Study on the mechanism of cantharidin-induced hepatotoxicity in rat using serum and liver metabolomics combined with conventional pathology methods

Cantharidin (CTD), a compound secreted from Mylabris species, exhibits strong antitumor properties; however, hepatotoxicity restricts its clinical application. The mechanism by which CTD induces toxicity remains unclear. In the present study, the hepatotoxicity of CTD in the rat was investigated using a metabolomic approach combined with conventional pathology methods. A total of 30 rats were intragastrically treated with two doses of CTD (0.75 and 1.5 mg/kg) for 15 days to evaluate hepatotoxicity. Serum and liver samples were collected for biochemical dynamics analyses, histopathological examination and metabolomic analysis. It was found that liver index and serum biochemical indices were significantly increased. Furthermore, the pathology results showed that hepatocytes and subcellular organelles were damaged. Metabolomics analysis found 4 biomarkers in serum and 15 in the liver that were associated with CTD-induced hepatotoxicity. In addition, these were responsible for CTD hepatotoxicity by glycerophospholipid metabolism, sphingolipid metabolism, and steroid hormone biosynthesis. In conclusion, conventional pathology and metabolomics for exploring hepatotoxicity can provide useful information about the safety and potential risks of CTD.     

内质网应激、自噬与凋亡在斑蝥素致大鼠肝毒性中的作用

目的 探讨斑蝥素(cantharidin，CTD)致大鼠药物性肝损伤(drug-induced liver injury，DILI)的毒理学机制。方法 采用不同剂量CTD(0.061 4，0.092 1，0.184 1 mg·kg⁻¹)连续灌胃SD大鼠 28 d，检测肝脏指数和血清肝功能指标，HE 染色评估肝脏病理变化。进一步采用免疫印迹法检测内质网应激(endoplasmic reticulum stress，ERS)、自噬和细胞凋亡通路蛋白。结果 CTD 干预后肝脏指数显著升高，生化指标ALT、AST、LDH、ALP和T-Bil显著升高， 且呈剂量依赖性，肝脏组织出现结构破坏和中央静脉扩张等病理变化；GRP78、CHOP、ATF4、Beclin-1、LC3、Caspase-3、Caspase-8和Bax/Bcl-2的蛋白表达水平显著升高。分子对接结果显示，GRP78、ATF4和Beclin-1与CTD对接结果良好。结论 CTD可激活大鼠ERS，进一步激活自噬，诱导下游凋亡，研究结果可为CTD诱导的DILI提供新的科学依据。     

Hepatotoxicity of cantharidin is associated with the altered bile acid metabolism

Cantharidin (CTD) is an effective antitumor agent. However, it exhibits significant hepatotoxicity, the mechanism of which remains unclear. In this study, biochemical and histopathological analyses complemented with ultra-high-performance liquid chromatography–tandem mass spectrometry (UHPLC-MS/MS)-based targeted metabolomic analysis of bile acids (BAs) were employed to investigate CTD-induced hepatotoxicity in rats. Sixteen male and female Sprague–Dawley rats were randomly divided into two groups: control and CTD (1.0 mg/kg) groups. Serum and liver samples were collected after 28 days of intervention. Biochemical, histopathological, and BA metabolomic analyses were performed for all samples. Further, the key biomarkers of CTD-induced hepatotoxicity were identified via multivariate and metabolic pathway analyses. In addition, metabolite–gene–enzyme network and Kyoto Encyclopedia of Genes and Genomes pathway analyses were used to identify the signaling pathways related to CTD-induced hepatotoxicity. The results revealed significantly increased levels of biochemical indices (alanine aminotransferase, aspartate aminotransferase, and total bile acid). Histopathological analysis revealed that the hepatocytes were damaged. Further, 20 endogenous BAs were quantitated via UHPLC-MS/MS, and multivariate and metabolic pathway analyses of BAs revealed that hyocholic acid, cholic acid, and chenodeoxycholic acid were the key biomarkers of CTD-induced hepatotoxicity. Meanwhile, primary and secondary BA biosynthesis and taurine and hypotaurine metabolism were found to be associated with the mechanism by which CTD induced hepatotoxicity in rats. This study provides useful insights for research on the mechanism of CTD-induced hepatotoxicity.     

