the achievement of MMR was characterized by a higher level of expression of regulatory molecules p53 and c-myc, as well as an increase in the IL-10 concentration and a decrease in the IL-1β, IL-2, IL-6 and IL-17 concentration. Analysis of the correlation between the level of expression of regulatory molecules and the single cytokine concentration showed a negative correlation between c-myc and p53 with IL-2, IL-1β, IL-17 and a positive (direct) correlation between c-myc and p53 with IL-10, a positive correlation between caspase-3 and IL-2, IL-1β, IL-6, IL-17 and a negative correlation between caspase-3 and IL-10. Thus, the achievement of MMR in patients with CML is more likely with a higher expression of regulatory molecules c-myc and p53 on the bone marrow cells, low expression of caspase-3, and low serum concentrations of IL-2, IL-1β, IL-17, IL-6 and a high concentration of IL-10, which indicates synergism of the biomarkers in the pathogenesis of CML and its tumor progression. The ROC analysis results showed the high quality of predictive models characterizing the achievement of MMR at the level of expression of c-myc &gt; 6%, p53 &gt; 4% in the bone marrow, which correlates with a low serum concentrations of IL-2, IL-1β, IL-17 and a high concentration of IL-10, and indicates the possibility of using these indicators as potential biomarkers of effectiveness of CML therapy and achievement of MMR. Conclusion. The results of the study showed that the concentration of cytokines in the blood serum of CML patients correlates with the intensity of expression of c-myc, p53 and caspase-3 proteins and is important in predicting the effectiveness of therapy.

S-Nitrosoglutathione protects acute kidney injury in septic rats by inhibiting the activation of NLRP3 inflammasome.

We aimed to study the effect of S-nitroso glutathione (SNG) on acute kidney injury (AKI) in septic rats by regulating nucleotide oligomerization domain-like receptor protein 3 (NLRP3). Sprague Dawley rats were used to construct the AKI model, and biochemical methods were used to detect the levels of inflammatory factors and anti-oxidant enzymes in renal tissue. We observed the ultrastructural changes of renal tissue by transmission electron microscopy and detected the protein and mRNA levels of NLRP3, apoptosis-associated speck-like protein containing a caspase recruitment domain foci (ASC) and caspase-1 by western-blotting and RT-qPCR. Cecal ligation and puncture induced renal tubular epithelial tissue damage in septic rats, resulting in decreased renal function, increased levels of inflammation and decreased levels of anti-oxidant enzymes in renal tissue, and aggravated mitochondrial damage, significantly decreased mitochondrial density and enzyme complex I/II/III/IV levels (all P<0.001), and increased the protein and mRNA expression of NLRP3, ASC, and caspase-1 (all P<0.001). However, after pretreatment with SNG, the pathological damage of renal tubular epithelial tissue was reduced, the renal function was improved, the level of inflammation in renal tissue decreased and the level of anti-oxidant enzymes increased, the density of mitochondria and the level of enzyme complex I/II/III/IV were significantly increased (all P<0.001), meanwhile the protein and mRNA levels of NLRP3, ASC, and caspase-1 were all decreased significantly (all P<0.05). SNG protects AKI in septic rats by inhibiting NLRP3 inflammasome activation.

Effects of Huangqin Tang on NLRP3/Caspase-1 pathway in mice model of ulcerative colitis

