Silibinin protects rat pancreatic ß-cell through up-regulation of estrogen receptors' signaling against amylin- or Aß1-42 -induced reactive oxygen species/reactive nitrogen species generation.

Amylin and amyloid-ß (Aß) were found to induce reactive oxygen species (ROS) and reactive nitrogen species (RNS) in rat pancreatic ß-cell line, INS-1 cells, leading to cell death. In this study, we report on reciprocal relationship between the expression of estrogen receptors (ERs) α and ß (ERα and ERß) and generation of ROS/RNS in amylin/Aß1-42 -treated INS-1 cells. That is, pharmacological activation of ERs in INS-1 cells significantly decreases ROS/RNS generation, but blockage of ERs increases ROS/RNS generation. Silibinin is a natural polyphenolic flavonoid isolated from milk thistle with phytoestrogen activities, also known as silybin. Treatment with silibinin down-regulated ROS/RNS production induced by treatment with amylin/Aß1-42 in the cells. Silencing ERs expression with siRNAs targeting ERs showed that the protective effect of silibinin was markedly weakened, indicating that silibinin protection is largely attributed to activation of ERs' signaling. The binding of silibinin to ERs implies that the protective effect of silibinin on amylin/Aß1-42 -treated INS-1 cells owes to down-regulation of ROS/RNS through the activation of ERs phosphorylation. Amylin and Aß1-42 cotreatment enhanced furthermore ROS/RNS generation and cytotoxicity through further down-regulation of ERs phosphorylation, and this was reversed by silibinin. Silibinin also protects INS-1 cells from amylin and Aß1-42 cotreatment. These results indicate that protective effect of silibinin is mediated by enhancement of ERs phosphorylation that depresses ROS/RNS generation in amylin/Aß1-42 -treated INS-1 cells.

Sub-lethal ultraviolet B irradiation and Poly I:C treatment synergistically induced apoptosis of HaCaT cells through NF-κB pathway.

Ultraviolet B (UVB) irradiation exerts multiple effects on skin cells, inducing apoptosis, senescence and carcinogenesis. Toll-like receptor 3, a member of pattern recognition receptors, is reported to initiate inflammation by recognizing double-strand RNA (dsRNA) released from UVB-irradiated cells. It has not been studied, however, whether apoptosis induction in UVB irradiation is attributed to TLR3 activation. Here, we report on the pro-apoptotic role of TLR3 in UVB-irradiated epidermal cells. Poly I:C, an analogue of dsRNA that activates TLR3, was used in combination with sub-lethal UVB (4.8 mJ/cm2) irradiation for investigating the effects of TLR3 activation on human immortalized keratinocyte HaCaT cells. Although sub-lethal dose of either Poly I:C or UVB alone did not induce cell death, UVB-Poly I:C co-treatment synergistically induced cell death by activation of caspase-3 and cleavages of ICAD and PARP, with apoptotic features when stained with Annexin V/PI or Hoechst 33342. Treatment with pan-caspase inhibitor, Z-VAD, attenuated UVB-Poly I:C-induced cell death. Silencing TLR3 by siRNA rescued HaCaT cells from UVB-Poly I:C-induced apoptosis. NF-κB, a major downstream component of TLR3 pathway, that usually negatively regulates the classical TLR3 apoptotic pathway, was analyzed by western blotting and immunofluorescence confocal microscopy. The results indicate to our surprise that NF-κB is translocated to nucleus in the cells co-treated with UVB-Poly I:C. The nuclear translocation of NF-κB is attenuated by TLR3 silencing. Treatment with BAY, an inhibitor of NF-κB pathway, blocked UVB-Poly I:C-induced apoptosis. Therefore, we conclude that NF-κB pathway plays a cytotoxic role in UVB-Poly I:C-treated HaCaT cells, mediating TLR3-related apoptosis.

Estrogen Receptors Are Involved in the Neuroprotective Effect of Silibinin in Aß1-42-Treated Rats.