Triptolide‐induced hepatotoxicity via apoptosis and autophagy in zebrafish

Previous research about the development of triptolide (TP) as a natural active compound has often focused on hepatotoxicity. Among its various mechanisms, autophagy and apoptosis are two important signaling pathways. In this study, we used zebrafish to establish a TP‐induced hepatotoxicity model, and investigated the roles of autophagy and apoptosis in the progress of liver injury. Zebrafish exposed to TP showed increased mortality and malformation because of the increased drug dose and duration of exposure. Meanwhile, we found that TP induced liver injury in a time‐ and dose‐dependent manner, which was observed as a reduction in liver area, slow yolk absorption, upregulation of transaminase and local neurosis. With the application of the high‐content imaging system (HCIS) technique in liver 3D imaging in vivo, clear imaging of the zebrafish liver was achieved. The results showed a decrease in volume and location of necrosis in the liver after TP exposure. Increased expression of inflammatory cytokines genes tumor necrosis factor (Tnf)α, Il1β and Il6 were shown, particularly Tnfα. The Fas‐Caspase8 signaling pathway was activated. The apoptosis‐related gene Bcl‐2 was increased, and Bax, Caspase9 and Caspase3 were increased. However, autophagy related genes Beclin1, Atg5, Atg3 and Lc3 were increased more significantly, and the changes of Beclin1 and Atg5 were the most severe. This study successfully established a TP‐induced zebrafish hepatotoxicity model and applied the HCIS technique in a zebrafish hepatotoxicity study. The result indicated Fas might be the main target of TP‐induced hepatotoxicity. Autophagy played a more important role than apoptosis and was characterized by the overexpression of Beclin1 and Atg5.     

Inhibition of GSK3β protects against collagen type II-induced arthritis associated with a decrease in synovial leukocyte infiltration and inhibition of endoplasmic reticulum stress and autophagy biomarkers

We sought to determine whether TDZD-8, the inhibitor of the glycogen synthase kinase-3β (GSK3β), can protect the synovial membrane of the knee joint against injuries induced by collagen type II immunization (CIA) possibly via the downregulation of synovial leukocyte infiltration, endoplasmic reticulum stress (ERS), and autophagy. The model group of rats (CIA) were immunized over a period of 3 weeks with collagen type II, whereas the treated group of rats (CIA + TDZD-8) were treated with TDZD-8 (1 mg/kg) for 21 days after the completion of the immunization regimen. All rats were then killed at week 6. Harvested synovial tissues were prepared for immunohistochemistry staining, and synovial homogenates were assayed for biomarkers of ERS, autophagy, apoptosis, and cell survival and proliferation. In addition, blood samples were assayed for biomarkers of arthritis. Synovial tissue images showed that CIA enhanced leukocyte recruitment as demonstrated by an increased CD45+ (leukocyte common antigen) immunostaining, which was markedly decreased by TDZD-8. TDZD-8 also significantly (P < .05) inhibited collagen-induced autophagy biomarkers Beclin-1 and LC3II, the ERS biomarkers GRP-78, IRE1-α, XBPIs, and eIF2a, and the survival protein Bcl-2. Whereas, the collagen-induced proliferative biomarkers Akt and mTOR were not inhibited by TDZD-8, and CIA inhibited the apoptotic proteins CHOP and cleaved caspase-3, which were augmented by TDZD-8. We further demonstrated a significant (P < .05) correlation between autoantibodies generated during the course of arthritis and biomarkers of ERS and autophagy. We conclude that TDZD-8 inhibits CIA and decreases synovial leukocyte infiltration, ERS, and autophagy, which is independent of Akt/mTOR signalling.     

