Bacopa monnieri‐Induced Protective Autophagy Inhibits Benzo[a]pyrene‐Mediated Apoptosis

Benzo[a]pyrene (B[a]P) is capable of inducing oxidative stress and cellular injuries leading to cell death and associates with a significant risk of cancer development. Prevention of B[a]P‐induced cellular toxicity with herbal compound through regulation of mitochondrial oxidative stress might protect cell death and have therapeutic benefit to human health. In this study, we demonstrated the cytoprotective role of Bacopa monnieri (BM) against B[a]P‐induced apoptosis through autophagy induction. Pretreatment with BM rescued the reduction in cell viability in B[a]P‐treated human keratinocytes (HaCaT) cells indicating the cytoprotective potential of BM against B[a]P. Moreover, BM was found to inhibit B[a]P‐mediated reactive oxygen species (ROS)‐induced apoptosis activation in HaCaT cells. Furthermore, BM was found to preserve mitochondrial membrane potential and inhibited release of cytochrome c in B[a]P‐treated HaCaT cells. Bacopa monnieri induced protective autophagy; we knocked down Beclin‐1, and data showed that BM was unable to protect from B[a]P‐induced mitochondrial ROS‐mediated apoptosis in Beclin‐1‐deficient HaCaT cells. Moreover, we established that B[a]P‐induced damaged mitochondria were found to colocalize and degraded within autolysosomes in order to protect HaCaT cells from mitochondrial injury. In conclusion, B[a]P‐induced apoptosis was rescued by BM treatment and provided cytoprotection through Beclin‐1‐dependent autophagy activation. Copyright © 2016 John Wiley & Sons, Ltd.     

1,7‐Bis(4‐hydroxyphenyl)‐4‐hepten‐3‐one from Betula platyphylla induces apoptosis by suppressing autophagy flux and activating the p38 pathway in lung cancer cells

Betula platyphylla (BP) is frequently administered in the treatment of various human diseases, including cancers. This study was undertaken to investigate the pharmacological function of the active components in BP and the underlying mechanism of its chemotherapeutic effects in human lung cancer cells. We observed that BP extracts and 1,7‐bis(4‐hydroxyphenyl)‐4‐hepten‐3‐one (BE1), one of the components of BP, effectively decreased the cell viability of several lung cancer cell lines. BE1‐treated cells exhibited apoptosis induction and cell cycle arrest at the G2/M phase. Further examination demonstrated that BE1 treatment resulted in suppression of autophagy, as evidenced by increased protein expression levels of both LC3 II and p62/SQSTM1. Interestingly, the pharmacological induction of autophagy with rapamycin remarkably reduced the BE1‐induced apoptosis, indicating that apoptosis induced by BE1 was associated with autophagy inhibition. Our data also demonstrated that BE1 exposure activated the p38 pathway resulting in regulation of the pro‐apoptotic activity. Taken together, we believe that BE1 is a potential anticancer agent for human lung cancer, which exerts its effect by enhancing apoptosis via regulating autophagy and the p38 pathway.     

补阳还五汤类方提取物对PC12细胞氧化应激损伤模型凋亡与自噬的调控

目的从凋亡与自噬的调控探讨补阳还五汤类方提取物对PC12细胞氧化应激模型的保护机制。方法以不同浓度H_2O_2刺激PC12细胞制备不同损伤程度的氧化应激模型,采用MTT法确定补阳还五汤类方提取物对氧化应激损伤初期和加剧期发挥保护作用的有效浓度,流式细胞术和TUNNEL法检测细胞凋亡情况,透射电镜和m RFP-GFP-LC3腺病毒双标法检测细胞自噬情况,蛋白质印迹法检测凋亡相关蛋白(Bcl-2和Bax)与自噬相关蛋白(Beclin1、LC3A和LC3B)的表达。结果分别以0.5、2.0 mmol/L H_2O_2处理PC12细胞制备氧化应激损伤的初期模型和加剧期模型;与对照组比较,模型组细胞凋亡率增加,自噬程度加剧,Bax/Bcl-2比例增加,Beclin1和LC3B表达上调,LC3A表达下调(P0.05);与氧化应激损伤初期的模型1组比较,补阳还五汤类方提取物均能下调Bax/Bcl-2,抑制凋亡率,并一定程度上调Beclin1和LC3B/LC3A的表达,激活自噬(P0.05);当氧化应激损伤程度加剧时,模型细胞中出现较高水平的凋亡与自噬,此时,两方则通过抑制凋亡和降低自噬,共同发挥保护作用。结论补阳还五汤类方提取物可通过对凋亡与自噬的交互动态调控对不同程度损伤的氧化应激模型细胞挥发保护作用。     

