Cardioprotective effects of calorie restriction against inflammation and apoptosis in ovariectomized obese rats: Role of classical estrogen receptors and SIRT1.

AIM: Obesity is a metabolic complication linked with bad eating habits and a sedentary lifestyle, and the heart is one of the target organs damaged by it. Estrogen deficiency during menopause worsens the situation. Calorie restriction (CR) can contribute to reducing cardiovascular disease (CVD) in postmenopausal conditions. Thus, the effects of CR on inflammation and apoptosis in ovariectomized rats' hearts with obesity were studied. METHOD: Female Wistar rats were categorized into Sham and OVX (ovariectomized) groups and received a standard diet (SD) or high-fat diet (60%HFD) or calorie restriction (30% CR) for 16 weeks. The real-time PCR method was used to evaluate the inflammatory markers and estrogen receptors gene expression. Western-blot and ELISA methods were respectively used for the measurement of apoptosis and SIRT1 protein expression. RESULTS: HFD led to the elevation of body weight, IL-6 (interleukin-6) and TNF-α (tumor necrosis factor-α) and reduction of IL-10 (interleukin-10) gene expressions, and also an increment in protein levels of cleaved caspase-3, Bax and Bax/Bcl2 ratio and decrement in Bcl-2 in OVX rats (P < 0.001). Additionally, HFD reduced SIRT1 (sirtuin1) protein levels, ERα (estrogen receptor α), and ERß (estrogen receptor ß) gene expressions (P < 0.001). In contrast, CR declined body weight, IL-6 and TNF-α (P < 0.001), increased IL-10 expressions (P < 0.05), decreased cleaved caspase-3 (P < 0.001), Bax (P < 0.01), and Bax/Bcl2 ratio (P < 0.05), enhanced Bcl-2 (P < 0.001), increased SIRT1 (P < 0.05) and ERα (P < 0.001) and ERß (P < 0.01) expressions. CONCLUSION: CR through the SIRT1 regulation and estrogen receptors attenuate obesity-induced-cardiac inflammation and apoptosis. CR can be a cardioprotective candidate in postmenopausal conditions.

Protective effects of calorie restriction and 17-ß estradiol on cardiac hypertrophy in ovariectomized obese rats.

Obesity and menopause lead to cardiovascular diseases. Calorie restriction (CR) can modulate estrogen deficiency and obesity-related cardiovascular diseases. The protective effects of CR and estradiol on cardiac hypertrophy in ovariectomized obese rats were explored in this study. The adult female Wistar rats were divided into sham and ovariectomized (OVX) groups that received a high-fat diet (60% HFD) or standard diet (SD) or 30% CR for 16 weeks, and then, 1mg/kg E2 (17-ß estradiol) was injected intraperitoneally every 4 days for four weeks in OVX-rats. Hemodynamic parameters were evaluated before and after each diet. Heart tissues were collected for biochemical, histological, and molecular analysis. HFD consumption led to weight gain in sham and OVX rats. In contrast, CR and E2 led to body weight loss in these animals. Also, heart weight (HW), heart weight/body weight (HW/BW) ratio, and left ventricular weight (LVW) were enhanced in OVX rats that received SD and HFD. E2 reduced these indexes in both diet conditions but reduction effects of CR were seen only in HFD groups. HFD and SD feeding increased hemodynamic parameters, ANP (atrial natriuretic peptide) mRNA expression, and TGF-ß1(transforming growth factor-beta 1) protein level in the OVX animals, while CR and E2 reduced these factors. Cardiomyocyte diameter and hydroxyproline content were increased in the OVX-HFD groups. Nevertheless, CR and E2 decreased these indicators. The results showed that CR and E2 treatment reduced obesity-induced-cardiac hypertrophy in ovariectomized groups (20% and 24% respectively). CR appears to have almost as reducing effects as estrogen therapy on cardiac hypertrophy. The findings suggest that CR can be considered a therapeutic candidate for postmenopausal cardiovascular disease.

Anti-tumor effects of Thymus Caramanicus Jalas extract in mice through oxidative stress, inflammation and apoptosis.