Cigarette smoke exposure leads to follicle loss via an alternative ovarian cell death pathway in a mouse model

Cigarette smoking among reproductive-aged women is increasing worldwide. Cigarette smoking is a lifestyle behavior associated with important adverse health effects including subfertility and premature ovarian failure. We previously demonstrated that cigarette smoke (CS) exposure in mice decreases the primordial follicle pool; however, the mechanism of action is largely unknown. Therefore, the present study was designed to elucidate the mechanisms underlying CS exposure-induced ovarian follicle loss. CS exposure induced a significant decrease in the relative ovarian weight and the number of primordial and growing follicles. Oxidative stress, as shown by increased Hsp25 and decreased superoxide dismutase 2 protein expression, was found in mice exposed to CS for 8 weeks. Exposure decreased Bcl-2 but failed to induce apoptosis. An increased number of autophagosomes in granulosa cells of ovarian follicles together with increased expression of Beclin-1 and microtubule-associated protein light chain 3, key regulatory proteins in the autophagy (Atg) pathway, was found in CS-exposed mice compared with the control group. Taken together, our results suggest that CS exposure does not induce apoptosis but rather activates the Atg pathway ultimately leading to ovarian follicle loss. We further postulate that Atg is a novel mechanism of toxicant-induced ovarian follicle loss.     

Rottlerin induces autophagy which leads to apoptotic cell death through inhibition of PI3K/Akt/mTOR pathway in human pancreatic cancer stem cells

Multiple lines of evidence support the idea that autophagy plays an essential role in the development of drug resistance, self-renewal, differentiation, and tumorigenic potentials of cancer stem cells (CSCs). Rottlerin (ROT) is widely used as a protein kinase C-delta (PKC-δ) inhibitor. Recent studies revealed that ROT induces apoptosis through engagement of mitochondria. However, it is not known whether ROT-induced apoptosis is associated with other mechanisms such as autophagy. Here we found that ROT induced autophagy followed by induction of apoptosis via inhibition of PI3K/Akt/mTOR pathway and activation of caspase cascade in human pancreatic CSCs. ROT induced a dose- and time-dependent inhibition of cell survival and induction of cytoplasmic vacuolations. The conversion of microtubule-associated protein LC3-I to LC3-II, and increased accumulations of Atg7 and Beclin-1 were also observed in CSCs treated with ROT. Prolonged exposure of CSCs to ROT eventually caused apoptosis which was associated with the suppression of phosphorylated Akt (Ser473) and mTOR (Ser2448), downregulation of XIAP, cIAP-1, Bcl-2 and Bcl-X_L, induction of Bax, activation of caspase-3 and -9, and concomitant degradation of PARP. ROT-induced apoptosis was enhanced by dominant negative AKT, Akt1/2 inhibitor, and rapamycin. Our study also demonstrates that gene silencing of Atg7 and Beclin1, or cotreatment of the autophagosome inhibitor, 3-methyladenine, inhibited ROT-induced autophagy and accelerated ROT-induced apoptosis. The knockdown of PKC-δ did not block ROT-induced autophagy and cell death, suggesting these effects of ROT were exerted through PKC-δ-independent pathway. In summary, our data demonstrate that ROT can induce autophagy which leads to cell death in pancreatic CSCs.     

Modulation of Atg5 expression by globular adiponectin contributes to autophagy flux and suppression of ethanol-induced cell death in liver cells

