Quercetin activates mitochondrial autophagy via PINK1/parkin pathway to alleviate cerebral ischemia-reperfusion injury in rats

AIM To analyze the regulatory effect of quercetin （QUE） on PTEN-induced putative kinase 1 （PINK1）/parkin mitochondrial autophagy pathway, and to explore the mechanism of quercetin in relieving cerebral ischemia/reperfusion （I/R） injury. METHODS Sixty SD male rats were randomly divided into sham operation group, model group （I/R group）, QUE group,3-methyladenine （3-MA） group and QUE+3-MA group. Administration started in each group 3 days before modeling, once a day, at 30 min after the last administration,except sham group, the other groups used 4-vessel blockage method to establish the whole brain I/R model. On the day after modeling, the neural function was evaluated by neuropathy disability score （NDS）. The volume of cerebral infarction was measured by 2,3,5-triphenyltetrazolium chloride （TTC） staining. The morphological changes of mitochondria in hippocampus were observed by transmission electron microscopy. The contents of interleukin-6 （IL-6） and tumor necrosis factor-α （TNF-α） in hippocampus were measured by ELISA. The activity of superoxide dismutase （SOD） and contents of malondialdehyde （MDA） in hippocampus were detected by xanthine oxidase method, thiobarbituric acid condensation method. Western blot was used to detect the proteinex pression of PINK1, parkin and LC3-II in brain tissue. RESULTS Compared with sham group, the hippocampus of the rats in I/R group and QUE+3-MA group showed swelling of mitochondria, destruction or disappearance of internal crista and other pathological damage,also the volume of cerebral infarction, the contents of IL-6, TNF-α and MDA, the protein expression levels of PINK1, parkin and LC3-II were increased （P<0.05）, while NDS score and activity of SOD were decreased （P<0.05）. Compared with I/R group and QUE+3-MA group, the pathological damage degree of hippocampus in QUE group was reduced, the volume of cerebral infarction, the contents of IL-6, TNF-α and MDA were decreased （P<0.05）, the proteinexpression levels of PINK1, parkin and LC3-II, and NDS score and activity of SOD were increased （P<0.05）.The above indexes in 3-MA group were opposite to QUE group. No significant difference in the above indexes between I/R group and QUE+3-MA group was observed （P>0.05）. CONCLUSION Quercetin activates mitochondrial autophagy and reduces cerebral I/R by regulating the expression of PINK1/parkin pathway proteins.     

Mechanism of quercetin improving rat coronary artery myogenic response under high glucose

AIM: To investigate the mechanism of quercetin improving rat coronary artery myogenic response under high glucose (HG) by measuring muscle tension of coronary arterial ring and recording voltage-gated K⁺ channel (Kv) current of coronary artery smooth muscle cells by whole cell patch clamp. METHODS: The coronary rings from the normal SD rats were acutely isolated, and then divided into 6 groups: (1) control group; (2) HG group; (3) HG+low dose (3 μmol/L) of quercetin group; (4) HG+moderate dose (10 μmol/L) of quercetin group; (5) HG+high dose (30 μmol/L) of quercetin group; (6) HG+C6303 (PKC inhibitor)+high dose of quercetin group. Determinations of coronary artery response to vasoconstrictor (60 mmol/L KCl or 0.1 mmol/L U46619) or vasodilator (ACh at 10^-9~10^-5 mol/L) were performed, and the percentage of coronary ring tension was calculated using the contraction as 100% caused by 60 mmol/L KCl. The rat coronary artery smooth muscle cells were acutely isolated for recording the Kv current using whole cell patch clamp. RESULTS: Compared with control group, the contraction amplitudes to 60 mmol/L KCl or 0.1 mmol/L U46619 were significantly increased under HG incubation. Quercetin intervention concentration-dependently reduced the coronary artery contraction amplitude. Incubation of PKC specific inhibitor C6303 attenuated the effect of quercetin. Compared with control group, the diastolic amplitude to ACh decreased significantly in HG group, and quercetin intervention concentration-dependently increased the coronary artery diastolic amplitude. Incubation of PKC specific inhibitor C6303 attenuated the effect of quercetin. Compared with control group, HG incubation inhibited Kv current of coronary artery vascular smooth muscle cells significantly, and quercetin intervention attenuated the inhibitory effect of HG on Kv current intensity. Incubation of PKC specific inhibitor C6303 attenuated the effect of quercetin. CONCLUSION: Quercetin has a protective effect on myogenic response of coronary artery under HG and the effects is related to the increase in Kv current and the activation of PKC in vascular smooth muscle cells.     

