Dayun Sui

20(S)-Protopanaxadiol Induces Human Breast Cancer MCF-7 Apoptosis through a Caspase-Mediated Pathway

20(S)-Protopanaxadiol (PPD), a ginsenoside isolated from Pananx quinquefolium L., has been shown to inhibit growth and proliferation in several cancer cell lines. The aim of this study was to evaluate its anticancer activity in human breast cancer cells. MCF-7 cells were incubated with different concentrations of 20(S)-PPD and cytotoxicity was evaluated by MTT assay. Occurrence of apoptosis was detected by DAPI and Annexin V-FITC/PI double staining. Mitochondrial membrane potential was measured with Rhodamine 123. The Bcl-2 and Bax expression were determined by Western blot analysis. Caspase activity was measured by colorimetric assay. 20(S)-PPD dose-dependently inhibited cell proliferation in MCF-7 cells, with an IC50 value of 33.3 μM at 24h. MCF-7 cells treated with 20(S)-PPD presented typical apoptosis, as observed by morphological analysis in cell stained with DAPI. The percentages of annexin V-FITC positive cells were 8.92%, 17.8%, 24.5% and 30.5% in MCF-7 cells treated with 0, 15, 30 and 60μM of 20(S)-PPD, respectively. Moreover, 20(S)-PPD could induce mitochondrial membrane potential loss, up-regulate Bax expression and down-regulate Bcl-2 expression. These events paralleled activation of caspase-9, -3 and PARP cleavage. Apoptosis induced by 20(S)-PPD was blocked by z-VAD-fmk, a pan-caspase inhibitor, suggesting induction of caspase-mediated apoptotic cell death. In conclusion, the 20(S)-PPD investigated is able to inhibit cell proliferation and to induce cancer cell death by a caspase-mediated apoptosis pathway.

Protective effect of Panax quinquefolium 20(S)‑protopanaxadiol saponins, isolated from Pana quinquefolium, on permanent focal cerebral ischemic injury in rats

Oxidative stress is significant in the pathogenesis of cerebral ischemia. Panax quinquefolium 20(S)-protopanaxadiol saponins (PQDS) have been demonstrated to exhibit a variety of biological effects in the cardiovascular system as a result of their antioxidant properties. However, little is known regarding the effect of PQDS on cerebral ischemia. The purpose of this study was to investigate whether PQDS exhibited protective effects against cerebral ischemia. A model of cerebral ischemia was induced by middle cerebral artery occlusion (MCAO) in Sprague-Dawley rats. Adult male rats were randomly divided into five groups: Sham, MCAO and PQDS treatment groups at doses of 12.5, 25.0 and 50.0 mg/kg. The effects of PQDS on neurological deficits, cerebral infarct area, brain water content, and the malondialdehyde (MDA) and Ca2+ levels and Na+-K+-ATPase and superoxide dismutase (SOD) activities in the brain tissue were analyzed, and the nitric oxide (NO) content and nitric oxide synthase (NOS) activity in the serum were evaluated. Moreover, the expression of Bcl-2 was analyzed using western blotting. Pretreatment with PQDS (25.0 and 50.0 mg/kg) significantly reduced the neurological deficit score, decreased the infarcted area and decreased the brain water content from 83.09 to 80.27% (P<0.05). In addition, PQDS pretreatment decreased the NOS activity and the NO levels in the serum compared with those in the MCAO group. Furthermore, pretreatment with PQDS (25.0 and 50.0 mg/kg) significantly increased the activities of SOD and Na+-K+-ATPase and decreased the levels of Ca2+ and MDA in the brain tissue (P<0.05) compared with those in the MCAO group. Pretreatment with PQDS (25.0 and 50.0 mg/kg) also increased the protein expression level of Bcl-2 compared with that in the MCAO group. The histopathological results demonstrated the protective effect of PQDS on ischemic injury. The results indicated that PQDS has protective effects against ischemic injury in rats. The mechanism may be associated with the inhibition of oxidative stress and apoptosis.