The aim of this study was to explore the effects of Huangqin Tang(HQT) on the NLRP3/Caspase-1 signaling pathway in mice with DSS-induced ulcerative colitis(UC). C57BL/6J mice were randomly divided into a blank group, a model group(DSS group), and low-, medium-and high-dose HQT groups(HQT-L, HQT-M, and HQT-H), and western medicine mesalazine group(western medicine group). The UC model was induced in mice. Subsequently, the mice in the HQT-L, HQT-M, HQT-H groups, and the western medicine group were given low-, medium-, high-dose HQT, and mesalazine suspension by gavage, respectively, while those in the blank and DSS groups were given an equal volume of distilled water by gavage. After 10 days of administration, the body weight, DAI scores, and colonic histopathological score of mice in each group were determined. The levels of IL-6, IL-10, IL-1β, and TNF-α in serum were determined by ELISA. The mRNA expression of NLRP3 and Caspase-1 in colon tissues was determined by RT-qPCR. The protein expression of NLRP3 and Caspase-1 in colon tissues was detected by immunohistochemistry. The results showed that compared with the blank group, the DSS group showed decreased body weight of mice and increased DAI scores and intestinal histopathological score. Compared with the DSS group, the HQT groups and the western medicine group showed improved DAI scores, especially in the HQT-M, HQT-H, and the western medicine groups(P&lt;0.05). The intestinal histopathological scores of the HQT groups and the western medicine group significantly decreased, especially in the HQT-M, HQT-H, and the western medicine groups(P&lt;0.05). In addition, compared with the blank group, the DSS group showed elevated expression of NLRP3 and Caspase-1 in colon tissues, increased serum levels of IL-6, IL-1β, and TNF-α, and decreased IL-10 level. Compared with the DSS group, the HQT groups and the western medicine group displayed decreased expression of NLRP3 and Caspase-1 in colon tissues, reduced serum levels of IL-6, IL-1β, and TNF-α, and increased IL-10 level. The improvement was the most significant in the HQT-H group and the western medicine group(P&lt;0.01). In conclusion, HQT may reduce the expression of NLRP3 and Caspase-1 in colon tissues, reduce the se-rum levels of IL-6, IL-1β, and TNF-α, and increase the expression of IL-10 by regulating the classic pyroptosis pathway of NLRP3/Caspase-1, thereby improving the symptoms of intestinal injury and inflammatory infiltration of intestinal mucosa in DSS mice to achieve its therapeutic effect.

Garlic Extract Participates in the Proliferation and Apoptosis of Nonsmall Cell Lung Cancer Cells Via Endoplasmic Reticulum Stress Pathway.

To investigate the effect of garlic extract (GE) on the proliferation and apoptosis of cell lines A549 and H1299 in lung cancer (LC). A549 and H1299 cells with well-developed logarithmic growth were added with GE at a concentration of 0 μg/ml, 25 μg/ml, 50 μg/M, 75 μg/ml, and 100 μg/ml, respectively. The inhibition of A549 cell proliferation was detected using CCK-8 after cultured for 24 h, 48 h, and 72 h. The apoptosis of A549 cells was analyzed via flow cytometry (FCM) after 24 h of cultivation. In vitro migration of A549 and H1299 cells was determined by cell wound scratch assay after 0 h and 24 h culture. The caspase-3 and caspase-9 protein expression levels in A549 and H1299 cells were evaluated through western blot after 24 h of cultivation. Colony formation and EdU assays revealed that Z-ajoene could inhibit cell viability and cell proliferation in NSCLC cells. After 24 h culture, there was no significant difference in the proliferation rate of A549 and H1299 cells with different GE concentrations (P > 0.05). A remarkable proliferation rate difference emerged between A549 and H1299 cells with different GE concentrations after 48 and 72 hours of cultivation. The proliferation rate of A549 and H1299 cells in the experiment group was significantly lower than that in the control group. With an elevated level of GE concentration, the proliferation rate of A549 and H1299 cells decreased (P < 0.05) while the apoptotic rate increased continuously. GE could exert toxic effects on A549 and H1299 cells, inhibit cell proliferation, promote apoptosis, and attenuate cell migration. Meanwhile, it might induce apoptosis of A549 and H1299 cells through the caspase signal pathway, which is positively correlated with the mass action concentration and is expected to be a new drug for LC treatment.

Hyperthermia Improves Doxorubicin-Based Chemotherapy by Activating Mitochondrial Apoptosis in Bladder Cancer

Purpose: Although intravesical chemotherapy has several antitumoral benefits, it can also have severe side effects. The development of novel therapeutic approaches for bladder cancer (BC) is thus warranted. Hyperthermia (HT) is a widely applicable adjuvant therapy in various cancers. Therefore, this study investigated the effect of HT on improving the chemosensitivity of BC. Materials and Methods: The BC cell lines 5637 and T24 were cultured and treated with HT (43°C) for 24 h. Then, cell viability and survival were assessed using resazurin reagent and colony formation assay, respectively. Western blot assay was used to analyze the levels of Bax, Bcl-2, cleaved caspase-3, and cleaved poly (ADP-ribose) polymerase (PARP) protein expression. Mitochondria degradation was observed by MitoTracker Green staining. Results: In BC cells, HT co-administered with various concentrations of doxorubicin significantly inhibited cell viability and survival. Moreover, HT combined with doxorubicin promoted mitochondrial apoptosis, which caused Bax upregulation and Bcl-2 downregulation. Levels of cleaved caspase-3 and PARP protein expression were also elevated after co-treatment. Conclusion: Taken together, HT improved the chemosensitivity of BC cells to doxorubicin. HT combined with chemotherapy further activated mitochondrial apoptosis in BC cells. The findings suggested that HT may serve as a potential adjunctive treatment for BC that is ready to be applied clinically.

