Protective Effects of a Lutein Ester Prodrug, Lutein Diglutaric Acid, against H2O2-Induced Oxidative Stress in Human Retinal Pigment Epithelial Cells.

Oxidative stress-induced cell damage and death of the retinal pigmented epithelium (RPE), a polarized monolayer that maintains retinal health and homeostasis, lead to the development of age-related macular degeneration (AMD). Several studies show that the naturally occurring antioxidant Lutein (Lut) can protect RPE cells from oxidative stress. However, the poor solubility and low oral bioavailability limit the potential of Lut as a therapeutic agent. In this study, lutein diglutaric acid (Lut-DG), a prodrug of Lut, was synthesized and its ability to protect human ARPE-19 cells from oxidative stress was tested compared to Lut. Both Lut and Lut-DG significantly decreased H2O2-induced reactive oxygen species (ROS) production and protected RPE cells from oxidative stress-induced death. Moreover, the immunoblotting analysis indicated that both drugs exerted their protective effects by modulating phosphorylated MAPKs (p38, ERK1/2 and SAPK/JNK) and downstream molecules Bax, Bcl-2 and Cytochrome c. In addition, the enzymatic antioxidants glutathione peroxidase (GPx) and catalase (CAT) and non-enzymatic antioxidant glutathione (GSH) were enhanced in cells treated with Lut and Lut-DG. In all cases, Lut-DG was more effective than its parent drug against oxidative stress-induced damage to RPE cells. These findings highlight Lut-DG as a more potent compound than Lut with the protective effects against oxidative stress in RPE cells through the modulation of key MAPKs, apoptotic and antioxidant molecular pathways.

An Ingenol Derived from Euphorbia kansui Induces Hepatocyte Cytotoxicity by Triggering G0/G1 Cell Cycle Arrest and Regulating the Mitochondrial Apoptosis Pathway in Vitro.

Natural product lingenol, a purified diterpenoid compound derived from the root of Euphorbia kansui, exerts serious hepatotoxicity; however, the molecular mechanisms remain to be defined. In the present study, cell counting Kit-8 (CCK-8), inverted phase contrast microscope and flow cytometry were used to demonstrate that lingenol significantly inhibited L-O2 cells proliferation, and induced cell cycle arrest and apoptosis. Moreover, the results investigated that lingenol markedly disrupted mitochondrial functions by high content screening (HCS). In addition, the up-regulation of cytochrome c, AIF and Apaf-1 and activation of caspases were found in L-O2 cells detected by Western blotting and ELISA assay, which was required for lingenol activation of cytochrome c-mediated caspase cascades and AIF-mediated DNA damage. Mechanistic investigations revealed that lingenol significantly down-regulated the Bcl-2/Bax ratio and enhanced the reactive oxygen species (ROS) in L-O2 cells. These data collectively indicated that lingenol modulation of ROS and Bcl-2/Bax ratio led to cell cycle arrest and mitochondrial-mediated apoptosis in L-O2 cells in vitro. All of these results will be helpful to reveal the hepatotoxicity mechanism of Euphorbia kansui and to effectively guide safer and better clinical application of this herb.

Grape Juice Concentrate Protects Rat Liver Against Cadmium Intoxication: Histopathology, Cytochrome C and Metalloproteinases Expression.

The aim of this study was to investigate if grape juice concentrate is able to protect rat liver against cadmium toxicity. For this purpose, histopathological analysis, cytochrome C expression and immunoexpresssion of metalloproteinases (MMP) 2 and 9 were investigated. A total of 15 Wistar rats weighing 250 g on the average, and 8 weeks age were distributed into 3 groups (n=5), as follows: Control group (non-treated group, CTRL); Cadmium group (Cd) and grape juice concentrate group (Cd+GJ). Histopathological analysis revealed that liver from animals treated with grape juice concentrate improved tissue degeneration induced by cadmium intoxication. Animals intoxicated with cadmium and treated with grape juice concentrate showed higher cytochrome C gene expression in liver cells. No significant statistically differences (p>0.05) were found to MMP 2 and 9 immunoexpression between groups. Taken together, our results demonstrate that grape juice concentrate is able to prevent tissue degeneration in rat liver as a result of increasing apoptosis.