Metformin ameliorates diabetic nephropathy in a rat model of low-dose streptozotocin-induced diabetes

The present study aimed to explore the renoprotective effect of metformin on diabetic nephropathy in type 2 diabetic rats. A rat model of type 2 diabetic nephropathy (T2DN) was successfully induced via a high-fat diet combined with a single low-dose of streptozotocin. Metformin was administered intragastrically for 13 weeks, and fasting blood glucose (FBG), total cholesterol (TC), triglycerides (TG), HDL-c, LDL-c, urinary and serum creatinine levels were subsequently examined at the end of administration. Renal function was determined after the treatment protocol. Expression levels of transforming growth factor (TGF)-β1 and connective tissue growth factor (CTGF) were assessed via immunohistochemical analysis. Superoxide dismutase activity, malondialdehyde content and glutathione peroxidase levels were assessed in kidney tissues using commercially available kits. The results of the present study demonstrated that metformin administration significantly decreased the levels of serum blood urea nitrogen, serum creatinine, creatinine clearance, urinary albumin excretion and fasting blood glucose in rats with T2DN. Furthermore, TG, TC and LDL-c levels were significantly decreased following metformin treatment, whereas HDL-c was increased. Metformin treatment significantly increased SOD activity and significantly decreased malondialdehyde levels, as compared with the model group. It was also demonstrated that metformin administration significantly decreased the expression levels of TGF-β1 and attenuated the morphological changes associated with T2DN in rats. These data clearly demonstrated the renoprotective effects of metformin against the development and progression of T2DN in rats. The underlying mechanism of this protective effect may be associated with glycemic control, lipid metabolism, and anti-oxidative and anti-inflammatory functions.

Neuroprotective effects of Kaempferide-7-O-(4″-O-acetylrhamnosyl)-3-O-rutinoside on cerebral ischemia-reperfusion injury in rats

In the present study, we aim to evaluate the potential neuroprotective effect and the underlying mechanism of Kaempferide-7-O-(4″-O-acetylrhamnosyl)-3-O-rutinoside (A-F-B) against cerebral I/R injury. Adult male rats were pretreated with A-F-B by intragastric administration once a day for 3 days. One hour after the third day administration, animals were subjected to 2h of transient middle cerebral artery occlusion (MCAO) followed by 24h of reperfusion. Neurological deficit, infarct volume, histopathological changes, oxidative stress-related biochemical parameters, neuronal apoptosis, apoptosis-related proteins and the expression of pro-inflammator cytokines genes were measured. A-F-B significantly decreased neurological and histological deficits, reduced the infarct volume, and decreased neuroapoptosis. Meanwhile, A-F-B inhibited the expression of Bax, cleaved caspase-3, cleaved caspase-9, and promoted Bcl-2 expression. In addition, the expression of pro-inflammator cytokines, including phospho-NF-kBp65, interleukin-1β, interleukin-6, tumor necrosis factor-α, intercellular adhesion molecule-1, cyclooxygenase-2 and inducible nitric oxide synthase, were also suppressed by A-F-B pretreatment. Furthermore, pretreatment with A-F-B could significantly increase the activities of superoxide dismutase, glutathione peroxidase, but decrease the content of malondiadehyde in blood serum. These results suggest that A-F-B has the neuroprotective effect in ischemic stroke by suppressing neuroinflammation, reactive oxygen species and neuroapoptosis.

Ginsenoside Rg3 induces ginsenoside Rb1-comparable cardioprotective effects independent of reducing blood pressure in spontaneously hypertensive rats

Ginsenoside Rg3 (Rg3) is a rare type of ginsenoside used as an anti-tumor medicine in China. Ginsenoside Rb1 (Rb1), which exhibits protective effects on the cardiovascular system, is similar to Rg3 in chemical structure. In the present study, Rb1 and Rg3 were administered for 6 weeks to spontaneously hypertensive rats (SHR) and their cardioprotective effects were assessed. According to echocardiography and histopathological examinations, the decrease in cardiac function and ventricular remodeling that occurred in SHR rats were attenuated by Rb1 and Rg3. However, tail-cuff blood pressure measurements indicated that Rb1 and Rg3 did not reduce blood pressure in SHR rats. The cardioprotective effects of Rb1 and Rg3 occurred independently of blood pressure reduction. Furthermore, immunohistochemistry (IHC) revealed that renin angiotensin system (RAS) activity in the myocardium of SHR was significantly attenuated by Rb1 and Rg3, whereas ELISA identified no significant changes of RAS activity in the serum. The results of IHC and reverse transcription-quantitative polymerase chain reaction demonstrated that levels of transforming growth factor β1, tumor necrosis factor-α, interleukin-6, interleukin-1 and endothelian-1 in the myocardium of SHR rats were reduced following Rb1 and Rg3 treatment. This may be due to the attenuation of RAS activity in the myocardium and the mechanisms of the cardioprotective effects of Rb1 and Rg3.