Alzheimer's disease (AD) is a progressive neurodegenerative disease that is characterized by a cascade of pathologic changes. A widely discussed theory indicates that amyloid ß (Aß) peptides are the causative agents of AD. Silibinin, a flavonoid derived from milk thistle, is well known for its hepato-protective activities and we have reported the neuroprotective effects of silibinin. In this study, we investigated the role of estrogen receptors (ERs) in silibinin's neuroprotective effect on Aß1-42-injected rats. Results of Morris water maze and novel object-recognition tests demonstrated that silibinin significantly attenuated Aß1-42-induced memory impairment. Silibinin attenuated ERs and PI3K-Akt pathways, as well as modulated mitogen-activated protein kinases in the hippocampus of Aß1-42-injected rats. Taken together, silibinin is a potential candidate in the treatment of Alzheimer's disease.

Enhanced migration of murine fibroblast-like 3T3-L1 preadipocytes on type I collagen-coated dish is reversed by silibinin treatment.

Migration of fibroblast-like preadipocytes is important for the development of adipose tissue, whereas excessive migration is often responsible for impaired adipose tissue related with obesity and fibrotic diseases. Type I collagen (collagen I) is the most abundant component of extracellular matrix and has been shown to regulate fibroblast migration in vitro, but its role in adipose tissue is not known. Silibinin is a bioactive natural flavonoid with antioxidant and antimetastasis activities. In this study, we found that type I collagen coating promoted the proliferation and migration of murine 3T3-L1 preadipocytes in a dose-dependent manner, implying that collagen I could be an extracellular signal. Regarding the mechanisms of collagen I-stimulated 3T3-L1 migration, we found that NF-κB p65 is activated, including the increased nuclear translocation of NF-κB p65 as well as the upregulation of NF-κB p65 phosphorylation and acetylation, accompanied by the increased expressions of proinflammatory factors and the generation of reactive oxygen species (ROS). Reduction of collagen I-enhanced migration of cells by treatment with silibinin was associated with suppression of NF-κB p65 activity and ROS generation, and negatively correlated with the increasing sirt1 expression. Taken together, the enhanced migration of 3T3-L1 cells induced on collagen I-coated dish is mediated by the activation of NF-κB p65 function and ROS generation that can be alleviated with silibinin by upregulation of sirt1, leading to the repression of NF-κB p65 function and ROS generation.

Silibinin ameliorates Aß25-35-induced memory deficits in rats by modulating autophagy and attenuating neuroinflammation as well as oxidative stress.

Alzheimer's disease (AD) is a progressive, neurodegenerative disease. Accumulating evidence suggests that inflammatory response, oxidative stress and autophagy are involved in amyloid ß (Aß)-induced memory deficits. Silibinin (silybin), a flavonoid derived from the herb milk thistle, is well known for its hepatoprotective activities. In this study, we investigated the neuroprotective effect of silibinin on Aß25-35-injected rats. Results demonstrated that silibinin significantly attenuated Aß25-35-induced memory deficits in Morris water maze and novel object-recognition tests. Silibinin exerted anxiolytic effect in Aß25-35-injected rats as determined in elevated plus maze test. Silibinin attenuated the inflammatory responses, increased glutathione (GSH) levels and decreased malondialdehyde (MDA) levels, and upregulated autophagy levels in the Aß25-35-injected rats. In conclusion, silibinin is a potential candidate for AD treatment because of its anti-inflammatory, antioxidant and autophagy regulating activities.

Timosaponin AIII induces apoptosis and autophagy in human melanoma A375-S2 cells.

Timosaponin AIII (AIII), a steroidal saponin isolated from Anemarrhena asphodeloides Bge. Our study showed that AIII induced both apoptosis and autophagy, and autophagy inhibited apoptosis in A375S2 cells. Furtherly, this study was carried out to investigate what kind of cytokines plays an important role in this process. The results revealed that AIII induced apoptosis through activating c-Jun N-terminal protein kinase (JNK) or extracellular signal related kinase (ERK) signaling pathway and generating NO. However, JNK or ERK inhibited autophagy, while NO had no effect on autophagy. Therefore, JNK, ERK or NO regulates two programmed death processes in different ways. AIII did not show obvious cytotoxic effect on human peripheral blood mononuclear cells, which indicated that AIII has less side effects on normal cells, and could be considered as a leading compound for developing novel anticancer drug.