Andrographolide Attenuates Oxidized LDL-Induced Activation of the NLRP3 Inflammasome in Bone Marrow-Derived Macrophages and Mitigates HFCCD-Induced Atherosclerosis in Mice.

Andrographolide (AND) is a bioactive component of the herb Andrographis paniculata and a well-known anti-inflammatory agent. Atherosclerosis is a chronic inflammatory disease of the vasculature, and oxidized LDL (oxLDL) is thought to contribute heavily to atherosclerosis-associated inflammation. The aim of this study was to investigate whether AND mitigates oxLDL-mediated foam cell formation and diet-induced atherosclerosis (in mice fed a high-fat, high-cholesterol, high-cholic acid [HFCCD] diet) and the underlying mechanisms involved. AND attenuated LPS/oxLDL-mediated foam cell formation, IL-1[Formula: see text] mRNA and protein (p37) expression, NLR family pyrin domain containing 3 (NLRP3) mRNA and protein expression, caspase-1 (p20) protein expression, and IL-1[Formula: see text] release in BMDMs. Treatment with oxLDL significantly induced protein and mRNA expression of CD36, lectin-like oxLDL receptor-1 (LOX-1), and scavenger receptor type A (SR-A), whereas pretreatment with AND significantly inhibited protein and mRNA expression of SR-A only. Treatment with oxLDL significantly induced ROS generation and Dil-oxLDL uptake; however, pretreatment with AND alleviated oxLDL-induced ROS generation and Dil-oxLDL uptake. HFCCD feeding significantly increased aortic lipid accumulation, ICAM-1 expression, and IL-1[Formula: see text] mRNA expression, as well as blood levels of glutamic pyruvic transaminase (GPT), total cholesterol, and LDL-C. AND co-administration mitigated aortic lipid accumulation, the protein expression of ICAM-1, mRNA expression of IL-1[Formula: see text] and ICAM-1, and blood levels of GPT. These results suggest that the working mechanisms by which AND mitigates atherosclerosis involve the inhibition of foam cell formation and NLRP3 inflammasome-dependent vascular inflammation as evidenced by decreased SR-A expression and IL-1[Formula: see text] release, respectively.

Asterarcys quadricellulare (Chlorophyceae) protects H9c2 cardiomyoblasts from H2O2-induced oxidative stress

Oxidative stress has recently been identified as an important mediator of cardiovascular diseases. The need to find efficient antioxidant molecules is essential in the disease’s prevention. Therefore, the present study aimed to evaluate the potential of microalgae bioactive in protecting H9c2 cardiomyoblasts from H2O2-induced oxidative stress. Four microalgal species were investigated for their antioxidant capacity. A qualitative assessment of oxidative stress in H9c2 cardiomyoblasts stained with DCFH-DA, treated with the highly active microalgae extracts, was performed. The protein expression of total caspase-3 was also examined to investigate whether the extract protects H9c2 cardimyoblasts from H2O2-induced apoptosis. High antioxidant activity was observed for the hexanoic extracts after 10 days of cultivation. Asterarcys quadricellulare exhibited the highest antioxidant capacity of 110.59 ± 1.75 mg TE g−1 dry weight and was tested against H9c2 cardiomyoblasts, which were initially subjected to H2O2-induced oxidative stress. This hexanoic extract protected against H2O2 induced oxidative stress with a similar scavenging capacity as N-Acetylcysteine. Furthermore, total caspase-3 was increased following treatment with the hexanoic extract, suggesting that A. quadricellulare also had anti-apoptotic properties. The outcome of our study highlighted the possible use of the local A. quadricellulare strain QUCCCM10 as a natural, safe, and efficient antioxidant to prevent cardiovascular diseases.