20(S)-Protopanaxadiol-Induced Apoptosis in MCF-7 Breast Cancer Cell Line through the Inhibition of PI3K/AKT/mTOR Signaling Pathway

20(S)-Protopanaxadiol (PPD) is one of the major active metabolites of ginseng. It has been reported that 20(S)-PPD shows a broad spectrum of antitumor effects. Our research study aims were to investigate whether apoptosis of human breast cancer MCF-7 cells could be induced by 20(S)-PPD by targeting the Phosphatidylinositol 3-kinase/Protein kinase B/Mammalian target of rapamycin (PI3K/AKT/mTOR) signal pathway in vitro and in vivo. Cell cycle analysis was performed by Propidium Iodide (PI) staining. To overexpress and knock down the expression of mTOR, pcDNA3.1-mTOR and mTOR small interfering RNA (siRNA) transient transfection assays were used, respectively. Cell viability and apoptosis were evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)-test and Annexin V /PI double-staining after transfection. The antitumor effect in vivo was determined by the nude mice xenograft assay. After 24 h of incubation, treatment with 20(S)-PPD could upregulate phosphorylated-Phosphatase and tensin homologue deleted on chromosome 10 (p-PTEN) expression and downregulate PI3K/AKT/mTOR-pathway protein expression. Moreover, G0/G1 cell cycle arrest in MCF-7 cells could be induced by 20(S)-PPD treatment at high concentrations. Furthermore, overexpression or knockdown of mTOR could inhibit or promote the apoptotic effects of 20(S)-PPD. In addition, tumor volumes were partially reduced by 20(S)-PPD at 100 mg/kg in a MCF-7 xenograft model. Immunohistochemical staining indicated a close relationship between the inhibition of tumor growth and the PI3K/AKT/mTOR signal pathway. PI3K/AKT/mTOR pathway-mediated apoptosis may be one of the potential mechanisms of 20(S)-PPD treatment.

20(S)-Ginsenoside Rg2 attenuates myocardial ischemia/reperfusion injury by reducing oxidative stress and inflammation: role of SIRT1

Previously we demonstrated that 20(S)-ginsenoside Rg2 protects cardiomyocytes from H2O2-induced injury by inhibiting reactive oxygen species (ROS) production, increasing intracellular levels of antioxidants and attenuating apoptosis. We explored the protective effect of 20(S)-ginsenoside Rg2 on myocardial ischemia/reperfusion (MI/R) injury and to clarify its potential mechanism of action. Rats were exposed to 20(S)-ginsenoside Rg2 in the presence/absence of the silent information regulator SIRT(1) inhibitor EX527 and then subjected to MI/R. 20(S)-Ginsenoside Rg2 conferred a cardioprotective effect by improving post-ischemic cardiac function, decreasing infarct size, reducing the apoptotic index, diminishing expression of creatine kinase-MB, aspartate aminotransferase and lactate dehydrogenase in serum, upregulating expression of SIRT1, B-cell lymphoma-2, procaspase-3 and procaspase-9, and downregulating expression of Bax and acetyl (Ac)-p53. Pretreatment with 20(S)-ginsenoside Rg2 also resulted in reduced myocardial superoxide generation, gp91phox expression, malondialdehyde content, cardiac pro-inflammatory markers and increased myocardial activities of superoxide dismutase, catalase and glutathione peroxidase. These results suggested that MI/R-induced oxidative stress and inflammation were attenuated significantly by 20(S)-ginsenoside Rg2. However, these protective effects were blocked by EX527, indicating that SIRT1 signaling may be involved in the pharmacological action of 20(S)-ginsenoside Rg2. Our results demonstrated that 20(S)-ginsenoside Rg2 attenuates MI/R injury by reducing oxidative stress and inflammatory responses via SIRT1 signaling.

Neuroprotective effects of pramipexole transdermal patch in the MPTP-induced mouse model of Parkinson's disease

Parkinsons disease (PD) is characterized by the selective death of dopaminergic neurons. To avoid inconvenience of frequent administration caused by short half life and recurrence of symptoms such as tremor and bradykinesia incurred by drug elimination, a novel long-acting pramipexole transdermal patch has been made. In the present study, we evaluated the neuroprotective effects and underlying mechanisms of pramipexole patch (PPX patch) in a subacute PD mouse model induced by 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). The results showed that PPX patch treatment improved dyskinesia. MPTP-induced reduction of DA as well as its metabolites DOPAC and HVA in the striatum were prevented by PPX patch in a dose-dependent manner. PPX patch also restored the activity of antioxidant enzymes including SOD, GSH-Px and CAT in the striatum while reduced the content of MDA. Furthermore, PPX patch upregulated Nrf2/HO-1 expression. The protective effects of PPX patch was also associated with downregulation of the Bax/Bcl-2 ratio and Apaf-1, inhibition of cytochrome c release and inactivation of caspase-9 and caspase-3. In conclusion, our studies demonstrated that the long-acting pramipexole patch exerts its neuroprotective effects, at least in part, by inhibiting oxidative stress and mitochondrial apoptosis pathway and holds promise as a candidate drug.