金槐米槲皮素对人鼻咽癌细胞的干预作用研究

[目的]探讨金槐米槲皮素对人鼻咽癌CNE2细胞的干预作用。[方法]通过微波辅助提取法从桂北金槐米中提取槲皮素并纯化,用浓度20、40、60和80μmol/L的槲皮素作用于人鼻咽癌细胞CNE2;分别在24、48和72 h后,采用MTT法初步检测人鼻咽癌细胞被抑制的情况,用流式细胞术分析其细胞周期和凋亡率、光学显微镜观察细胞形态学变化。[结果]从槐米中提取的槲皮素对鼻咽癌细胞有明显的抑制作用,且呈浓度、时间明显的依赖性,药物干预作用的最适浓度为60μmol/L,药物干预后形态学观察的最适宜时间为48 h。细胞周期分析显示,槲皮素能阻滞鼻咽癌细胞于G1期。[结论]从桂北金槐米中提取的槲皮素对人鼻咽癌细胞有较强的干预作用。     

In vitro suppression of quercetin on hypertrophy and extracellular matrix accumulation in rat glomerular mesangial cells cultured by high glucose

Quercetin's protective effects on the glomerulosclerosis of diabetic nephropathy (DN) in rat mesangial cells were investigated. The cell cycles, type IV collagen and laminin, TGF-β₁ mRNA, Smad 2/3 and Smad 7, and activities of cell antioxidases were measured. Compared with the high glucose group, quercetin may decrease the cell percentages of G₀/G₁ phase, Smad 2/3 expression, laminin and type IV collagen, and TGF-β₁ mRNA level significantly. The antioxidant capacity, the cell percentages of S phase and Smad 7 expression was significantly increased by quercetin. These results suggest that quercetin is a protective agent against glomerulosclerosis in DN.     

植物雌激素槲皮素对非洲爪蟾生长和骨骼发育的影响

用两栖动物非洲爪蟾作模型动物,来揭示植物雌激素槲皮素对非洲爪蟾生长和骨骼发育的影响。非洲爪蟾从46/47期开始,分别暴露50μg/L,100μg/L,200μg/L槲皮素,直至变态后1个月。结果表明槲皮素处理一方面促进蝌蚪体重的增加,另一方面导致尾的畸形。200μg/L槲皮素还导致脊柱的畸形,表现为少一块脊椎骨。     

Subacute chlorpyrifos-induced oxidative stress in rat erythrocytes and the protective effects of catechin and quercetin

This study examined the effects of chlorpyrifos in the rat erythrocyte antioxidant system and evaluated the ameliorating effects of catechin and quercetin on the oxidative damage induced by chlorpyrifos. Sexually mature male Wistar rats were given chlorpyrifos (5.4 mg/kg, 1/25 of the oral LD₅₀), catechin (20 mg/kg), quercetin (20 mg/kg), catechin plus chlorpyrifos, and quercetin plus chlorpyrifos daily via gavage for four weeks. No statistical differences were found in the catechin-only and quercetin-only groups compared with the control group. By the end of the fourth week, chlorpyrifos alone increased the levels of malondialdehyde (MDA) and decreased superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) activities compared with the control group in rat erythrocytes. In the catechin-plus-chlorpyrifos and quercetin-plus-chlorpyrifos groups, there were statistically significantly decreased MDA levels and increased SOD, CAT, and GPx activities compared with the chlorpyrifos-only group. Thus, it appears that catechin and quercetin ameliorate chlorpyrifos-induced oxidative stress in rat erythrocytes in vivo.     