Xiangshao Granules reduce the aggressive behavior and hippocampal injury of premenstrual irritability in rats by regulating JIK/JNK/p38 signal pathway

Ethnopharmacological relevanceAs a typical prescription for soothing the liver, Xiangshao granule has a good effect on the symptoms of irritability and anxiety. Clinical evidence suggests that it has significant efficacy in the treatment of Premenstrual dysphoria disorder (PMDD). However, the underlying mechanism remains unclear. Aim of the studyPMDD is a common disease in women of childbearing age, seriously affecting their family, society, and daily work life. The registered herbal medicine, Xiangshao granules, is used for relieving PMDD dysphoria and irritability symptoms with excellent efficacy in China. This study was focused on the deep intervention mechanism of Xiangshao granules in treating PMDD. Materials and methodsThe vaginal smear and open field test were used to screen rats in nonreception phase of estrus cycle with similar macroscopic behaviors and regular estrus cycle. The rat model of PMDD irritability was established through social isolation and residential invasion, with which, the irritability symptoms of PMDD patients with menstrual cycle dependence was also well simulated. Elevated plus Maze Test and Social interaction activities were used to measure the anxiety-like behavior of rats. TUNEL Staining and Hematoxylin-Eosin staining were used to measure apoptosis of hippocampal neurons. RT-PCR, Western blot and immunofluorescence were used to measure the expression of GR, JIK, p-JIK, p38, P–P38, JNK, caspase 3, and caspase 12. ResultsIn this study, Xiangshao granules showed consistent therapeutic effects similar with those in clinic, significantly reducing aggressive and anxiety-like behaviors with improved social skills in PMDD rats. In mechanism, Xiangshao granules lowered the apoptosis of hippocampal neurons and weakened the morphological damage of the hippocampal brain evidenced by the decreased mRNA and protein expression of glucocorticoid receptor, caspase-3, and caspase-12. In addition, administration of Xiangshao granules led to the decreased expression of JIK in the PMDD irritability rat model which agreed well with the previous studies. The JNK/p38 mitogen-activated protein kinases (MAPKs) signaling pathway is abnormally activated in the hippocampal brain region of PMDD rats, while treated with Xiangshao granules could increase JIK expression and inhibit the abnormal activation of the JNK/p38 MAPK signaling pathway, effectively reducing the stress damage in the hippocampus. ConclusionsXiangshao Granules Reduce the Aggressive Behavior and Hippocampal Injury of Premenstrual Irritability in Rats by Regulating JIK/JNK/p38 Signal Pathway.

Neohesperidin dihydrochalbazone protects against septic acute kidney injury in mice

BackgroundNeohesperidin dihydrochalbazone (NHDC) shows a range of pharmacological actions, however, in septic acute kidney injury (AKI), the effect of NHDC is little known. PurposeTo assess the role of NHDC against AKI and the possible mechanisms. MethodsIn vivo, we used different concentration of NHDC (50, 100, and 200 mg/kg) treated septic AKI model of mice. Moreover, in vitro, in HK-2 cells, a lipopolysaccharide (LPS) induced cell model was treated with 10, 20, and 30 μM NHDC. Next, kidney tissue pathologic change, marker of renal injury, apoptosis, and inflammatory factors were assessed using hematoxylin and eosin staining, enzyme-linked immunosorbent assay, terminal deoxynucleotidyl transferase dUTP nick end labeling, and western blot. HK-2 cell apoptosis and viability were assessed via flow cytometry and cell counting kit-8. In HK-2 cells and tissues, NLRP3, caspase 1, ASC, and P38/ERK 1/2/JNK pathway related protein levels were tested using western blot. ResultsNHDC (100 and 200 mg/kg) significantly attenuated kidney injury in caecal ligation and puncture (CLP)-treated mice. In CLP-treated mice, the level of BUN, Scr, KIM-1, and NAGL was reduced by 100 and 200 mg/kg NHDC. Furthermore, 100 and 200 mg/kg NHDC inhibited inflammation by reducing the production of IL-6, TNF-α, and IL-1β, and inhibited oxidative stress by regulating the change of MDA, SOD, GSH, and CAT. NHDC (100 and 200 mg/kg) inhibited renal cell apoptosis by increasing Bcl2 protein expression and inhibiting Bax and cleaved caspase-3 protein expression. Additionally, NHDC (100 and 200 mg/kg) inhibited the protein levels of phosphorylated (p)-P38, p-JNK, p-ERK 1/2, NLRP3, caspase 1, ASC. In vitro, in LPS-stimulated HK-2 cells, NHDC (20 and 30 μM) increased cell viability, reduced cell apoptosis, restrained inflammation by reducing the content of IL-6, TNF-α, and IL-1β, and inhibited the protein expression of caspase 1, NLRP3, ASC, p-P38, p-JNK, and p-ERK1/2. Importantly, the promotive effect of NHDC on HK-2 cell viability was reversed by DHR (an activator of P38 MAPK signaling pathway), and DHR reversed the inhibitive effects of NHDC on HK-2 cell apoptosis and inflammation. ConclusionFor the first time, NHDC was found to inhibit oxidative stress, inflammation, and apoptosis in AKI model, which was related to the inhibition of P38 MAPK pathways. Our findings provided the theoretical basis for NHDC on the prevention of AKI.