Protective Effect of Silibinin on Learning and Memory Impairment in LPS-Treated Rats via ROS-BDNF-TrkB Pathway.

Silibinin, a flavonoid derived from the herb milk thistle (Silybum marianum), has been used as a hepato-protectant in the clinical treatment of liver disease. In the present study, the effect of silibinin on lipopolysaccharide (LPS)-induced neuroinflammatory impairment in rats is investigated. Injection of LPS into lateral ventricle caused learning and memory impairment. Rats were treated with silibinin to see the effect in comparison with resveratrol as a positive control. Y-maze and Morris water maze tests showed that silibinin significantly attenuated memory damage caused by LPS treatment. At the molecular analysis, the levels of IL-1ß and of IL-4 in the hippocampus were decreased and enhanced, respectively, by the treatment with silibinin. NF-κB expression was attenuated by silibinin treatment. Furthermore, generation of total reactive oxygen species (ROS) in the hippocampus was elevated in silibinin-treated groups, and so were the expressions of brain-derived neurotrophic factor (BDNF) and tyrosine receptor kinase B (TrkB). At the same time, LPS-induced reduction of neurons in hippocampus was reversed by silibinin. In conclusion, silibinin ameliorated the impairment of learning and memory of LPS-injection rats, possibly due to the activation of ROS-BDNF-TrkB pathway in the hippocampus as well as the suppression of inflammatory response. This study gives an insight on the beneficial consequences of ROS in central nervous system. Silibinin might be a potential candidate drug for neurodegenerative diseases.

Inhibition of caspase-9 by oridonin, a diterpenoid isolated from Rabdosia rubescens, augments apoptosis in human laryngeal cancer cells.

Rabdosia rubescens, a commonly used traditional Chinese medicine, has increasingly gained attention for its use as an antitumor herb. Oridonin, a bioactive diterpenoid isolated from Rabdosia rubescens, has been reported to induce apoptosis in human laryngeal cancer HEp-2 cells by our group. Here, we made unexpected observations that the caspase-9 inhibitor (C9i) enhanced apoptosis in response to selected stimuli, and HEp-2 cells which were made deficient in caspase-9 using siRNA exhibited no resistance to apoptotic signals and actually demonstrated increased apoptotic sensitivity to oridonin. The results were reversed by the transfection of an exogenous caspase-9 expression vector. Caspase-9 reduced sensitivity to apoptotic stimuli through reactive oxygen species (ROS)-suppressing and autophagy-promoting methods. ROS triggered the progression of apoptosis through activation of both the caspase-9-independent mitochondrial pathway and death receptor pathways, and the autophagy had an anti-apoptotic function in oridonin-treated HEp-2 cells. These collective results suggest that oridonin targets caspase-9 to alter ROS production and autophagy situation to promote HEp-2 cell apoptosis. Therefore, oridonin has the potential to be developed as an anticancer agent, and the combination of oridonin with those agents leading to reduction of caspase-9 expression in tumor cells could represent a novel approach to human laryngeal cancer treatment.

Nitric oxide induces apoptosis and autophagy; autophagy down-regulates NO synthesis in physalin A-treated A375-S2 human melanoma cells.

Physalin A is an active withanolide isolated from Physalis alkekengi var. franchetii, a traditional Chinese herbal medicine named Jindenglong, which has been used for the treatment of sore throat, hepatitis, eczema and tumors in China. Our previous study demonstrated that physalin A induced apoptosis and cyto-protective autophagy in A375-S2 human melanoma cells. Induction of reactive oxygen species (ROS) with physalin A triggered apoptosis. In this study, NO generated by physalin A induced apoptosis and autophagy in A375-S2 cells, since physalin A induced the expression of inducible nitric oxide synthase (iNOS) in the cells. Generation of NO partially promoted both apoptosis and autophagy in A375-S2 cells. NO suppressed mTOR expression, which led to autophagy induction. An autophagic inhibitor, 3-methyladenine (3MA) promoted NO production, while acceleration of autophagy with an autophagic agonist rapamycin repressed NO production, suggesting that autophagy and NO production form a negative feedback loop that eventually protects the cells from apoptosis. The results together with the previous study indicate apoptosis and autophagy induced by physalin A in A375-S2 cells; the autophagy, repressing production of reactive nitrogen species (RNS) and ROS, protects the cells from apoptosis.