Globular adiponectin (gAcrp) protects liver cells from ethanol-induced apoptosis via induction of autophagy. However, the underlying mechanisms are unknown. The present study aims to investigate the potential role of autophagy-related protein 5 (Atg5), an essential Atg for the elongation of autophagosomes, in suppression of ethanol-induced cytotoxicity by gAcrp. Here, we demonstrated that suppression of Atg5 expression by ethanol was restored by pretreatment with gAcrp both in primary rat hepatocytes and human hepatoma cell line (HepG2). Moreover, ethanol-induced accumulation of p62 (sequestosome1), a marker of autophagic flux, was restored by gAcrp treatment, implying that gAcrp modulates autophagic flux in liver cells. Further, Atg5 silencing prevented p62 degradation by gAcrp, suggesting that Atg5 plays a critical role in induction of autophagic flux by gAcrp. Interestingly, gene silencing of Atg5 by siRNA abrogated restoration of autophagosome formation by gAcrp in ethanol-treated cells. Finally, protection of liver cells by gAcrp from ethanol-induced apoptosis was also significantly attenuated by knocking-down of Atg5 expression, suggesting an important role of Atg5 in autophagy induction and cellular apoptosis modulated by gAcrp. Taken together, our data demonstrated that Atg5 expression, at least in part, is implicated in gAcrp-induced autophagy and subsequent anti-apoptotic effects in ethanol-treated liver cells.     

Nickel sulfate induced apoptosis via activating ROS‐dependent mitochondria and endoplasmic reticulum stress pathways in rat Leydig cells

Nickel can induce apoptosis of testicular Leydig cells in mice, whereas the mechanisms remain unclear. In this study, we investigated the role of nickel‐induced reactive oxygen species (ROS) generation in mitochondria and endoplasmic reticulum stress (ERS) mediated apoptosis pathways in rat Leydig cells. Fluorescent DCF and Annexin‐V FITC/PI staining were performed to measure the production of ROS and apoptosis in Leydig cells. RT‐qPCR and Western blot were conducted to analyze the key genes and proteins involved in mitochondria and ERS apoptotic pathways. The results showed that nickel sulfate induced ROS generation, consequently resulted in nucleolus deformation and apoptosis in testicular Leydig cells, which were then attenuated by ROS inhibitors of N‐acetylcysteine (NAC) and 2,2,6,6‐tetramethyl‐1‐piperidinyloxy (TEMPO). Nickel sulfate‐triggered Leydig cells apoptosis via mitochondria and ERS pathways was characterized by the upregulated mRNA and proteins expression of Bak, cytochrome c, caspase 9, caspase 3, GRP78, GADD153, and caspase 12, which were inhibited by NAC and TEMPO respectively. The findings indicated that nickel‐induced ROS generation was involved in apoptosis via mitochondria and ERS pathways in rat Leydig cells.     

β-Elemene induces apoptosis as well as protective autophagy in human non-small-cell lung cancer A549 cells

Objectives β‐Elemene, a novel traditional Chinese medicine, has been shown to be effective against a wide range of tumours. In this study, the antitumour effect of β‐elemene on human non‐small‐cell lung cancer (NSCLC) A549 cells and the mechanism involved have been investigated. Methods Cell viability and apoptosis were measured by the 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) assay and flow cytometry, respectively. Protein expression was assayed by Western blotting. Autophagy was evaluated under fluorescence microscopy and transmission electron microscopy. Key findings β‐Elemene inhibited the viability of A549 cells in a dose‐dependent manner. This suppression of cell viability was due to the induction of apoptosis. Further study showed that β‐elemene inhibited the activity of the PI3K/Akt/mTOR/p70S6K1 signalling pathway, and at the same time it triggered a robust autophagy. The autophagy was characterized by the accumulation of punctate LC3 dots in the cytoplasm, morphological changes, and the increased levels of LC3‐II as well as Atg5‐Atg12 conjugated proteins. Inhibition of autophagy with chlorochine significantly enhanced the antitumour effect of β‐elemene. Conclusions Our data indicated that β‐elemene inhibited the activity of the PI3K/Akt/mTOR/p70S6K1 signalling pathway in human NSCLC A549 cells, which resulted in apoptosis as well as protective autophagy. A combination of β‐elemene with autophagy inhibitor might be an effective therapeutic option for advanced NSCLC.     