Graveoline Isolated from Ethanolic Extract of Ruta graveolens Triggers Apoptosis and Autophagy in Skin Melanoma Cells: A Novel Apoptosis‐Independent Autophagic Signaling Pathway

Anti‐cancer drugs generally kill cancer cells by apoptosis but fail to do so when they become resistant and escape apoptosis signals. But these resistant cells can still be killed by autophagy. Therefore, drugs having both apoptotic and autophagic abilities are solicited in effective cancer management. In search of such a drug, we examined the efficacy of graveoline, a bioactive compound isolated from Ruta graveolens on skin melanoma A375 cells through the use of specific signaling cascades and their inhibitors. Cytotoxicity of graveoline was tested by conducting MTT assay. Induction of autophagy and apoptosis was checked. Expression of related proteins and their localization were studied by conducting immunoblot assay and through confocal microscopy, respectively. We found graveoline‐induced Beclin‐1 associated autophagy in A375 cells and 3‐methyladenine, an inhibitor of autophagy did not affect apoptosis. Conversely, caspase inhibitor that blocked apoptosis did not affect autophagic cell death, suggesting thereby that these two were independent events. Use of reactive oxygen species (ROS) scavengers inhibited cell death, but blocking autophagy did not affect graveoline‐induced ROS generation, suggesting that ROS generation ensued autophagy. Thus, graveoline‐induced both apoptotic and autophagic cell death in skin melanoma cells, a desirable quality in effective anti‐cancer drug design. Copyright © 2013 John Wiley & Sons, Ltd.     

活血消瘿方含药血清调控AMPK/mTOR通路对脂多糖诱导的甲状腺滤泡上皮细胞自噬和凋亡的影响

目的 探讨活血消瘿方含药血清对脂多糖(LPS)诱导的甲状腺滤泡上皮细胞(TFECs)自噬和凋亡的影响以及作用机制。方法 光学显微镜观察分离培养的SD大鼠TFECs细胞的形态;免疫荧光染色鉴定TFECs中特异抗原甲状腺球蛋白(Tg)。取对数生长期的TFECs,分为对照组、LPS组、含药血清组、含药血清+compound C(AMPK抑制剂)组、含药血清+MHY1485(mTOR激活剂)组,MTT法检测各组细胞活力;绿色荧光蛋白(GFP)标记质粒转染检测各组细胞自噬小体变化;流式细胞术检测各组细胞凋亡;western blot检测各组细胞中微管相关蛋白1轻链3(LC3)I、LC3II、Beclin1、Bcl-2相关X蛋白(Bax)、B淋巴细胞瘤-2(Bcl-2)及AMP活化蛋白激酶(AMPK)/哺乳动物雷帕霉素靶蛋白(mTOR)通路相关蛋白表达。结果 光学显微镜观察显示,TFECs形态为梭形、形态均一,且可成簇生长。免疫荧光结果显示,TFECs细胞质中可见较强的Tg荧光染色。与对照组比较,LPS组TFECs细胞活力、自噬小体数量、LC3II/LC3I、Beclin1、Bcl-2、p-AMPK/AMPK蛋白相对表达量显著降低,炎性因子IL-6 、TNF-α水平、细胞凋亡率、Bax、p-mTOR/mTOR蛋白相对表达量显著升高(P< 0.05);与LPS组比较,含药血清组TFECs细胞活力、自噬小体数量、LC3II/LC3I、Beclin1、Bcl-2、p-AMPK/AMPK蛋白相对表达量显著升高,炎性因子IL-6 、 TNF-α水平、细胞凋亡率、Bax、p-mTOR/mTOR蛋白相对表达量显著降低(P< 0.05);与含药血清组比较,含药血清+compound C组、含药血清+MHY1485组TFECs中相应指标与上述趋势相反。结论 活血消瘿方含药血清对LPS诱导的TFECs自噬的促进作用及细胞凋亡的抑制作用可能与激活AMPK/mTOR通路有关。     