Inhibition of c-Met promoted apoptosis, autophagy and loss of the mitochondrial transmembrane potential in oridonin-induced A549 lung cancer cells.

OBJECTIVE: Herein, inhibition of hepatocyte growth factor receptor, c-Met, significantly increased cytochrome c release and Bax/Bcl-2 ratio, indicating that c-Met played an anti-apoptotic role. The following experiments are to elucidate this anti-apoptotic mechanism, then the effect of c-Met on autophagy has also been discussed. METHODS: Investigated was the influence of c-Met on apoptosis, autophagy and loss of mitochondrial transmembrane potential (Δψm), and the relevant proteins were examined. KEY FINDINGS: First, we found that activation of extracellular signal-regulated kinase (ERK), p53 was promoted by c-Met interference. Subsequent studies indicated that ERK was the upstream effector of p53, and this ERK-p53 pathway mediated release of cytochrome c and up-regulation of Bax/Bcl-2 ratio. Secondly, the inhibition of c-Met augmented oridonin-induced loss of mitochondrial transmembrane potential (Δψm), resulting apoptosis. Finally, the inhibition of c-Met increased oridonin-induced A549 cell autophagy accompanied by Beclin-1 activation and conversion from microtubule-associated protein light chain 3 (LC3)-I to LC3-II. Activation of ERK-p53 was also detected in autophagy process and could be augmented by inhibition of c-Met. Moreover, suppression of autophagy by 3-methyladenine (3-MA) or small interfering RNA against Beclin-1 or Atg5 decreased oridonin-induced apoptosis. Inhibition of apoptosis by pan-caspase inhibitor (z-VAD-fmk) decreased oridonin-induced autophagy as well and Loss of Δψm also occurred during autophagic process. CONCLUSION: Thus, inhibiting c-Met enhanced oridonin-induced apoptosis, autophagy and loss of Δψm in A549 cells.

Physalin A induces apoptosis via p53-Noxa-mediated ROS generation, and autophagy plays a protective role against apoptosis through p38-NF-κB survival pathway in A375-S2 cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Physalin A is a bioactive withanolide isolated from natural plant Physalis alkekengi L. var. franchetii (Mast.) Makino, a traditional Chinese herbal medicine named Jindenglong which has long been used for the treatment of cough, sore throat, hepatitis, eczema, dysuria and tumors in China. AIM OF THE STUDY: Based on the previous study that physalin A induced cytotoxic effect in human melanoma A375-S2 cells, this study was designed to further illustrate the molecular mechanisms underlying. MATERIALS AND METHODS: Cell viability was evaluated in A375-S2 cells by MTT assay, and the mechanisms involved in physalin A-induced A375-S2 cell death were investigated by phase contrast microscopy and fluorescence microscopy, siRNA transfection, flow cytometry and western blot analysis. RESULTS: We demonstrated that physalin A decreased the proportion of viable A375-S2 cells in a time- and dose-dependent manner, and exposure of A375-S2 cells to physalin A led to both apoptosis and autophagy. Moreover, physalin A-induced apoptosis was triggered by activation of p53-Noxa pathway and intracellular reactive oxygen species (ROS) generation. The administration of ROS scavengers NAC and GSH resulted in the complete inhibition of physalin A-induced ROS generation and apoptosis. Application of p53 inhibitor PFT-α or transfection with Noxa-siRNA could also lead to the same results. Autophagy, demonstrated by the punctuate distribution of monodansylcadaverine staining, as well as the change of LC3-II/LC3-I proportion and Beclin 1 activation, played a protective role against apoptosis via up-regulation of the p38-NF-κB survival pathway in A375-S2 cells. Additionally, inhibition of autophagy by the specific autophagic inhibitor 3MA or blocking the p38-NF-κB pathway with p38 inhibitor SB203580 or NF-κB inhibitor PDTC obviously promoted physalin A-induced apoptosis. CONCLUSIONS: Physalin A induced apoptotic cell death via p53-Noxa-mediated ROS generation, and autophagy played a protective role against apoptosis through up-regulating the p38-NF-κB survival pathway in A375-S2 cells. These results stated the possibility that physalin A would be a potential agent for the treatment of melanoma in the future.