Protective effect of quercetin on ER stress-related apoptosis induced by thapsigargin in macrophages

AIM: To investigate the effects and possible mechanisms of quercetin (Que) on endoplasmic reti-culum stress (ERS)-related apoptosis induced by thapsigargin (TG) in RAW264.7 cells. METHODS: ER stress of RAW264.7 cells were induced by TG at concentration of 1 μmol/L for 24 h. After treated with different concentrations of Que (80, 120 and 160 μmol/L), the cell viability was determined by MTT assay.The apoptotic rate and the changes of intracellular Ca²⁺ concentration ([Ca²⁺]_i) were determined by flow cytometry, and the cell apoptotic morphology was observed under laser scanning confocal microscope.The protein levels of glucose-regulated protein 78 (GRP78) and C/EBP homologous protein (CHOP) were detected by Western blotting. The effect of Que on GRP78 and CHOP induced by TG with phosphatidylinositol 3-kinase (PI3K) inihibitor LY294002 at concentration of 15 nmol/L was measured by Western blotting. RESULTS: Que suppressed ER stress-related injury induced by TG in RAW264.7 cells. Compared with TG group, the cell viability increased (P<0.05), apoptotic rate and [Ca²⁺]_i decreased (P<0.05) and the changes of apoptotic morphology were alleviated. The increase in GRP78 and CHOP induced by TG as an ER stress marker was suppressed by Que (P<0.05). The suppressive effect of Que on GRP78 and CHOP was reproduced by LY294002 (P<0.05), but they failed to exhibit additive suppression. CONCLUSION: Que suppresses the ER stress induced by TG in RAW264.7 cells. The protective effect may be related to its suppression on PI3K signaling pathway.     

桑叶中黄酮化合物的提取工艺及不同品种不同时期的含量变化

探讨了不同单因子对桑叶中黄酮成分浸提效果的影响。用80%乙醇作溶剂,原料质量浓度50.00 g/L,于70℃、pH 8的条件下浸提3 h并抽提2次的提取效果较好。以毛细管电泳仪测定、分析了不同品种、不同时期桑叶中的芦丁、槲皮素等成分,湖桑9001叶片中的芦丁含量高,湖桑32号的槲皮素含量高,10月份采摘桑叶中的芦丁和槲皮素的含量最高。芦丁、槲皮素等成分在桑叶中的动态变化:以10 mmol/L磷酸二氢钠-20 mmol/L的硼砂溶液(pH 8.62)作缓冲液,在25℃,20 kV的压力下电泳,245 nm波长处检测,线性关系良好,在8 m in内完全分离。     

月月红中槲皮素·山奈酚的定性与定量研究

[目的]通过对月月红中槲皮素、山奈酚的定性定量研究,拟建立其HPLC分析方法,为进一步研究民族民间药物月月红的药效学奠定基础。[方法]采用化学定性法确定月月红不同部位中的主要化学组分;色谱条件：Hypersil DJZ C18色谱柱（4.6 mm×200.0 mm,5μm）;流动相：甲醇-0.4%磷酸（60∶40,V/V）;检测波长：368 nm;流速：1.000 ml/min;柱温：30℃;理论塔板数按山奈酚和槲皮素峰计算不低于3 000,外标法计算。[结果]槲皮素在0.081 6~0.489 6μg线性关系良好（R2=0.999 6）,平均回收率为101.6%,RSD为0.87%;山奈酚在0.069 6~0.417 6μg线性关系良好（R2=0.999 6）,平均回收率为96.8%,RSD为1.01%。月月红叶中槲皮素为0.038 2%,茎为0.005 2%,全株为0.010 5%;月月红叶中山奈酚含量为0.027 6%,茎为0.002 1%,全株为0.009 6%。[结论]HPLC分析方法稳定、重复性较好;月月红不同部位均含有槲皮素、山奈酚,且叶含量高于全株和茎。     

金樱子中芦丁和槲皮素不同提取方法的比较研究

[目的]研究不同提取方法对金樱子（Rosa laevigata Michx）果实芦丁和槲皮素提取的影响,为金樱子（FRUCTUS ROSAE LAEVI-GATAE）药材的合理开发利用提供科学依据。[方法]采用热水浸提、乙醇回流和乙醇超声波辅助法提取金樱子果实中芦丁和槲皮素,用HPLC法测定含量,并进行方法学考察。[结果]HPLC法测定中芦丁和槲皮素能较好地分离,分别在0.03～15μg/ml（r=0.999 0）和0.04～20μg/ml（r=0.999 4）的浓度范围内与峰面积成良好线性关系,检测限分别为12.6和15.2 ng/ml。3种提取方法得到的芦丁和槲皮素趋势为乙醇超声波辅助法﹥乙醇回流﹥热水浸提,分别为芦丁0.62、1.04和1.52μg/ml;槲皮素0.78、1.18和2.24μg/ml。[结论]乙醇-超声波提取法的效果较好。