MiR-34a alleviates podocyte injury in mice with diabetic nephropathy by targeted downregulation of Notch signaling pathway

To explore the effects of miR-34a on injury and apoptosis of podocytes in diabetic nephropathy (DN) and the role of Notch signaling pathway in mediating its effects. The expression of miR-34a in podocytes exposed to high glucose (30 mmol/L) was detected using RT-PCR. A podocyte line with miR-34a overexpression was constructed, and the miRNA-target relationship between miR-34a and Notch 1 was verified with luciferase assay. The effects of overexpression of Notch 1 and both miR-34a and Notch 1 on podocyte survival and apoptosis were evaluated using CCK-8 and flow cytometry and by detecting apoptosis-related proteins using Western blotting. In a DN mouse model established by high-fat diet and streptozotocin, the effect of tail vein injection of agomir-34a and agomir-NC on pathology and apoptosis in the renal tissues were observed with HE staining and TUNEL staining, and the renal expressions of apoptosis-related proteins and Notch 1 protein were detected with Western blotting. High glucose exposure significantly lowered miR-34a expression in cultured human podocytes (P < 0.05). The expression of Notch 1 was significantly lowered in miR-34a-overexpressing podocytes as compared with the cells with miR-NC transfection (P < 0.05). Luciferase assay confirmed the mRNA-target relationship between miR-34a and Notch 1 (P < 0.05). MiR-34a overexpression obviously promoted podocyte survival (P < 0.05), reduced Notch 1 expression, and lowered apoptosis rate and the protein expressions of caspase-3, caspase-9 and Bax/Bcl-2 levels in the cells (P < 0.05), while the reverse changes were observed in Notch 1-overexpressing podocytes (P < 0.05). In DN mouse models, treatment with miR-34a obviously alleviated renal pathologies. Compared with that in the control group, the expression level of miR-34a in the renal tissues was significantly lowered in DN model group (P < 0.05) and increased in miR-34a group (P < 0.05). The mice in the model group showed significantly higher apoptosis index of the renal tissues with increased expressions of caspase-3, caspase-9 and Notch 1 (P < 0.05), which were lowered by treatment with miR-34a (P < 0.05). MiR-34a is capable of improving podocyte injury and apoptosis in DN mice by targeted downregulation of Notch 1.

Sidaxue, a traditional Guizhou Miao herbal medicine formula, reduces necroptosis and synovial vascular proliferation in rats with collagen-induced arthritis by inhibiting the RIPK1/RIPK3/MLKL pathway

To explore the inhibitory effect of Sidaxue (SX), a traditional Guizhou Miao herbal medicine formula, on necrotic apoptosis and synovial angiogenesis in rats with collagen-induced arthritis (CIA) and the role of the RIPK1/RIPK3/MLKL pathway in mediating this effect. Forty-two SD rats were randomized into 6 groups (n=7), including a normal control group, a CIA model group, 3 SX treatment groups at low (10 g/kg), moderate (20 g/kg) and high (40 g/kg) doses, and a GTW treatment group. CIA rat models were established by subcutaneous injections of bovine type II collagen, and the treatments were administered daily by gavage for 21 days. The rats were observed for swelling of the hind limb joints, which was rated using the arthritis index (AI) score on a weekly basis. Serum levels of TNF-α, IL-1β and IL-17 in the rats were detected using ELISA, and the pathological changes in the synovium were observed with HE staining. Real-time PCR was performed to detect the mRNA expression levels of VEGF, MMP-9, Ang-1, RIPK1, RIPK3, and caspase-8 in the synovial tissues, and the protein expressions of VEGF, MMP9, Ang-1, Stat-3, RIPK1, RIPK3, MLKLl, p-MLKL and caspase-8 were detected using Western blotting. Compared with those in CIA model group, the rats receiving treatment with GTW and SX showed milder swelling of the hind limb joints with significantly lower AI scores (P < 0.05). In CIA model group, a large number of inflammatory cells were observed in the synovium with obvious damages of the tissue structure. In the drug treatment groups, inflammatory cell infiltration, synovial angiogenesis and synovial hyperplasia were alleviated, and the therapeutic effects were obviously enhanced as SX dose increased. Compared with those in the model group, the rats treated with GTW and high-dose SX showed significantly decreased serum levels of IL-1β, IL-17 and TNF-α (P < 0.05), lower mRNA and protein expressions of RIPK1, RIPK3, VEGF, Ang-1, and MMP9 (P < 0.05), higher expressions of caspase-8 (P < 0.01), and obviously lowered expression of Stat-3 protein and phosphorylation level of MLKL (P < 0.05). SX can improve synovial angiogenesis in CIA rats possibly by inhibiting the activation of RIP1/RIP3/MLKL signaling pathway and down-regulating the expression of the vascular growth factors VEGF, Ang-1, MMP9, and Stat-3.