OBJECTIVE: Breast cancer causes death in women. Thymus Caramanicus Jalas (TCJ) as a polyphenolic plant has an antiproliferative effect. Accordingly, this investigation studied the TCJ extract anti-tumor effects in a breast cancer model. METHODS: Twenty-four female BALB/c mice were used in 4 groups including (1) breast cancer (control); (2), (3) and (4) breast cancer + 100, 300 and 500 mg/kg of TCJ extract (once daily for 20-days after breast tumor induction). The breast tumour was induced by 4T1 cell carcinoma injection. Then tumor size and weight were measured. Tumor necrosis factor-α (TNF-α), nuclear factor κ-B (NF-κB), interleukin-6 (IL-6) as inflammatory markers and also Bcl-2, Bax, cytosolic cytochrome-c, apoptosis-inducing factor, and cleaved caspase-3 as biochemical apoptosis markers were evaluated in tumor tissue with western blotting analysis. Also, malondialdehyde (MDA) concentration, hydrogen peroxidase (H2O2), catalase, glutathione peroxidase (GPx) and superoxide dismutase (SOD) activities were exanimated. KEY FINDINGS: Treatment with TCJ extract (500 mg/kg) decreased the tumor volume, tumor weight, GPx, SOD, and catalase enzyme activity versus the control group (P < 0.05). Also, TCJ (500 mg/kg) extract increased MDA, H2O2, inflammatory and apoptosis markers versus control (P < 0.05). CONCLUSIONS: Current study showed that TCJ can induce anti-tumour effects via promoting inflammation, apoptosis, and oxidative stress in breast tumour tissue.

Substitution of calorie restriction for protective effects of estrogen on cardiometabolic risk factors and oxidative stress in obese postmenopausal rat model.

Estrogen has an anti-obesity effect and plays an important role in improving cardiometabolic disorders. Weight loss and reduction in calorie intake impede the development of obesity-related cardiometabolic risk factors. Therefore, we investigated the substitution of calorie restriction for effects of estrogen on cardiometabolic risk factors and oxidative stress in obese postmenopausal rat model. In this study, adult female Wistar rats were allocated into Sham and ovariectomized (OVX) groups and were given standard diet (SD) or 60% high-fat diet (HFD) or 30% calorie restriction (CR) for 16 weeks, following this, animals received E2 (17-ß estradiol; 1 mg/kg; i.p.) every four days for 4 weeks. Results showed that HFD elevated the body weight, BMI, food intake, and blood glucose (BG) level in both sham and OVX groups. In addition, HFD had negative effects on lipid profile and oxidative stress in these groups. Whereas CR decreased these indices in both Sham and OVX groups fed an HFD, it could not diminish the BG level in the OVX-HFD group. E2 treatment in OVX animals with or without CR reduced body weight, BMI, food intake, and BG level, and also had positive effects on lipid profile alterations and oxidative stress reduction. In comparison, no significant differences were observed regarding the effects of E2 with CR between two groups for body weight, lipid profile, BG, and oxidative stress in the OVX-HFD rats. Overall, CR prevents and ameliorates cardiometabolic risk factors induced by obesity in postmenopausal conditions and is also a good candidate for E2 substitution.

The protective effects of 17-ß estradiol and SIRT1 against cardiac hypertrophy: a review.

One of the major causes of morbidity and mortality worldwide is cardiac hypertrophy (CH), which leads to heart failure. Sex differences in CH can be caused by sex hormones or their receptors. The incidence of CH increases in postmenopausal women due to the decrease in female sex hormone 17-ß estradiol (E2) during menopause. E2 and its receptors inhibit CH in humans and animal models. Silent information regulator 1 (SIRT1) is a NAD+-dependent HDAC (histone deacetylase) and plays a major role in biological processes, such as inflammation, apoptosis, and oxidative stress responses. Probably SIRT1 because of these effects, is one of the main suppressors of CH and has a cardioprotective effect. On the other hand, estrogen and its agonists are highly efficient in modulating SIRT1 expression. In the present study, we review the protective effects of E2 and SIRT1 against CH.

Estrogen receptor agonists induce anti­edema effects by altering α and ß estrogen receptor gene expression.