Hydroxyl radical (·OH) played a pivotal role in oridonin-induced apoptosis and autophagy in human epidermoid carcinoma A431 cells.

Oridonin, a diterpenoid compound extracted and purified from Rabdosia rubescen, has been reported to induce tumor cell apoptosis through tyrosine kinase pathway. To further examine the mechanism of oridonin, we selected human epidermoid carcinoma A431 cell as a test object. Besides apoptosis, oridonin also induced A431 cell autophagy, and this autophagy antagonized apoptosis and played a protective role for A431 cells. Reactive oxygen species (ROS) played a pivotal role in induction of cytotoxicity. Therefore, a ROS scavenger, N-acetylcysteine (NAC) combined with oridonin was appiled. Results of morphologic observation, flow cytometric analysis and Western blot analysis showed that NAC could significantly reverse both ROS generation and down-regulation of mitochondrial membrane potential in oridonin treated cells. NAC inhibited oridonin induced apoptosis through both the intrinsic and extrinsic apoptotic pathways. NAC effectively inhibited both oridonin-induced apoptosis and autophagy by reducing intracellular oxidative stress. To further examine the mechanism of ROS, exogenous enzyme antioxidants (superoxide dismutase (SOD), catalase (CAT)) and non-enzyme antioxidants (glutathione (GSH)) were applied to detect the effect of oridonin on ROS generation. Only GSH exerted a similar role with NAC, suggesting that hydroxyl radical (·OH) played the major role in oridonin-induced cell death. Oridonin could decrease the GSH level in A431 cells in a dose-dependent manner. In addition, after treatment with ·OH donor, Fenton reagent, the changes in A431cells were similar to the results of oridonin treatment. All the results proved that ·OH played the pivotal role in oridonin induced apoptosis and autophagy in A431 cells.

Dual effects of silibinin treatment on autophagy-regulated dermal apoptosis retardation and epidermal apoptosis up-regulation in UVB-induced skin inflammation.

Skin inflammation induced by ultraviolet B (UVB) radiation is characterized by migration and chemotaxis of inflammatory cells, epidermic thickening and erythema. Apoptosis and autophagy of epidermal and dermal cells are involved in its development through the adjustment of balance between cell survival and death. In this study, the role of balance between cell survival and apoptosis in dermis and epidermis in UVB-induced skin inflammation and the effect of autophagy on the balance were elucidated, and the protective mechanism of silibinin was investigated through the examination of the influence of autophagy activation or inhibition on erythema, migration, and chemotaxis of inflammatory cells as well as apoptosis adjustments. In UVB-irradiated controls, dermal apoptosis was retarded and the survival of inflammatory cells was promoted through the up-regulation of dermal autophagic level; epidermal apoptosis was increased through the down-regulation of epidermal autophagic level, causing migration and chemotaxis of neutrophils and mast cells as well as skin erythema. In silibinin-treated group (50 mg/kg/day for 4 days), dermal apoptosis was increased through inhibiting dermal autophagy; improper adjustment of epidermal apoptosis was attenuated through promoting epidermal autophagy, presenting dual effects on the balance between autophagy and apoptosis of epidermal and dermal cells and the protection.

In vivo recovery effect of silibinin treatment on streptozotocin-induced diabetic mice is associated with the modulations of Sirt-1 expression and autophagy in pancreatic ß-cell.