Emodin protects against homocysteine-induced cardiac dysfunction by inhibiting oxidative stress via MAPK and Akt/eNOS/NO signaling pathways

Elevated levels of plasma homocysteine (Hcy) causes severe cardiac dysfunction, which is closely associated with oxidative stress. Emodin, a naturally occurring anthraquinone derivative, has been shown to exert antioxidant and anti-apoptosis activities. However, whether emodin could protect against Hcy-induced cardiac dysfunction remains unknown. The current study aimed to investigate the effects of emodin on the Hcy-induced cardiac dysfunction and its molecular mechanisms. Rats were fed a methionine diet to establish the animal model of hyperhomocysteinemia (HHcy). H9C2 cells were incubated with Hcy to induce a cell model of Hcy-injured cardiomyocytes. ELISA, HE staining, carotid artery and left ventricular cannulation, MTT, fluorescence staining, flow cytometry and western blotting were used in this study. Emodin significantly alleviated the structural damage of the myocardium and cardiac dysfunction from HHcy rats. Emodin prevented apoptosis and the collapse of MMP in the Hcy-treated H9C2 cells in vitro. Further, emodin reversed the Hcy-induced apoptosis-related biochemical changes including decreased Bcl-2/Bax protein ratio, and increased protein expression of Caspase-9/3. Moreover, emodin suppressed oxidative stress in Hcy-treated H9C2 cells. Mechanistically, emodin significantly inhibited the Hcy-activated MAPK by reducing ROS generation in H9C2 cells. Furthermore, emodin upregulated NO production by promoting the protein phosphorylation of Akt and eNOS in injured cells. The present study shows that emodin protects against Hcy-induced cardiac dysfunction by inhibiting oxidative stress via MAPK and Akt/eNOS/NO signaling pathways.

Tropisetron improved testicular inflammation in the streptozotocin-induced diabetic rats: The role of toll-like receptor 4 (TLR4) and mir146a.

As a serotonin antagonist, tropisetron positively affects blood glucose lowering, insulin synthesis, pancreas inflammation, and apoptosis in diabetes. Reproductive disorders are one of the diabetes-induced chronic complications. The present study aimed to evaluate the effect of tropisetron on diabetes-induced testicular inflammation, its signaling pathway, and mir146a. To this end, animals were assigned to the control, tropisetron, diabetes (DM), DM-tropisetron, and DM-glibenclamide groups. Streptozotocin (50 mg/kg) was intraperitoneally injected to provide diabetes. Tropisetron and glibenclamide were then administrated intraperitoneally for 2 weeks after diabetes induction. Testes histology, real-time polymerase chain reaction, western blot analysis, ELISA, and immunohistochemistry assays were also performed. The finding revealed that tropisetron significantly improved diabetes-induced testis damages, lowered TLR4, TRAF6, IRAK1, NF-κB, and caspase3 protein expressions, and decreased TNF-α and IL-1 levels. Moreover, the mir146a expression declined following the tropisetron treatment. This study demonstrated that the significant role of tropisetron in lowering testicular inflammation and apoptosis might have been due to the inhibition of the TLR4/IRAK1/TRAF6 signaling pathway and thereby the attenuation of NF-κB and caspase3 expression and inflammatory cytokines. Furthermore, the downregulation of mir146a, as an inflammatory microRNA interacting with TLR4, showed another pathway, through which tropisetron improved diabetes-induced testicular injuries.