The present study aimed to examine whether the attenuation of estrogen receptor expression is prevented by propyl pyrazole triol (PPT), an agonist for estrogen receptor α (ERα) or and diarypropiolnitrile (DPN), an agonist for estrogen receptor ß (ERß) after traumatic brain injury (TBI). The tests performed on ovariectomized female Wistar rats included sham group, vehicle group, and treated groups: PPT, DPN, and PPT+DPN 30 minutes after TBI. Blood­brain barrier (BBB) disruption and brain water content were estimated. RT­PCR and\r\nwestern blotting were utilized to evaluate ESR1 and ESR2 gene and protein expression. The data indicated that PPT, DPN, and PPT+DPN attenuated TBI­induced brain edema. Also, BBB disruption after TBI was prevented in PPT, DPN, and PPT+DPN­treated TBI animals. Estrogen agonist­treated animals showed a significant elevation in Esr1 mRNA and protein expression levels in the brain tissue of TBI rats. In addition, the data indicated a significant elevation of Esr2 mRNA and protein expression levels in the brain tissue of estrogen agonist­treated TBI rats. The data shows that both ESR1 and ESR2 agonists can enhance ER mRNA and protein levels in TBI animals' brain. It appears that this effect contributes to the neuroprotective function of ER agonists.

Anticancer and cytotoxic effects of troxerutin on HeLa cell line: an in-vitro model of cervical cancer.

Cervical cancer is one of the grave uterine tumors which leads to death in women worldwide. Troxerutin (TRX) as a bioflavonoid compound has many pharmacological effects such as anti-neoplastic, radioprotective, and anti-cancer. The present study was designed to examine the cytotoxic effect of TRX on human HeLa tumor cells. Human HeLa cells were cultured and treated with different doses of TRX (20-640 mg/ml) to evaluate the effective half-maximal inhibitory concentration (IC50) after 24 h. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) test was used for cell proliferation assay. Also, the Bax, Bcl-2, cleaved caspase-3, and tumor necrosis factor-α (TNF-α) protein expression levels were detected with immunoblotting analysis. The malondialdehyde (MDA) concentration, glutathione peroxidase (GPx) and superoxide dismutase (SOD) activity levels were measured via their commercial kits. Data were analyzed using one-way ANOVA. The result showed that TRX at 320 mg/ml concentration (IC50) has a growth inhibitory effect against HeLa cells at 24 h treatment (P ˂ 0.01). Moreover, it increased the MDA concentration and also decreased the GPx and SOD activity levels at 320 mg/ml concentration versus control (P < 0.001). Also, TRX significantly up-regulated the Bax, cleaved caspase-3 and TNF-α proteins expression levels (P  < 0.01) and down-regulated the Bcl-2 protein expression in HeLa tumor cells at 320 mg/ml concentration compared to control (P < 0.05). Our study showed that 24 h of treatment with TRX (320 mg/ml) has apoptotic and growth inhibitory effects against HeLa cells. It can induce inflammation (at least via up-regulating the TNF-α protein expression) and oxidative stress in human HeLa cells.

Neuroprotective and antihyperalgesic effects of orexin-A in rats with painful diabetic neuropathy.

AIM OF STUDY: Diabetes mellitus is related to the development of neuronal tissue injury in different peripheral and central nervous system regions. A common complication of diabetes is painful diabetic peripheral neuropathy (PDN). We have studied the neuroprotective and anti-nociceptive properties of neuropeptide orexin-A in an animal experimental model of diabetic neuropathy. METHODS: All experiments were carried out on male Wistar rats (220-250 g). Diabetes was induced by a single intraperitoneal injection of 55 mg/kg (i.p.) streptozotocin (STZ). Orexin-A was chronically administrated into the implanted intrathecal catheter (0.6, 2.5 and 5 nM/L, daily, 4 weeks). The tail-flick and rotarod treadmill tests were used to evaluate the nociceptive threshold and motor coordination of these diabetic rats, respectively. Cleaved caspase-3, Bax, Bcl2 and the Bax/Bcl-2 ratio, as the biochemical indicators of apoptosis, were investigated in the dorsal half of the lumbar spinal cord tissue by western blotting method. RESULTS: Treatment of the diabetic rats with orexin-A (5 nM/L) significantly attenuated the hyperalgesia and motor deficit in diabetic animals. Furthermore, orexin-A (5 nM/L) administration suppressed pro-apoptotic cleaved caspase-3 and Bax proteins. Also, orexin-A (5 nM/L) reduced the expression of Bax/Bcl-2 ratio in spinal cord dorsal half of rats with PDN. CONCLUSIONS: Altogether our data suggest that the orexin-A has anti-hyperalgesic and neuroprotective effects in rats with PDN. Cellular mechanisms underlying the observed effects may, at least partially, be related to reducing the neuronal apoptosis.