Pseudo‑Ginsenoside Rh2 induces A549 cells apoptosis via the Ras/Raf/ERK/p53 pathway

Ginsenoside Rh2, a major effective constituent of ginseng, has been suggested to have a pro-apoptotic effect in a variety of cancer cells. Pseudo-Ginsenside-Rh2 (pseudo-G-Rh2) is a novel derivative of ginsenoside Rh2. The aim of the present study was to evaluate the effect of pseudo-G-Rh2 on the apoptosis of lung adenocarcinoma A549 cells. The cytotoxicity of pseudo-G-Rh2 on A549 cells was evaluated using an MTT assay. Apoptosis was detected using DAPI staining and flow cytometry. The expression of apoptosis associated proteins was identified by western blot analysis. The results demonstrated that pseudo-G-Rh2 inhibits the proliferation of A549 cells in a dose-dependent manner. DAPI staining revealed topical morphological changes in apoptotic bodies following pseudo-G-Rh2 treatment. Flow cytometric analysis revealed that the percentage of Annexin V-fluorescein isothiocyanate-positive cells, which are apoptotic, increased with pseudo-G-Rh2 treatment in a dose-dependent manner. Furthermore, treatment with pseudo-G-Rh2 increased the level of reactive oxygen species in A549 cells as well as the activation of caspase-9, caspase-3 and poly ADP-ribose polymerase. Pseudo-G-Rh2 treatment was observed to induce mitochondrial membrane potential loss. Furthermore, the results of western blotting revealed that B-cell lymphoma 2 (Bcl-2) expression was significantly decreased while Bcl-2-associated X protein expression was significantly upregulated in A549 cells with pseudo-G-Rh2 treatment. Pseudo-G-Rh2-induced apoptosis was accompanied by sustained phosphorylation of Ras, Raf, extracellular signal-regulated kinase (ERK) and p53. In conclusion, the results of the present study suggest that pseudo-G-Rh2 induces mitochondrial apoptosis in A549 cells and is responsible for excessive activation of the Ras/Raf/ERK/p53 pathway.

20(S)-Protopanaxadiol induces apoptosis in human hepatoblastoma HepG2 cells by downregulating the protein kinase B signaling pathway

Hepatoblastoma is the most common primary liver tumor for children aged <5 years old. 20(S)-Protopanaxadiol (PPD) is a ginsenoside extracted from Pananx quinquefolium L., which inhibits tumor growth in several cancer cell lines. The purpose of the present study was to assess the anticancer activities of 20(S)-PPD in human hepatoblastoma HepG2 cells. The cytotoxicity of 20(S)-PPD on HepG2 cells was evaluated using an MTT assay. Apoptosis was detected using DAPI staining and flow cytometry. The expression of apoptosis-associated proteins was identified by western blotting. The results demonstrated that 20(S)-PPD inhibited the viability of HepG2 cell in a dose and time-dependent manner. The IC50 values were 81.35, 73.5, 48.79 µM at 24, 48 and 72 h, respectively. Topical morphological changes of apoptotic body formation following 20(S)-PPD treatment were detected by DAPI staining. The percentage of Annexin V-fluoroscein isothyiocyanate positive cells were 3.73, 17.61, 23.44 and 65.43% in HepG2 cells treated with 0, 40, 50 and 60 µM of 20(S)-PPD, respectively. Furthermore, 20(S)-PPD upregulated the expression of Bax and downregulated the expression of Bcl-2 and also activated caspases-3 and −9, and Poly [ADP-ribose] polymerase cleavage. In addition, 20(S)-PPD inhibited the phosphorylation of protein kinase B (Akt; Ser473). The results indicate that 20(S)-PPD inhibits the viability of HepG2 cells and induces apoptosis in HepG2 cells by inhibiting the phosphoinositide-3-kinase/Akt pathway.