天麻素对毛果芸香碱诱发的癫痫大鼠TLR4/NF-κB信号通路影响的研究

目的:探讨天麻素对毛果芸香碱诱发的癫痫大鼠的脑保护作用及其作用机制。方法:72只大鼠随机分为空白组、模型组、阳性药组、天麻素组、Toll样受体4 (TLR4)激活剂脂多糖(LPS)组、天麻素+LPS组,每组12只。采用腹腔注射毛果芸香碱建立癫痫大鼠模型。双抗体夹心酶联免疫法检测大鼠血清和海马组织肿瘤坏死因子-α(TNF-α)与白细胞介素-1β(IL-1β)水平,TUNEL染色法检测海马组织细胞凋亡数,Western blot分别检测海马组织活化半胱氨酸基天冬氨酸酶-3 (CleavedCaspase-3)、B淋巴细胞瘤-2 (Bcl-2)、Bcl-2相关X蛋白(Bax)、TLR4、兔抗大鼠p-核因子-κB亚基p65亲和肽(p-NF-κBp65)和磷酸化核因子κB抑制蛋白α(p-IκB-α)表达。结果:与空白组比较,模型组大鼠脑电频率显著降低(P<0.05),波幅显著升高(P<0.05);血清和海马组织TNF-α和IL-1β水平,海马组织细胞凋亡率、Cleaved-Caspase-3、Bax、TLR4和p-NF-κBp65蛋白明显升高(P<0.05);IL-10水平,Bcl-2和p-IκB-α蛋白表达显著降低(P<0.05)。与模型组比较,阳性药组、天麻素组和天麻素+LPS大鼠脑电频率显著升高(P<0.05),波幅显著降低(P<0.05),血清和海马组织TNF-α和IL-1β水平,海马组织细胞凋亡率、Cleaved-Caspase-3、Bax、TLR4和p-NF-κBp65蛋白明显降低(P<0.05),IL-10水平,Bcl-2和p-IκB-α蛋白表达显著升高(P<0.05);LPS组大鼠脑电频率、血清及海马组织IL-10水平、Bcl-2蛋白显著降低(P<0.05),血清与海马组织TNF-α与IL-1β水平、海马组织细胞凋亡率、Cleaved-Caspase-3、Bax、TLR4、p-NF-κBp65和p-IκB-α蛋白表达明显升高(P<0.05)。与LPS组比较,天麻素+LPS组大鼠脑电频率显著升高(P<0.05),波幅显著降低(P<0.05);血清及海马组织TNF-α与IL-1β水平、海马组织细胞凋亡率、Cleaved-Caspase-3、Bax、TLR4、p-NF-κBp65蛋白表达均明显降低(P<0.05),IL-10水平、Bcl-2及p-IκB-α蛋白表达明显升高(P<0.05)。结论:天麻素可通过抑制TLR4/NF-κB信号通路对毛果芸香碱诱发的癫痫大鼠发挥脑保护作用。     

从调节Cajal细胞自噬探讨芍药甘草汤改善Oddi括约肌功能障碍的机制

基于芍药甘草汤具有显著改善Oddi括约肌功能障碍(SOD)患者腹痛的临床疗效和抑制自噬相关蛋白(GPR78、Be-clin1)的工作基础,结合Cajal间质细胞(ICC)过度自噬将引起平滑肌收缩紊乱、进一步导致SOD的研究进展,探讨芍药甘草汤调节ICC自噬水平及治疗SOD的分子药理机制.首先,复制豚鼠SOD模型,观察芍...     