Neuroprotective and antinociceptive effects of rosemary (Rosmarinus officinalis L.) extract in rats with painful diabetic neuropathy.

Diabetes mellitus is associated with the development of neuronal tissue damage in different central and peripheral nervous system regions. A common complication of diabetes is painful diabetic peripheral neuropathy. We have explored the antihyperalgesic and neuroprotective properties of Rosmarinus officinalis L. extract (RE) in a rat model of streptozotocin (STZ)-induced diabetes. The nociceptive threshold and motor coordination of these diabetic rats was assessed using the tail-flick and rotarod treadmill tests, respectively. Activated caspase-3 and the Bax:Bcl-2 ratio, both biochemical indicators of apoptosis, were assessed in the dorsal half of the lumbar spinal cord tissue by western blotting. Treatment of the diabetic rats with RE improved hyperglycemia, hyperalgesia and motor deficit, suppressed caspase-3 activation and reduced the Bax:Bcl-2 ratio, suggesting that the RE has antihyperalgesic and neuroprotective effects in this rat model of STZ-induced diabetes. Cellular mechanisms underlying the observed effects may, at least partially, be related to the inhibition of neuronal apoptosis.

Leptin attenuates oxidative stress and neuronal apoptosis in hyperglycemic condition.

One of the main pathological mechanisms of neurotoxicity in diabetic situation is oxidative stress promoted by hyperglycemia. It has been shown that leptin has neuroprotective effects and may provide neuronal survival signals. This study was designed to reveal the possible neuroprotective effects of leptin in hyperglycemic conditions. Pheochromocytoma (PC12) cell viability was assessed via the MTT test. Cellular reactive oxygen species (ROS) generation was determined by DCFH-DA analysis. The malondialdehyde (MDA) and glutathione (GSH) levels were measured in high-glucose-treated PC12 cells with and without leptin cotreatment. Western blotting was performed to measure apoptosis markers (Cleaved caspase-3 and Bax/Bcl2 ratio). Elevation of glucose levels (100 mmol/L) consecutively increased intracellular ROS and MDA level, and apoptosis in PC12 cells after 24 h leptin administration (12 and 24 nmol/L) decreased the high-glucose-induced cell toxicity, caspase-3 activation, and Bax/Bcl-2 ratio. Also, cotreatment with leptin (12 and 24 nmol/L) significantly reduced oxidative damage to PC12 cells in high-glucose condition, as reflected by the diminution in MDA and ROS levels and the increase in GSH content. Our finding demonstrates that leptin has protective effects against hyperglycemia-induced neural damage. This could be related to the attenuation of oxidative stress and neural apoptosis.

Cellular Preoxygenation Partially Attenuates the Antitumoral Effect of Cisplatin despite Highly Protective Effects on Renal Epithelial Cells.

Our previous in vitro studies demonstrated that oxygen pretreatment significantly protects human embryonic renal tubular cell against acute cisplatin- (CP-) induced cytotoxicity. The present study was designed to investigate whether this protective effect is associated with decreasing therapeutic effects of cisplatin on malignant cells. For this purpose, cultured human embryonic kidney epithelial-like (AD293), cervical carcinoma epithelial-like (Hela), and ovarian adenocarcinoma epithelial-like (OVCAR-3) cells were subjected to either 2-hour pretreatment with oxygen (≥90%) or normal air and then to a previously determined 50% lethal dose of cisplatin for 24 hours. Cellular viability was evaluated via MTT and Neutral Red assays. Also, activated caspase-3 and Bax/Bcl-2 ratio, as the biochemical markers of cell apoptosis, were determined using immunoblotting. The hyperoxic preexposure protocol significantly protects renal AD293 cells against cisplatin-induced toxicity. Oxygen pretreatment also partially attenuated the cisplatin-induced cytotoxic effects on Hela and OVCAR-3 cells. However, it did not completely protect these cells against the therapeutic cytotoxic effects of cisplatin. In summary, the protective methods for reducing cisplatin nephrotoxic side effects like oxygen pretreatment might be associated with concurrent reduction of the therapeutic cytotoxic effects of cisplatin on malignant cells like cervical carcinoma (Hela) and ovarian adenocarcinoma (OVCAR-3) cells.