右美托咪定通过lncRNA H19介导香烟提取物诱导的肺泡巨噬细胞效应研究

目的探索右美托咪定(Dexmedetomidine,DEX)通过长链非编码RNA(long noncoding RNA,lncRNA)H19对香烟烟雾提取物(cigarette smoke extract,CSE)诱导的RAW264.7巨噬细胞活力、凋亡和自噬的影响。方法制备CSE及CSE干预巨噬细胞模型,运用体外细胞培养,DEX以不同浓度(2、4和8μmol/L)作用于CSE诱导的巨噬细胞,采用MTT法检测细胞存活率,流式细胞术检测模型细胞的凋亡率,采用蛋白质印迹法(Western blot)检测细胞中凋亡相关蛋白Bax和Bcl-2及自噬相关蛋白LC3Ⅱ和ATG7表达情况,实时荧光定量PCR(qRT-PCR)检测H19表达水平;为了进一步验证DEX对CSE诱导的巨噬细胞增殖、凋亡和自噬影响,运用双酶切法构建pcDNA3.1-H19表达载体。结果一定剂量的DEX可明显拮抗CSE诱导的巨噬细胞活力下降作用(P<0.05),具有浓度依赖性;DEX抑制CSE诱导的巨噬细胞凋亡,且细胞中凋亡蛋白Bax明显下调(P<0.05),抗凋亡蛋白Bcl-2上调(P<0.05),自噬相关蛋白LC3Ⅱ和ATG7表达上调(P<0.05),H19表达下调(P<0.05);成功构建pcDNA3.1-H19表达载体,DEX作用于CSE诱导的巨噬细胞,细胞活力明显降低(P<0.05),且细胞中凋亡蛋白Bax上调(P<0.05),抗凋亡蛋白Bcl-2下调(P<0.05),自噬相关蛋白LC3Ⅱ和ATG7表达下调(P<0.05)。结论DEX抑制H19表达,增强CSE诱导的巨噬细胞活力,抑制细胞凋亡和自噬。     

Arsenic-induced autophagy regulates apoptosis in AML-12 cells

Arsenic (As), a potent toxicant, is known to be a hepatotoxicant. Although As induced liver apoptosis and autophagy, the relationship between apoptosis and autophagy of hepatocytes caused by As remains largely unknown. 3-methyladenine (3-MA) and rapamycin can inhibit and promote autophagy of AML-12 cells, respectively. Hence, in this study, AML-12 cells were treated with different concentrations (0, 2, 4, 6, 8, 10 and 12 μmol/L) of As₂O₃, and 5 mmol/L 3-MA or 100 nmol/L rapamycin were applied to distinguish the effect of autophagy on apoptosis in AML-12. Results showed that exposure to As induced cell apoptosis and autophagy, which were mediated by the significantly altered expression levels of autophagy markers (mTOR, LC3, PI3K and P62), and apoptosis markers (Bcl-2 and caspase-3). Further analysis indicated that a certain dosage of 3-MA and rapamycin decreased apoptosis and the caspase-3 expression, which suggested that As-induced autophagy regulated AML-12 cells apoptosis through the expressions of PI3K, mTOR, P62 and Bcl-2.     