黄芪多糖抑制缺氧/复氧损伤乳鼠心肌细胞凋亡与自噬机制研究

目的:探讨黄芪多糖(APS)对缺氧/复氧(H/R)所致乳鼠心肌细胞凋亡与自噬抑制作用的相关机制。方法:分离并体外培养乳鼠心肌细胞3 d后制备H/R损伤细胞模型,设正常对照组、H/R组、APS低、中、高剂量(20、40、80 μmol/ml)组,各组于造模前30 min给药处理。复氧2 h后,CCK-8法检测细胞增殖抑制率,Annexin V-FITC/PI染色法检测细胞凋亡水平,Western blot法检测p-Akt、Cleaved Caspase-3、Bcl-2、Bax、p-mTOR、Beclin1、LC3、P62蛋白表达。结果:与H/R组比较,APS中、高剂量细胞增殖抑制率和凋亡率显著降低(P<0.01),p-Akt、p-mTOR、Bcl-2表达量显著升高而Cleaved Caspase-3、Bax、Beclin1、LC3-Ⅰ、LC3-Ⅱ、P62表达量显著降低(P<0.05),Bcl-2/Bax比值显著升高、LC3-Ⅱ/LC3-Ⅰ比值显著降低(P<0.01)。结论:APS可能通过激活Akt/mTOR通路调控凋亡和自噬相关蛋白表达,对H/R所致乳鼠心肌细胞凋亡与自噬起到抑制作用。     

Chrysophanol attenuates airway inflammation and remodeling through nuclear factor‐kappa B signaling pathway in asthma

Chrysophanol (CHR), a purified active constituent extracted from Rheum palmatum L., possesses anti‐inflammatory activity. This study aimed to evaluate its effects on asthma‐associated airway inflammation and remodeling. BALB/c mice were sensitized and challenged by ovalbumin (OVA) and administrated with different doses of CHR. We found that CHR decreased OVA‐induced pulmonary inflammation: the levels of interleukin (IL)‐4, IL‐5, and IL‐13, tumor necrosis factor (TNF)‐α, and inducible nitric oxide synthase were downregulated. CHR also attenuated airway remodeling induced by OVA challenge—CHR inhibited pulmonary α‐smooth muscle actin expression. Moreover, both the nuclear translocation and activity of NF‐κB p65 were inhibited by CHR in the asthmatic lung. Enhanced autophagy was initiated in the lung by OVA challenge as evidenced by upregulated light chain 3 beta, autophagy‐related protein 5, and Beclin 1. CHR suppressed OVA‐induced alterations in these autophagy‐related molecules. In vitro, CHR (2 or 20 μM) was used to treat human pulmonary epithelial BEAS‐2B cells in the presence of 10 ng/ml recombinant TNF‐α. CHR not only exhibited the antiproliferation effect but also inhibited the activation of nuclear factor‐kappa B (NF‐kB) signaling pathway in TNF‐α‐treated BEAS‐2B cells. In conclusion, our study indicates that CHR has the potential to ameliorate asthma.     

Tectorigenin protects against experimental fulminant hepatic failure by regulating the TLR4/mitogen‐activated protein kinase and TLR4/nuclear factor‐κB pathways and autophagy

Tectorigenin has received attention due to its antiproliferation, anti‐inflammatory, and antioxidant activities. In this study, we investigated the effects of tectorigenin on lipopolysaccharide (LPS)/D‐galactosamine(D‐GalN)‐induced fulminant hepatic failure (FHF) in mice and LPS‐stimulated macrophages (RAW 264.7 cells). Pretreatment with tectorigenin significantly reduced the serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), histological injury, apoptosis, and the mortality of FHF mice, by suppressing the production of inflammatory cytokines such as TNF‐α and IL‐6. Tectorigenin also suppressed the activation of the inflammatory response in LPS‐stimulated RAW 264.7 cells. Tectorigenin‐induced protection is mediated through its mitigation of TLR4 expression, inhibition of mitogen‐activated protein kinase (MAPK) and nuclear factor‐κB (NF‐κB) pathway activation, and promotion of autophagy in FHF mice and LPS‐stimulated RAW 264.7 cells. Therefore, tectorigenin has therapeutic potential for FHF in mice via the regulation of TLR4/MAPK and TLR4/NF‐κB pathways and autophagy.     