Improper adjustments of autophagy and silent information regulator 1 (Sirt-1) expression were reported to be closely associated with metabolic disorders. In this study, we examined the roles of Sirt-1 and autophagy in streptozotocin-induced diabetes mellitus, assessed the relationship between autophagy and Sirt-1, and investigated the protective mechanism of silibinin. Diabetes was induced in 6-week-old mice by intravenous injection of streptozotocin (150 mg/kg/day, for 2 weeks). In the treatment groups, silibinin (50 mg/kg/day, intramuscular injection, for 8 weeks) or inhibitors (50 mg/kg/day, subcutaneous injection, for 8 weeks) were given. Diabetic control animals received vehicle for the same time. Compared with diabetic controls, silibinin or autophagy inhibitor, 3-methyladenine, treated mice showed decreased levels of glycosylated hemoglobin A1C (P < 0.01), serum triglyceride (P < 0.01), cholesterol (P < 0.01), blood glucose (P < 0.05), autophagy (P < 0.05), and apoptosis ratio (P < 0.05) of pancreatic ß-cells. Systemic administration of silibinin reversed streptozotocin-induced downregulation of Sirt-1 expression. Sirt-1 may play a role in regulating the physiological level of autophagy and is associated with loss of pancreatic ß-cells and metabolic biochemical disorders. Through promoting Sirt-1 expression and recovering autophagy physiologically, silibinin may reverse hyperglycemia and repair damaged pancreatic ß-cells.

Silibinin induces protective superoxide generation in human breast cancer MCF-7 cells.

The pharmacological activity of polyphenolic silibinin from milk thistle (Silybum marianum) is primarily due to its antioxidant property. However, this study found that silibinin promoted sustained superoxide (O(2)(.-)) production that was specifically scavenged by exogenous superoxide dismutase (SOD) in MCF-7 cells, while the activity of endogenous SOD was not changed by silibinin. Previous work proved that silibinin induced MCF-7 cell apoptosis through mitochondrial pathway and this study further proved that O(2)(.-) generation induced by silibinin was also related to mitochondria. It was found that respiratory chain complexes I, II and III were all involved in silibinin-induced O(2)(.-) generation. Moreover, it was found that silibinin-induced O(2)(.-) had protective effect, as exogenous SOD markedly enhanced silibinin-induced apoptosis.

Nitric oxide activated by p38 and NF-kappaB facilitates apoptosis and cell cycle arrest under oxidative stress in evodiamine-treated human melanoma A375-S2 cells.

Nitric oxide (NO) has been identified as a fundamental molecule that interplays with reactive oxygen species (ROS) in determining cell fate. As a previous study indicated that ROS was stimulated in evodiamine-induced human melanoma A375-S2 cell apoptosis, the goal of this study was to investigate the role of NO in the cells. In this study, it was found that evodiamine has a strong inductive effect on NO production synthesized by inducible NOS (iNOS) enzyme in a positive-feedback manner. The generated NO was further showed to induce apoptosis and cell cycle arrest and linked to the activation of p53 and p21. After interruption of p38 and nuclear factor-kappaB (NF-kappaB) by pre-treatment with SB203580 and PDTC, iNOS expression, NO synthesis and cell damage were all significantly blocked. It was concluded that p38 and NF-kappaB were critical to the NO producing system, which contributed greatly to the apoptosis and cell cycle arrest in evodiamine-incubated cells.

Efficacy of Tokishakuyakusan on the anemia in the iron-deficient pregnant rats.

Iron-deficiency anemia not only causes insufficient oxygen delivery to the body of the mother, but creates serious conditions in the fetus, such as insufficient oxygen delivery and poor development, and its treatment has become an important issue. To elucidate the mechanism of the anemia-ameliorating action of Tokishakuyakusan, we investigated the effect of administration of Tokishakuyakusan on anemia-related parameters and the serum transferrin and erythropoietin levels, erythrocyte morphology, and bone marrow cells in pregnant rats and in pregnant rats with iron-deficiency anemia. The results showed that Tokishakuyakusan significantly improved the low erythrocyte count, hemoglobin, and hematocrit values of the pregnant rats in the iron-deficient state, increased the proportion of normal erythrocytes in terms of erythrocyte morphology, increased the proportion of erythroblasts among bone marrow cells, and also significantly increased the erythropoietin and transferrin levels in the blood. These findings suggested that Tokishakuyakusan promotes erythrocyte differentiation in iron-deficiency anemia, and that it possesses anemia-ameliorating efficacy.