Effects of MFG-E8 expression on the biological characteristics of ovarian cancer cells via the AKT/mTOR/S6K signalling pathway

In this study, we assessed the effects of MFG-E8 on the biological characteristics of ovarian cancer cells and explored the underlying mechanisms. Human ovarian cancer SKOV3 cells were transfected with MFG-E8 siRNA or NC siRNA. CCK-8, cell adhesion, scratch-wound, and Transwell assays were used to detect changes in cell metastatic processes. Effects of MFG-E8 silencing on the proteins involved in AKT/mTOR/S6K signalling pathway were assessed using qRT-PCR and Western blotting. Transient silencing of MFG-E8 in SKOV3 cells decreased cell proliferation and downregulated the expression of CDK4, cyclin D1, and caspase-3 proteins. Cell adhesion, migration, and invasion were also suppressed. p-AKT, p-mTORC1, and p-p70S6K levels decreased following MFG-E8 knockdown. Hence, MFG-E8 enhances carcinogenesis and affects the AKT/mTOR/S6K signalling pathway in ovarian cancer cells. In conclusion, our results suggested that MFG-E8 could promote ovarian cancer via AKT/mTOR/S6K signalling pathway which improved our understanding of the molecular mechanisms involved in ovarian cancer. IMPACT STATEMENT What is already known on this subject? Milk fat globule-epidermal growth factor 8 (MFG-E8) is expressed in several types of cancers such as oesophageal, breast, and liver. However, the mechanism of MFG-E8 involving in EOC remains unknown. We previously found that MFG-E8 expression was related to pathological staging, tissue differentiation, platinum sensitivity, ascites state, and other clinicopathological characteristics. What the results of this study add? Due to a series of in vitro studies, we confirmed that MFG-E8 is involved in the process of proliferation, invasion and metastasis. Our results show that silencing MFG-E8 can significantly inhibit the expression of cyclin D1 and CDK4 in EOC SKOV3 cells. MFG-E8 enhances carcinogenesis and affects the AKT/mTOR/S6K signaling pathway in ovarian cancer. What the implications are of these findings for clinical practice and/or further research? Taken together, our findings suggest that MFG-E8 may be an oncogene in EOC and provide new insights into the mechanism of MFG-E8 in the progression of EOC.

Aidi injection enhances the anti-tumor impact of doxorubicin in H22 tumor-containing mice

Ethnopharmacological relevanceAidi injection (AD) is a traditional medical preparation that has a Chinese origin. It is extensively used particularly in combination with doxorubicin (DOX) for the management of hepatocellular carcinoma (HCC). However, the combination's synergistic mechanism has not yet been clarified. Aim of the studyTo investigate the anti-tumor impact of AD in combination with DOX and their synergistic mechanism in HCC. Materials and methodsAn H22 mouse xenograft model was utilized to study the impact of AD, DOX, and their combination on HCC in vivo. Their effects on cell vitality, apoptosis, mitochondrial membrane potential, reactive oxygen species (ROS) production, caspase-3, and cleaved caspase-3 protein expression were also investigated in H22 cells in vitro. Subsequently, human umbilical vein endothelial cells (HUVECs) were utilized to investigate the impacts of AD, DOX, and their combination on cell viability, migration, invasion, tube formation, and vascular endothelial growth factor (VEGF) protein expression. ResultsThe study established that the tumor inhibition rate of AD combined with DOX reached 79.51%, which was significantly higher than that of AD (25.14%) or DOX (49.48%) alone. Additionally, the Q-value characterizing the synergy between AD and DOX was 1.72, demonstrating a strong synergistic effect. Furthermore, compared to AD or DOX administration alone, the combined administration group significantly decreased the alpha-fetoprotein (AFP) level in the serum, increased the tumor necrosis area, increased the Bax/Bcl-2, Cyt-c, caspase-9, Fas, Fasl, caspase-8, and caspase-3 protein expression, and significantly increased the CD31 and Ki67 protein expression in tumor tissue. Compared to AD or DOX alone, AD combined with DOX treatment had a synergistic effect on H22 cells (combination index values < 0.9), which inhibited cell viability, reduced mitochondrial membrane potential (MMP), induced apoptosis, promoted MMP loss, and increased ROS generation, cleaved caspase-3/caspase-3 levels, and caspase-3 activity. Moreover, combined administration showed a more pronounced inhibition of cell viability, migration, invasion, tube formation, and VEGF protein expression in HUVECs. ConclusionsAD enhances the anti-tumor effect of DOX by promoting apoptosis and inhibiting angiogenesis and cell proliferation. The findings of this study lay experimental foundations for the clinical combination of AD and DOX.