Autophagy Activation and Antiviral Activity by a Licorice Triterpene

The triterpene glycyrrhizic acid (GRA), the main product from the Glycyrrhiza glabra medicinal plant, is known for its antiinflammatory and antimicrobial activity. In this work, GRA was studied for its ability to induce the autophagic process activator Beclin 1 in epithelial cells and to observe how this property could influence its antiviral activity. After 24 h of treatment, GRA induced a Beclin 1 production that was more than twofold higher than that produced by rapamycin, used as a reference compound. When the compounds were added to HeLa cells together with the viruses, GRA demonstrated a strong antiherpes simplex virus type 1 (HSV1) activity, whereas rapamycin had no activity. However, if the compounds were added to the cells 24 h before the viruses, GRA induced the production of an even higher amount of Beclin 1 and showed an improved antiviral effect; under these conditions, rapamycin was also able to exert a significant anti‐HSV1 activity. In conclusion, GRA is a strong inducer of the autophagy activator Beclin 1, which establishes a resistance state to HSV1 replication. Copyright © 2014 John Wiley & Sons, Ltd.     

褐藻素诱导肝癌HepG2细胞凋亡和自噬的机制

目的：通过c-Jun氨基末端激酶（c-jun N-terminal kinase,JNK）信号通路来研究蹄叶橐吾醇化乙酸乙酯萃取物的抗炎作用机制。方法：采用噻唑蓝比色法（MTT法）测定倍比稀释的蹄叶橐吾醇化乙酸乙酯萃取物（浓度区间为0~640 mg·L^-1）作用于密度为5×10⁴个/mL的RAW264.7细胞24 h后的细胞活力,计算出药物对细胞的无毒浓度;测定JNK抑制剂SP600125（终浓度分别为25,12.5,6.25 μmol·L^-1）作用于细胞密度为5×10⁴个/mL的RAW264.7细胞24 h后的细胞活力,计算出SP600125作用的安全有效浓度;测定蹄叶橐吾醇化乙酸乙酯萃取物（终质量浓度分别为5,2.5,1 mg·L^-1）作用于LPS诱导细胞密度为5×10⁴个/mL的RAW264.7细胞12,24,36,48 h后的细胞活力,计算出药物抑制细胞增殖的浓度。采用蛋白质印迹（Western-blot）法测定蹄叶橐吾醇化乙酸乙酯萃取物（5 mg·L^-1）作用于LPS诱导的RAW264.7细胞24 h后p-JNK（磷酸化c-Jun氨基末端激酶）蛋白、JNK蛋白和环氧化酶-2（COX-2）蛋白的表达变化。结果：蹄叶橐吾醇化乙酸乙酯萃取物作用于LPS诱导的RAW264.7细胞,测定p-JNK,JNK,COX-2相对灰度值分别为：0.12±0.03,0.48±0.03,0.18±0.04,无药物作用的LPS诱导的RAW264.7细胞,测定p-JNK,JNK,COX-2,相对灰度值分别为：0.68±0.05,0.83±0.04,0.58±0.04,两组数据比较具有明显差异（P<0.01）。蹄叶橐吾醇化乙酸乙酯萃取物抑制LPS诱导的RAW264.7细胞增殖,下调p-JNK,JNK,COX-2等炎性蛋白的表达。结论：蹄叶橐吾醇化乙酸乙酯萃取物抗炎机制通过JNK信号通路来完成。     

Genistein protects against amyloid‐beta‐induced toxicity in SH‐SY5Y cells by regulation of Akt and Tau phosphorylation