P53-mediated cell cycle arrest and apoptosis through a caspase-3- independent, but caspase-9-dependent pathway in oridonin-treated MCF-7 human breast cancer cells.

AIM: To study the caspase-3-independent mechanisms in oridonin-induced MCF-7 human breast cancer cell apoptosis in vitro. METHODS: The viability of oridonin-treated MCF-7 cells was measured by MTT (thiazole blue) assay. Apoptotic cells with condensed nuclei were visualized by phase contrast microscopy. Nucleosomal DNA fragmentation was assayed by agarose gel electrophoresis. The apoptotic ratio was determined by lactate dehydrogenase assay. Cell cycle alternation and mitochondrial membrane potential were measured by flow cytometric analysis. Bax, Bcl-2, caspase-3, caspase-9, heat shock protein (Hsp)90, p53, p-p53, p21, Poly (ADP-ribose) polymerase (PARP), and the inhibitor of caspase-activated DNase (ICAD) protein expressions were detected by Western blot analysis. RESULTS: Oridonin inhibited cell growth in a time- and dose-dependent manner. Cell cycle was altered through the upregulation of p53 and p21 protein expressions. Pancaspase inhibitor Z-VAD-fmk and calpain inhibitor II both decreased cell death ratio. Nucleosomal DNA fragmentation and the downregulation of DeltaPhimit were detected in oridonin-induced MCF-7 cell apoptosis, which was involved in a postmitochondrial caspase-9-dependent pathway. Decreased Bcl-2 and Hsp90 expression levels and increased Bax and p21 expression levels were positively correlated with elevated levels of phosphorylated p53 phosphorylation. Moreover, PARP was partially cleaved by calpain rather than by caspase-3. CONCLUSION: DNA damage provoked alternations in the mitochondrial and caspase-9 pathways as well as p53-mediated cell cycle arrest, but was not related to caspase-3 activity in oridonin-induced MCF-7 cells.

[Mechanism of downregulation of apoptosis by autophagy induced by oridonin in HeLa cells].

To study the mechanism of downregulation of apoptosis by autophagy induced by oridonin in HeLa cells, the cell viability was measured by MTT method. DNA fragmentation was assayed by agarose gel electrophoresis. Autophagic and apoptotic ratio was determined by flowcytometric analysis. Protein expression was detected by Western blotting analysis. Oridonin induced both apoptosis and autophagy in HeLa cells. Apoptosis was upregulated by introduction of the inhibitor of autophagy, 3-methyladenine (3-MA). Addition of oridonin increased Bax/Bcl-2 expression ratio and cytochrome c, whereas the expression of SIRT-1 was decreased, and 3-MA pre-application enhanced these changes. Oridonin-induced autophagy antagonized apoptosis in HeLa cells through mitochondrial pathway.

[Protective effect of silibinin against isoproterenol-induced injury to cardiac myocytes and its mechanism].

Silibinin is a polyphenolic flavanoid derived from fruits and seeds of milk thistle (Silybum marianum). To investigate the effect and mechanism of silibinin on beta-isoproterenol-induced rat neonatal cardiac myocytes injury, the viability, the activation of lactate dehydrogenase (LDH) and the content of maleic dialdehyde (MDA) were chosen for measuring the degree of cardiac myocytes injury. Superoxide dismutase (SOD) activity, mitochondrial membrane potential (deltapsi) detected by flow cytometric analysis, and Western blotting analysis were applied to determine the related proteins. Silibinin protected isoproterenol-treated rat cardiac myocytes from death and significantly decreased LDH release and MDA production. Silibinin increased superoxide dismutase (SOD) activity, and increased mitochondrial membrane potential (deltapsi). Furthermore, the release of pro-apoptotic cytochrome c from mitochondria was reduced by silibinin. Silibinin increased the expression of anti-apoptotic Bcl-2 family protein Bcl-2, and up-regulation of SIRT1 inhibited the translocation of Bax from cytoplasm to mitochondria, which caused mitochondrial dysfunction and cell injury. Silibinin protects cardiac myocytes against isoproterenol-induced injury through resuming mitochondrial function and regulating the expression of SIRT1 and Bcl-2 family members.