不同浓度米诺环素对人胶质瘤细胞增殖、自噬、凋亡的影响及其机制

摘要：目的 探讨不同浓度米诺环素对人类胶质瘤 U87 和 LN229 细胞增殖及凋亡的影响和作用机制。方法 （1）U87和LN229细胞设对照组（DMSO处理），5 μmol/L米诺环素组和10 μmol/L米诺环素组，分组处理72 h 后采用 MTT法检测细胞增殖水平，免疫荧光标记检测细胞自噬蛋白微管相关蛋白l轻链3B亚基（LC3B）表达水平，Western blot法检测细胞自噬和凋亡相关蛋白的表达变化。（2）构建沉默信息调节因子2相关酶1（SIRT1）-shRNA载体，观察 敲低SIRT1表达后，不同浓度米诺环素对U87和LN229细胞自噬的影响。结果 （1）与对照组相比，5 μmol/L和10 μmol/L米诺环素组细胞增殖能力下降，免疫荧光标记显示胞浆内自噬标记蛋白LC3B的表达增多，Western blot结果 显示哺乳动物雷帕霉素靶蛋白（mTOR）水平显著降低，自噬基因相关蛋白5（Atg5）、磷酸化AMP依赖的蛋白激酶α亚 基（p-AMPKα）、SIRT1水平显著增加（P＜0.05）。与对照组相比，10 μmol/L米诺环素组B淋巴细胞瘤-2（Bcl-2）、磷 酸化 p70 核糖体蛋白 S6 激酶（p-p70s6k）水平显著降低，活化形式的 Caspase-3（Cleaved caspase-3）水平显著增加 （P＜0.05），而5 μmol/L米诺环素组除Cleaved Caspase-3表达水平下降外其他凋亡相关蛋白水平未见显著变化（P＞ 0.05）。（2）敲低SIRT1表达后，shSIRT1组mTOR和LC3B表达水平较对照组明显下降；而经过米诺环素处理后，mTOR 的表达出现明显升高，而LC3B表达水平仅部分恢复。结论 5、10 μmol/L的米诺环素均能有效抑制人类胶质瘤U87 和LN229细胞的生长，其机制可能与AMPK/SIRT1通路诱导细胞自噬和p70s6k/Bcl-2通路诱导细胞凋亡有关。     

Inhibition of autophagy protects against PAMAM dendrimers-induced hepatotoxicity

Abstract

Toxicity of nanomaterials is one of the biggest challenges in their medicinal applications. Although toxicities of nanomaterials have been widely reported, the exact mechanisms of toxicities are still not well elucidated. Consequently, the exploration of approaches to attenuate toxicities of nanomaterials is limited. In this study, we reported that poly-amidoamine (PAMAM) dendrimers, a widely used nanomaterial in the pharmaceutical industry, caused toxicity of human liver cells by inducing cell growth inhibition, mitochondria damage, and apoptosis. Meanwhile, autophagy was activated in PAMAM dendrimers-induced toxicity and inhibition of autophagy-rescued viability of hepatic cells, indicating that autophagy played a key role in PAMAM dendriemrs-induced hepatotoxicity. To further explore approaches to attenuate PAMAM dendrimers-induced liver injury, effects of autophagic inhibitors on PAMAM dendrimers’ hepatotoxicity were investigated in an in vivo model. Autophagy blockage in PAMAM dendrimers-administered mice led to weight restoration, damage reversion of liver tissue, and protection against changes of serum biochemistry parameters. Moreover, inhibition of Akt/mTOR and activation of Erk1/2 signaling pathways were involved in PAMAM dendrimers-induced autophagy. Collectively, these findings indicated that autophagy was associated with PAMAM dendrimers-induced hepatotoxicity, and supported the possibility that autophagy inhibitors could be used to reduce hepatotoxicity of PAMAM dendrimers.     

Di‐2‐ethylhexyl phthalate (DEHP) induces apoptosis and autophagy of mouse GC‐1 spg cells

As a widely used plasticizer in industry, di‐2‐ethylhexylphthalate (DEHP) can cause testicular toxicity, yet little is known about the potential mechanism. In this study, DEHP exposure dramatically inhibited cellviability and induced apoptosis of mouse GC‐1 spg cells. Furthermore, DEHP significantly increased the levels of autophagy proteins LC3‐II, Beclin1 and Atg5, as well as the ratio ofLC3‐II/LC3‐I. Transmission electron microscopy (TEM) further confirmed that DEHP induced autophagy of mouse GC‐1 spg cells. DEHP was also shown to induceoxidative stress; while inhibition of oxidative stress with NAC could increase cell viability and inhibit DEHP‐induced apoptosis and autophagy. These results suggested that DEHP induced apoptosis and autophagy of mouse GC‐1 spg cells via oxidative stress. 3‐MA, an inhibitor of autophagy, could rescue DEHP‐induced apoptosis. In summary, DEHP induced apoptosis and autophagy of mouse GC‐1 spg cells via oxidative stress, and autophagy might exert a cytotoxic effect on DEHP‐induced apoptosis.     