The effects of ulipristal on Bax/Bcl-2, cytochrome c, Ki-67 and cyclooxygenase-2 expression in a rat model with surgically induced endometriosis.

OBJECTIVE: To evaluate the effect of ulipristal on Bax/Bcl-2, cytochrome C, Ki-67 and cyclooxygenase-2 expression in surgically induced endometriosis in a rat model. STUDY DESIGN: We conducted a prospective, randomized, controlled, experimental study at the Experimental Research Center of the Iuliu Hatieganu University of Medicine and Pharmacy in Cluj-Napoca, Romania. Endometriosis was induced in 40 female Wistar albino rats by transplanting two autologous fragments of uterine horn onto bowel mesentery. After a 4-week induction period, we formed two groups: the first group was treated with ulipristal (UPA+) for 8 weeks, while the second group was treated only with the vehicle used for ulipristal (UPA-). We measured the volumes and masses of the implants both before and after treatment. A pathologist examined the sections microscopically for histological hallmarks of endometriosis. Immunostaining for Bax/Bcl-2, cytochrome C, Ki-67 and cyclooxygenase-2 (COX-2) was assessed in both groups. RESULTS: Ulipristal reduced the average implant volume and mass, indicating that the drug is effective (P=0.01). The treatment induced a greater than 50% reduction in the volume and mass of endometrial implants, and the histological findings correspond to this result. The overall Bax positivity rate was higher in the group treated with ulipristal (42.37% vs. 21.05% for UPA+ and UPA-, respectively) (P=0.0062). The overall Bcl-2 positivity rate was smaller in the group treated with ulipristal (15% vs. 40% for UPA+ and UPA-, respectively) (P=0.0593). The cytochrome C global positivity rate was 5% in the UPA- group and increased to 50% in the UPA+ treatment group (P<0.0001). The COX-2 positivity rate decreased from 75% in the UPA- treatment group to 10% in the UPA+ treatment group (P<0.0001) and the Ki67 positivity rate also decreased from 55% in the UPA- group to 10% in the UPA+ treatment group (P<0.0002). CONCLUSIONS: Treatment with ulipristal contributed to the regression and atrophy of endometriotic lesions in rats. The immunohistochemical expression profiles of Bax/Bcl-2 and cytochrome C revealed a pro-apoptotic effect of ulipristal. We also observed a reduced cellular proliferation, indicated by a decrease in Ki-67 expression and an anti-inflammatory effect, shown by a decrease in COX-2 expression after treatment with ulipristal.

Sarsasapogenin induces apoptosis via the reactive oxygen species-mediated mitochondrial pathway and ER stress pathway in HeLa cells.

Sarsasapogenin is a sapogenin from the Chinese medical herb Anemarrhena asphodeloides Bunge. In the present study, we revealed that sarsasapogenin exhibited antitumor activity by inducing apoptosis in vitro as determined by Hoechst staining analysis and double staining of Annexin V-FITC/PI. In addition, cell cycle arrest in G2/M phase was observed in sarsasapogenin-treated HeLa cells. Moreover, the results revealed that perturbations in the mitochondrial membrane were associated with the deregulation of the Bax/Bcl-2 ratio which led to the upregulation of cytochrome c, followed by activation of caspases. Meanwhile, treatment of sarsasapogenin also activated Unfolded Protein Response (UPR) signaling pathways and these changes were accompanied by increased expression of CHOP. Salubrinal (Sal), a selective inhibitor of endoplasmic reticulum (ER) stress, partially abrogated the sarsasapogenin-related cell death. Furthermore, sarsasapogenin provoked the generation of reactive oxygen species, while the antioxidant N-acetyl cysteine (NAC) effectively blocked the activation of ER stress and apoptosis, suggesting that sarsasapogenin-induced reactive oxygen species is an early event that triggers ER stress mitochondrial apoptotic pathways. Taken together, the results demonstrate that sarsasapogenin exerts its antitumor activity through both reactive oxygen species (ROS)-mediate mitochondrial dysfunction and ER stress cell death.