Alzheimer's disease is a neurodegenerative disorder characterized by extracellular deposition of amyloid‐β (Aβ) peptide and hyperphosphorylation of Tau protein, which ultimately leads to the formation of intracellular neurofibrillary tangles and cell death. Increasing evidence indicates that genistein, a soy isoflavone, has neuroprotective effects against Aβ‐induced toxicity. However, the molecular mechanisms involved in its neuroprotection are not well understood. In this study, we have established a neuronal damage model using retinoic‐acid differentiated SH‐SY5Y cells treated with different concentrations of Aβ_25–35 to investigate the effect of genistein against Aβ‐induced cell death and the possible involvement of protein kinase B (PKB, also termed Akt), glycogen synthase kinase 3β (GSK‐3β), and Tau as an underlying mechanism to this neuroprotection. Differentiated SH‐SY5Y cells were pre‐treated for 24 hr with genistein (1 and 10 nM) and exposed to Aβ_25–35 (25 μM), and we found that genistein partially inhibited Aβ induced cell death, primarily apoptosis. Furthermore, the protective effect of genistein was associated with the inhibition of Aβ‐induced Akt inactivation and Tau hyperphosphorylation. These findings reinforce the neuroprotective effects of genistein against Aβ toxicity and provide evidence that its mechanism may involve regulation of Akt and Tau proteins.     

Cordyceps sobolifera extract ameliorates lipopolysaccharide-induced renal dysfunction in the rat

Cordyceps Sobolifera (CS), an economic traditional Chinese herb, may ameliorate nephrotoxicity-induced renal dysfunction in the rat via antioxidant, anti-apoptosis, and anti-autophagy mechanisms. We investigated the water extract of fermented whole broth of CS on lipopolysaccharide (LPS)-induced renal cell injury in vitro and in vivo. CS effect on LPS-induced epithelial Lilly pork kidney (PK1) and Madin-Darby canine kidney epithelial (MDCK) cell death was detected with MTT assay. Two-month treatment of CS effects on renal blood flow (RBF), glomerular filtration rate (GFR), plasma blood urea nitrogen, creatinine level and leukocytes (WBC) count were determined in the LPS-treated rats. We further examined the effects of CS supplement on renal tubular oxidative stress, endoplasmic reticulum stress, apoptosis and autophagy by Western blot analysis. LPS dose-dependently induced PK1 and MDCK cell death, which can be ameliorated by CS treatment. LPS significantly decreased RBF and GFR and increased blood leukocyte counts, plasma blood urea nitrogen and creatinine level in the rat after 24 hours of injury. LPS enhanced renal tubular ER stress, autophagy and apoptosis via by increase protein expressions of GRP78, caspase 12, Beclin-1 and Bax/Bcl-2 ratio. These findings are associated with the significant staining in renal proximal and distal tubular ED-1, GRP78, Beclin-1 autophagy, and TUNEL apoptosis in the LPS-treated kidneys. Two months of CS supplement significantly improved RBF, GFR and WBC values and reduced ED-1, GRP78, Beclin-1 autophagy and TUNEL apoptosis in the LPS-treated kidneys. Long-term CS treatment reduced LPS-induced stress responses and tissue damage possibly via blocking LPS-triggered signaling pathways.     

Baicalein Triggers Autophagy and Inhibits the Protein Kinase B/Mammalian Target of Rapamycin Pathway in Hepatocellular Carcinoma HepG2 Cells

Baicalein (BA), isolated from the Chinese medicinal herb Scutellariae radix (Huangqin in Chinese), is a flavonoid with various pharmacological effects. Herein, we found that BA only slightly reduced the cell viability on HepG2 cells after 24‐h treatment as determined by 3‐(4, 5‐dimethylthiazol‐2‐yl)‐2, 5‐diphenyl tetrazolium bromide (MTT) assay. However, BA (50 μM) effectively blocked the colony formation. Meanwhile, BA remarkably induced the formation of autophagosomes after 24‐h treatment as determined by immunofluorescence with monodansylcadaverine staining as well as transmission electron microscopy, respectively. Moreover, BA obviously up‐regulated the expression of microtubule‐associated protein 1A/1B‐light chain 3‐II in concentration‐dependent and time‐dependent manners in HepG2 cells. When combined with the autophagy inhibitor chloroquine and BA, the cell viability and colony formation were significantly decreased, indicating that BA triggered protective autophagy, which prevented cell death. Further study showed that BA concentration‐dependently and time‐dependently decreased the expression of p‐AKT (S473), p‐ULK1 (S757) and p‐4EBP1 (T37 and S65), suggesting the involvement of protein kinase B (AKT)/mammalian target of rapamycin (mTOR) in BA‐triggered autophagy. Copyright © 2015 John Wiley & Sons, Ltd.     