Roles of endoplasmic reticulum stress,apoptosis and autophagy in 2,2′,4,4′-tetrabromodiphenyl ether-induced rat ovarian injury

Endocrine disruptor 2,2′,4,4′-tetrabromodiphenylether (PBDE-47) can harm the female reproductive system. Recent studies showed that PBDE-47 neurotoxicity is associated with endoplasmic reticulum stress (ERS); however, the role of ERS in PBDE-47-induced ovarian injury is unclear. New-born female Sprague-Dawley rats were orally exposed to PBDE-47 (1, 5, or 10 mg/kg bw) on postnatal day 10. An additional 10 mg/kg bw PBDE-47 group was given the ERS inhibitor 4-PBA intraperitoneally for three weeks beginning on postnatal day 8. At 2 months of age, PBDE-47 exposure significantly reduced the ovarian coefficients, increased the expression of ERS and autophagy markers, including GRP78, IRE1, Caspase-12, Beclin1, LC3 and P62. In the 10 mg/kg bw PBDE-47 group, PARP and Caspase-3 were markedly activated, indicative of apoptosis. These were accompanied by histopathological damage. Intriguingly, 4-PBA attenuated all these effects. Thus, these results suggest that ERS plays a vital role in PBDE-47-induced ovarian injury by regulating autophagy and apoptosis.     

Bioactive peptides attenuate cardiac apoptosis in spontaneously hypertensive rat hearts through activation of autophagy and mitochondrial biogenesis pathway

Alcalase potato protein hydrolysate (APPH) might have a very important role in therapeutic effects. This study aims to examine the beneficial effects of bioactive peptides (DIKTNKPVIF [DI] and IF) from APPH supplement in the regulation of cardiac apoptosis, autophagy, and mitochondrial biogenesis pathway in spontaneously hypertensive rats (SHR). We have investigated ejection fraction, fractional shortening, Tunel assay, apoptosis, autophagy, and mitochondrial biogenesis pathway marker expression to show the efficacy of bioactive peptides in an SHR model. Bioactive peptides significantly upregulate ejection fraction and fractional shortening in SHR rats. SHR rats exhibited higher protein expression of apoptotic markers such as BAD, cytochrome c, and caspase 3. Finally, the bioactive peptides upregulate survival proteins (p‐AKT/p‐PI3K), autophagy (Beclin1/LC3B), and mitochondrial biogenesis (p‐AMPKα/SIRT1/PGC1α/p‐Foxo3a/Nrf2/CREB) marker expressions compared with the SHR groups. In summary, the bioactive peptides protect the heart tissues through the activation of autophagy and mitochondrial biogenesis pathway and thereby attenuate cardiac apoptosis in a spontaneously hypertensive rat model.     

16-hydroxy-cleroda-3,13-dien-16,15-olide induced glioma cell autophagy via ROS generation and activation of p38 MAPK and ERK-1/2

16-hydroxy-cleroda-3,13-dien-16,15-olide (HCD), a natural product isolated from medicinal plant Polyalthia longifolia exhibits anticancer activity through caspase-independent apoptosis in brain tumors, as previously reported. This study further attempted to investigate the involvement of HCD-induced autophagy in brain tumor cell lines neuroblastoma N18 and glioma C6 through the induction of reactive oxygen species (ROS) and the activation of p38 and ERK-1/2 pathway. The results demonstrated that HCD increased the hyper-generation of ROS and decreased cellular antioxidant enzymes, such as superoxide dismutase (SOD), glutathione (GSH), glutathione peroxidase (GPx), and glutathione s transferase (GST). Furthermore, HCD increased the expressions of autophagic marker proteins LC3-II and Beclin-1 in a time- and dose-dependent manner. Additionally, HCD was found to significantly induce p-p38 MAPK and p-ERK-1/2 proteins by Western blot, which implies that HCD is a potential therapeutic anticancer agent that exerts its activity through inducing ROS-mediation for the autophagy of brain tumor cells.