Sanguisorbae radix protects against 6-hydroxydopamine-induced neurotoxicity by regulating NADPH oxidase and NF-E2-related factor-2/heme oxygenase-1 expressions.

6-Hydroxydopamine (6-OHDA) produces neuronal cell damage by generating reactive oxygen species (ROS). The major mechanisms of protection against ROS-induced stress are inhibiting expression of ROS generating genes such as NADPH oxidase (NOX) and increasing expression of endogenous antioxidant genes such as heme oxygenase-1 (HO-1). This study investigated whether a standardized Sanguisorbae Radix extract (SRE), a medical herb commonly used in Asian traditional medicine, has a protective effect on 6-OHDA-induced cell toxicity by regulating ROS in SH-SY5Y cells. SRE at 10 and 50 µg/mL significantly reduced 6-OHDA-induced cell damage dose dependently in the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and by Hoechst 33342 staining. SRE increased the B-cell lymphoma 2 (Bcl-2)/Bcl-2-associated X ratio and decreased cytochrome C release and caspase-3 activity. SRE also abolished 6-OHDA-induced ROS by inhibiting NOX expression and by inducing HO-1 expression via NF-E2-related factor-2 activation. Taken together, these results demonstrate that SRE has protective effects against 6-OHDA-induced cell death by regulating ROS in SH-SY5Y cells.

Antihyperglycemic potentials of a threatened plant, Helonias dioica: antioxidative stress responses and the signaling cascade.

Helonias dioica (HD) is a threatened species of herb growing in North America. It is used as a traditional medicine for treating various ailments particularly related to reproductive issues. The root is reported to contain approximately 10% of a saponin (chamaelirin; C(36)H(62)O(18)) apart from certain other fatty acids. As saponins are known to have hypoglycemic effects, we suspected its possible antihyperglycemic potentials. We injected intraperitoneally alloxan (ALX) at the dose of 200 mg/kg body weight (bw) to induce hyperglycemia in mice and tested possible hypoglycemic effects of HD in vivo by deploying two doses (100 and 200 mg/kg bw, respectively). We also tested its effects on the isolated pancreatic islets cells in vitro. We used various standard protocols like reactive oxygen species (ROS) generation and DNA damage, activities of biomarkers like catalase (CAT), superoxide dismutase (SOD), lipid peroxidase (LPO), reduced glutathione (GSH) of the pancreas tissue and glucokinase and glycogen content of the liver of hyperglycemic mice. With a mechanistic approach, we also tracked down the possible signaling pathway involved. We found an elevated level of ROS generation, LPO and overexpression of inducible nitric oxide synthase (iNOS), tumor necrosis factor α (TNF-α), p38 Map kinase (p38 MAPK), nuclear factor (NF)-κß, interferon gamma (IFN-γ), cytochrome c, caspase 3, poly [ADP ribose] polymerase (PARP) and cyclo oxygenase 2 (COX2) in ALX-induced diabetic mouse. Treatment of hyperglycemic mice with both the doses of HD showed a significant decrease with respect to all these parameters of study. Thus, our results suggest that HD prevents ALX-induced islet cell damage and possesses antihyperglycemic and antioxidative potentials.

Solanum incanum extract (SR-T100) induces human cutaneous squamous cell carcinoma apoptosis through modulating tumor necrosis factor receptor signaling pathway.