Ethanol Extract of Magnolia officinalis Prevents Lipopolysaccharide‐Induced Memory Deficiency via Its Antineuroinflammatory and Antiamyloidogenic Effects

Magnolia bark contains several compounds such as magnolol, honokiol, 4‐O‐methylhonokiol, obovatol, and other neolignan compounds. These compounds have been reported to have various beneficial effects in various diseases. There is sufficient possibility that ethanol extract of Magnolia officinalis is more effective in amyloidogenesis via synergism of these ingredients. Neuroinflammation has been known to play a critical role in the pathogenesis of Alzheimer's disease (AD). We investigated whether the ethanol extract of M. officinalis (10 mg/ kg in 0.05% ethanol) prevents memory dysfunction and amyloidogenesis in AD mouse model by intraperitoneal lipopolysaccharide (LPS, 250 µg/ kg/day for seven times) injection. We found that ethanol extract of M. officinalis prevented LPS‐induced memory deficiency as well as inhibited the LPS‐induced elevation of inflammatory proteins, such as inducible nitric oxide synthase and cyclooxygenase 2, and activation of astrocytes and microglia. In particular, administration of M. officinalis ethanol extract inhibited LPS‐induced amyloidogenesis, which resulted in the inhibition of amyloid precursor protein, beta‐site amyloid‐precursor‐protein‐cleaving enzyme 1 and C99. Thus, this study shows that ethanol extract of M. officinalis prevents LPS‐induced memory impairment as well as amyloidogenesis via inhibition of neuroinflammation and suggests that ethanol extract of M. officinalis might be a useful intervention for neuroinflammation‐associated diseases such as AD. Copyright © 2012 John Wiley & Sons, Ltd.     

化痰通络方对急性脑梗死大鼠rt-PA溶栓后神经细胞自噬途径中Beclin1与LC3蛋白表达的影响

[目的]观察化痰通络方对急性脑梗死大鼠重组组织纤溶酶原激活剂(rt-PA)溶栓后神经细胞自噬途径中自噬相关蛋白(Beclin1)及微管相关蛋白轻链3(LC3)蛋白表达的影响。[方法]选择160只健康SD大鼠,随机分为假手术组、模型组、rt-PA组、化痰通络方联合rt-PA组(简称中药组),每组采用6 h、1 d、3 d和7 d 4个时相。采用自身栓子法制备大鼠大脑中动脉栓塞模型(MCAO),rt-PA组与中药组分别给予尾静脉注入rt-PA(浓度为5.67 mg/kg)及联合化痰通络中药(浓度为7.2 g/kg)干预,每日2次。于相应时间点采用Western blot检测各组大鼠大脑皮质梗死区组织中Beclin1及LC3蛋白的表达。[结果]Western blot结果显示:与假手术组相比,模型组在4个时相中Beclin1和LC3蛋白的相对丰度值均明显增高(P0.05);采用rt-PA和rt-PA联合化痰通络法干预后,4个时相内Beclin1与LC3蛋白的相对丰度值均明显下降(P0.05);且rt-PA联合化痰通络组下降更加显著,与rt-PA组比较差异有统计学意义(P0.05)。[结论]化痰通络方可通过降低神经细胞自噬途径中Beclin1与LC3蛋白的表达,部分抑制神经细胞的过度自噬,进而保护神经细胞损伤,从而更好的防治急性脑梗死溶栓后缺血再灌注的发生与发展。     