Anti-proliferative and Apoptotic Activities of Kasturi Tobacco (Nicotiana tabacum L.) Leaf Resinoid on Cervical Cancer Cell

BACKGROUND: Cervical cancer has a high rate of morbidity and mortality in women with cancer. Recent studies have found that tobacco (Nicotiana tabacum L.) is a potential source of anti-cancer agents. Hence, this study was conducted to determine the potential of Kasturi tobacco leaf resinoids as apoptotic agents against cervical cell malignancies, since it has not been fully elucidated before.METHODS: The phytochemical diversity of Kasturi tobacco resinoids was generated by gas chromatography-mass spectrometry (GC-MS) analysis followed by spectral similarity to National Institute of Standards and Technology (NIST) database. Cytotoxicity and proliferative activity of HeLa cells treated with Kasturi tobacco resinoids at various concentrations were evaluated by MTT assay. The expression of Caspase-3, cyclooxygenase-2 (COX-2) and heat shock protein 90 (HSP-90) in HeLa cells was analyzed by immunocytochemistry. Next, the migration ability of HeLa cells was observed by the scratch method.RESULTS: Kasturi tobacco resin contains 4,8,13-cyclotetradecatriene-1,3-diol, 1,5,9-trim with α-2,7,11-cembratriene-4,6-diol (α-CBD) structure in the form of a diterpenoid compound with the chemical formula C20H34O2 and a molecular weight of 306 Da. Kasturi tobacco resinoid with IC50 value of 2500 μg/mL inhibited proliferative activity during 72 hours. At a concentration of 1¼ IC50 and incubation for 48 hours, Caspase-3 expression increased by 74.1%, while COX-2 and HSP-90 expression decreased by 28.3% and 26.1%, respectively. HeLa cell migration was inhibited by Kasturi tobacco resinoid at 24 hours incubation.CONCLUSION: Kasturi tobacco resinoids with a concentration of 1¼ IC50 have potential as cervical anti-cancer agents by increasing Caspase-3 expression and decreasing COX-2 and HSP-90 expression within 48 hours.KEYWORDS: Kasturi tobacco resinoids, cervical cancer, anti-cancer agent, proliferative activity

Cell Cycle Arrest and Apoptosis Induced by Atovaquone in Non-Hodgkin's Lymphoma Raji Cells

To investigate the effect of atovaquone on the cell cycle and apoptosis of non-Hodgkin's lymphoma Raji cells, and clarify the related mechanisms. MTT assay and trypan blue dye exclusion method were used to evaluate the effect of atovaquone on the proliferation of Raji cells. After the cells were stained by PI staining, the cell cycle distribution was detected by flow cytometry. Cell apoptosis was analyzed by Annexin V/PI double binding assay. The intracellular alterations of reactive oxygen species were detected by 2', 7'-dichlorofluorescein diacetate (DCFH-DA). The protein expression of cell cycle and apoptosis related molecules were detected by Western blot. Various concentrations of atovaquone (5-40 μmol/L) inhibited the growth of Raji cells in a concentration-dependent manner (r=0.951). The proliferation of Raji cells was significantly inhibited after treated by atovaquone (20 and 30 μmol/L) for 24, 48 and 72 h, which showed statistically different with that in the control group (P<0.01, P<0.001, P<0.001). G1 phase arrest (P<0.01, P<0.001) and apoptosis (P<0.01) of Raji cells was induced by atovaquone (20 and 30 μmol/L) significantly for 24 h and 48 h, respectively. The expression of p-JAK2 and p-STAT3(Y705) protein were down-regulated significantly induced by atovaquone (P<0.001, P<0.05). Furthermore, atovaquone treatment could induce the decreasing of antiapoptotic protein Mcl-1, Bcl-2, and Bcl-xl expression level (P<0.05) and increasing of cleaved caspase-3 protein expression level. In addition, atovaquone could also induce the down-regulation of c-Myc (P<0.001, P<0.01) and cell cycle related molecules Cyclin D1, CDK4, and CDK6 (P<0.01, P<0.05) protein expression. Atovaquone effectively inhibits cell proliferation and induces cell cycle arrest and apoptosis by suppression of STAT3 signaling pathway in Raji cells. It can be a potential therapeutic agent against non-Hodgkin's lymphoma.