BACKGROUND: The Solanum species herbs have been used to treat cancer for centuries; however, the underlying mechanisms and effectiveness in vivo remain unclear. OBJECTIVES: SR-T100, extracted from the Solanum incanum, contains solamargine alkaloid as the main active ingredient. Here, we investigated the apoptosis-inducing effects of SR-T100 for targeting squamous cell carcinoma (SCC) in vitro and in vivo. METHODS: We elucidated the mechanism by which SR-T100 induces apoptosis of human SCCs (A431, SCC4, SCC9, and SCC25) cells. The efficacy and safety issues were addressed regarding topical treatment of SR-T100 on UVB-induced cutaneous SCC of hairless mice and actinic keratoses (AKs) of human. RESULTS: SR-T100 induces apoptosis in human SCCs cell lines by up-regulating the expressions of tumor necrosis factor receptors (TNFRs) and Fas, and downstream adaptors FADD/TRADD of the TNF-α and Fas ligand signaling cascades. SR-T100 also triggered the mitochondrial apoptotic pathway, as up-regulated cytochrome c and Bax, down-regulated Bcl-X(L). Animal experiments showed that all papillomas (35/35) and 27 of 30 UVB-induced microinvasive SCCs in hairless mice disappeared within 10 weeks after once-daily application of topical SR-T100. Furthermore, 13 patients, who suffered with 14 AKs, were treated with once-daily topical SR-T100 gel and 10 AKs cured after 16 weeks, showing negligible discomforts. CONCLUSION: Our studies indicate that SR-T100 induces apoptosis of SCC cells via death receptors and the mitochondrial death pathway. The high efficacy of SR-T100 in our preclinical trial suggests that SR-T100 is a highly promising herb for AKs and related disorders.

Protective role of benfotiamine, a fat-soluble vitamin B1 analogue, in lipopolysaccharide-induced cytotoxic signals in murine macrophages.

This study was designed to investigate the molecular mechanisms by which benfotiamine, a lipid-soluble analogue of vitamin B1, affects lipopolysaccharide (LPS)-induced inflammatory signals leading to cytotoxicity in the mouse macrophage cell line RAW264.7. Benfotiamine prevented LPS-induced apoptosis, expression of the Bcl-2 family of proapoptotic proteins, caspase-3 activation, and PARP cleavage and altered mitochondrial membrane potential and release of cytochrome c and apoptosis-inducing factor and phosphorylation and subsequent activation of p38-MAPK, stress-activated kinases (SAPK/JNK), protein kinase C, and cytoplasmic phospholipase A2 in RAW cells. Further, phosphorylation and degradation of inhibitory kappaB and consequent activation and nuclear translocation of the redox-sensitive transcription factor NF-kappaB were significantly prevented by benfotiamine. The LPS-induced increased expression of cytokines and chemokines and the inflammatory marker proteins iNOS and COX-2 and their metabolic products NO and PGE(2) was also blocked significantly. Thus, our results elucidate the molecular mechanism of the anti-inflammatory action of benfotiamine in LPS-induced inflammation in murine macrophages. Benfotiamine suppresses oxidative stress-induced NF-kappaB activation and prevents bacterial endotoxin-induced inflammation, indicating that vitamin B1 supplementation could be beneficial in the treatment of inflammatory diseases.

[Effect of Neiyi Kangfu suppository on expressions of cytochrome C and survivin in ectopic and eutopic endometrium in rats with endometriosis].

OBJECTIVE: To explore the different effects of rectal application of Neiyi Kangfu Suppository (NYKFS) on the expressions of cytochrome C (Cyt C) and Survivin in ectopic and eutopic endometrium in endometriosis (EMT) model rats. METHODS: EMT rats were randomly divided into 6 groups, the high- and low-dose NYKFS groups, the Danazol Capsule (DN) group, the Dan'e Fukang Soft Extract (DFE) group, the model group and the blank group. The expressions of Cyt C and Survivin were measured using immunohistochemical SP staining method. RESULTS: (1) After treatment, the IOD value of Cyt C in ectopic endometrium significantly increased in the high-dose and low-dose NYKFS groups respectively to 6.08 +/- 0.35 and 6.23 +/- 0.35, which was significantly higher than that in the model group (5.07 +/- 0.70) and DFE group (5.98 +/- 1.02) respectively (P < 0.05, P < 0.01); while those of Survivin in ectopic endometrium was significantly decreased to 5.73 +/- 0.93 and 5.62 +/- 0.93 and was significantly lower than that in the model group (6.01 +/- 1.16, P < 0.05 or P < 0.01). (2) The lowered eutopic endometrial level of Cyt C in EMT rats after treatment was significantly higher in the two NYKFS groups than that in the model group (P < 0.05); while that of eutopic endometrial Survivin was insignificantly different between the NYKFS groups and the model group or the blank group (P > 0.05). CONCLUSION: NYKFS plays its role in inducing apoptosis by increasing the expression of Cyt C and decreasing the expression of Survivin in ectopic endometrium, but it up-regulates expression of Cyt C, has no obvious effect on Survivin expression in eutopic endometrium.