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??OBJECTIVE To investigage the effects of celastrol-triggered HeLa cells autophagy and the molecular mechanisms in vitro and in vivo. METHODS The antiproliferative effect of celastrol was detected using MTT assay. Apoptotic rate and cell cycle were evaluated using flow cytometric analysis. Autophagy was detected using fluorescence microscope. Protein expression was evaluated using Western blotting. Tumor growth was evaluated by subcutaneous xenograft model in vivo. RESULTS Celastrol inhibited HeLa cells proliferation and induced HeLa cells autophagy and cell cycle arrest at G₀/G₁ phase, but not induced HeLa cell apoptosis in vitro. The protein expression of Beclin 1 was up-regulated and the conversion from LC3 ?? to LC3 ?? was increase in HeLa cells in vitro after treatment with celastrol. Moreover, celastrol promoted the protein expression of PTEN??p-ERK1/2??p-MEK1/2 and inhibited the phosphorylated of Akt, p70S6K and mTOR in HeLa cells. After pretreatment with 3-methyladenine (5 mmol??L^-1), the antiproliferative and induced-autophagy effects of celastrol were reversed. Furthermore, celastrol inhibited tumor growth and the protein expression of p-Akt and p-mTOR, but up-regulated the protein expression of LC3 ?? and Beclin 1 in vivo. CONCLUSION Antitumor effect of celastrol dependent on cells autophagy in HeLa cells via inhibition of Akt/mTOR signaling pathway in vitro and in vivo.     

Dihydroartemisinin,an antimalarial drug,induces absent in melanoma 2 inflammasome activation and autophagy in human hepatocellular carcinoma HepG2215 cells

As an effective antimalarial drug, Dihydroartemisinin (DHA) is readily isolated from the traditional Chinese medicine of Artemisia annua. DHA is not only an autophagy promoter but also a substance with strong antitumor efficiency. The relationship between autophagy and inflammasomes has been suggested in hepatocellular carcinoma (HCC). However, there are few reports describing relationships between inflammasomes and autophagy in HCC therapy. The present study demonstrated that DHA suppressed cell proliferation in HepG2215 cells in a dose‐ and time‐dependent manner. The inhibitory activity is mediated by autophagy, in which reactive oxygen species (ROS) production induced nuclear and mitochondrial DNA damage. Then, DHA were first shown to promote AIM2/caspase‐1 inflammasome. Compared with the DHA group, the autophagy inhibitor 3‐MA significantly inhibited the expressions of activated Caspase‐1, a pyroptotic marker proteins. Meanwhile, repression of mTOR by rapamycin promoted autophagy and AIM2/caspase‐1 activation. The caspase‐1 inhibitor Z‐YVAD‐FMK also notably blocked autophagy cell death characterized by the downexpression of Beclin‐1 and LC3‐II. Additionally, the study demonstrated that DHA suppressed pseudopodium formation and cell mobility. Therefore, we first reveal a novel mechanism that DHA promotes AIM2/caspase‐1 inflammasome, which contributes to autophagy in HepG2215 cells. Moreover, nuclear and mitochondrial DNA damage was also involved in this process via ROS production.     

Musclide-A1: A novel Ca2+-dependent protein kinase activator derived from musk and its cardiotonic potentiating action in guinea-pig cardiac muscles

Musclide-A1 (6-methyl-2, 5-heptanediol 5-(hydrogen sulfate)) was isolated from musk, a dried secretion from the preputial follicle of musk deer, Moschus moschiferus Linn. Musclide (100 μg/mL), a water-soluble extract, and musclide-A1 (30 μg/mL) potentiated β-adrenergic cardiotonic action in guinea-pig papillary muscles. Musclide-A1 at 130 μM or 1,2-dioctanoylglycerol at 200 μM potentiated the positive inotropic action induced by the maximal concentration of isoproterenol (258 nM). The potentiation was suppressed by 3 nM staurosporine or 1 μM H-7, an inhibitor of Ca²⁺-dependent protein kinase. Musclide-A1 at 4 and 8 μM activated protein kinase C and other Ca²⁺-dependent protein kinase in ventricular muscles of guinea-pig. The activation of Ca²⁺-dependent protein kinase was completely suppressed by 3 nM saturosporine. The activity of musclide-A1 (4 μM) was comparable to that of 1,2-dioctanoylglycerol (4 μM, the optimum concentration to stimulate protein kinase C), and were inhibited completely by 30 nM staurosporine. These results indicate that one mechanism of cardiotonic potentiation induced by musk, may be in part due to the activation of protein kinase C and other Ca²⁺-dependent protein kinases.