Expression in periplasmic space of Shewanella oneidensis.

A Shewanella expression system has been used for an overproduction of c-type multiheme proteins. The proteins were exported to the periplasmic space for the maturation. Since the periplasmic expression system is attractive, especially for protease-sensitive proteins, an expression vector containing a signal peptide was constructed for expressions in the periplasmic space of Shewanella oneidensis. To evaluate the system, two eukaryotic proteins which originally do not have signal sequences and are difficult to express in Escherichia coli, were selected. The first is human cytochrome c. Properties of the recombinant cytochrome c were identical to those previously reported, indicating the protein is intact. The other was potato calcium-dependent protein kinase. The protein was expressed in periplasmic space. These results indicated that the system is generally applicable for any protein expression including c-type cytochromes, protease-sensitive proteins and those with multi-disulfide bonds because of transportation to the periplasmic space.

Effect of hyperbaric oxygen on cytochrome C, Bcl-2 and Bax expression after experimental traumatic brain injury in rats.

OBJECTIVE: To explore the effects of hyperbaric oxygen (HBO) treatment on the neuronal apoptosis at an earlier stage and the expressions of Cytochrome C (Cyt C), Bcl-2 (B-cell lymphoma-2 family) and Bax (Bcl-2 associated X protein) in rat brain tissues after traumatic brain injury (TBI). METHODS: Forty adult rats were divided into two groups, i.e., Group A (the rats with untreated TBI) and Group B (rats with HBO treatment after TBI). Sections of brain tissues of these two groups were then detected at 3, 6, 12, 24, 72 hours after TBI by immunohistochemistry and electronmicroscope, respectively. RESULTS: HBO treatment could up-regulate the expression of Bcl-2 within 72 hours, reduce the release of Cyt C from mitochondria, attenuate the formation of dimeric Bax and alleviate the mitochondrial edema within 24 hours after TBI. CONCLUSIONS: HBO treatment can alleviate neuronal apoptosis after TBI by reducing the release of Cyt C and the dimers of Bax and up-regulating the expression of Bcl-2.

Tetrandrine-induced apoptosis in rat primary hepatocytes is initiated from mitochondria: caspases and endonuclease G (Endo G) pathway.

Tetrandrine, a bisbenylisoquinoline alkaloid isolated from the dried root of Stephenia tetrandra (S Moore), possesses a remarkable pharmacological profile. However, the mechanisms of tetrandrine hepatotoxicity remain to be elucidated. In this study, we first proved apoptosis and mitochondrial dysfunction induced by tetrandrine in Sprague-Dawley rat liver in vivo. By further assuming apoptosis as an important mechanism in tetrandrine-induced hepatotoxicity, we focused on mitochondria-initiated apoptosis in primary hepatocytes isolated from Sprague-Dawley male rats. Tetrandrine treatment led to significant release of cytochrome c and downregulation of Bcl-X(L) accompanied by caspase 3 activation, and ultimately, DNA fragmentation. Caspase 3 activation was markedly inhibited by cyclosporin A (CsA) and Ac-DEVD-CHO. Furthermore, Endo G, a caspase-independent apoptotic protein, was detected for its expression and DNase activity. CsA blocked the release both of Endo G and cytochrome c significantly. Additionally, the generation of reactive oxygen species (ROS) increased in a time-dependent manner corresponding with a fall in intracellular GSH content after 10 microM tetrandrine treatment in 4h. Tetrandrine also induced mitochondrial dysfunction indicated by transition of mitochondrial transmembrane potential and decrease of intracellular ATP level. The findings indicated that the caspase-dependent mitochondrial apoptosis pathway was primarily involved in tetrandrine-induced apoptosis in rat primary hepatocytes. In addition, a caspase-independent pathway indicated by Endo G also contributed to apoptosis caused by tetrandrine. Meanwhile, ROS was proved an important inducer in this apoptosis process.