Effects of sex steroid hormones on neuromedin S and neuromedin U2 receptor expression following experimental traumatic brain injury.

OBJECTIVES: Neuroprotective effects of female gonadal steroids are mediated through several pathways involving multiple peptides and receptors after traumatic brain injury (TBI). Two of these peptides are including the regulatory peptides neuromedin U (NMU) and neuromedin S (NMS), and their common receptor neuromedin U2 receptor (NMUR2). This study investigates the effects of physiological doses of estradiol and progesterone on brain edema, NMS and NMU as well as NMUR2 expression following TBI. MATERIALS AND METHODS: Ovariectomized female rats were given high-and low-dose of female sex steroid hormones through implantation of capsules for a week before trauma. The brain NMUR2 expression, prepro-NMS expression, NMU content, and water content (brain edema) were evaluated 24 hr after TBI induced by Marmarou's method. RESULTS: Percentage of brain water content in high- and low-dose estradiol, and in high- and low- dose progesterone was less than vehicle (P<0.01). Results show high expression of prepro-NMS in high dose progesterone (TBI-HP) rats compared to the high dose estrogen (TBI-HE), as well as vehicle (P<0.01). NMU content in low-dose progesterone (TBI-LP) group was more than that of vehicle group (P<0.001). Furthermore a difference in NMU content observed between TBI-HP compared to TBI-HE, and vehicle (P<0.05). The NMUR2 mRNA expression revealed an upregulation in TBI-HP rats compared to the TBI-HE group (P<0.001). CONCLUSION: Findings indicate that progesterone attenuates brain edema and induces an increase in NMS and its receptor which may mediate the anti-edematous effect of progesterone after TBI.

Noxious Stimulation of the Rat Tooth Pulp May Impair Learning and Memory Through the Induction of Hippocampal Apoptosis.

AIMS: To determine whether noxious stimulation of the rat tooth pulp induces learning and memory impairment through the induction of apoptosis in the hippocampus. METHODS: Thirty-five adult rats were divided randomly into five groups (each n=7) as follows: control, sham-operated, sham-vehicle, capsaicin-treated, and capsaicin plus ibuprofen-treated group. After preparing dental cavities via cutting 2 mm of the distal extremities of the mandibular incisors, polyethylene crowns were placed on the teeth. Based on the study groups, different injections were administered into the cavities. Nociceptive scores for each block were obtained by measuring the number of seconds that the animal spent rubbing and flicking the lower jaw. After recording the nociceptive behaviors, spatial learning and memory were assessed by using the Morris Water Maze (MWM) test. The hippocampal levels of Bcl-2, Bax, and caspase-3 protein were determined by immunoblotting. Statistical analyses were performed using one- or two-way analysis of variance. RESULTS: Noxious pulp stimulation induced by intradental application of capsaicin significantly increased time and traveled distance in the MWM test. Capsaicin stimulation of the pulp also significantly increased the Bax:Bcl-2 ratio and activated caspase-3 in the hippocampus (P<.01), which was inhibited by ibuprofen pretreatment (P<.05). CONCLUSION: Memory and learning impairment induced by noxious stimulation of the rat tooth pulp may be correlated with activation of apoptotic pathways in the hippocampus.

Changes in the gene expression of estrogen receptors involved in the protective effect of estrogen in rat's trumatic brain injury.

It has been demonstrated that estradiol has neuroprotective effects after traumatic brain injury (TBI) in female rats. Since estrogen receptors have an important role in estradiol effects at the cellular level and the exact mechanism(s) of estradiol-induced neuroprotection has not yet been fully clarified, the present study was designed to determine the changes in the levels of estrogen receptors mRNAs and proteins involved in this phenomenon. All experiments were carried out on female Wistar rats. The brain edema and blood-brain-barrier (BBB) disruption were assessed. The TBI method was diffuse type and induced by the Marmarou method. Semiquantitative RT-PCR and immunoblotting were used to assess ERα and ERß gene expression. The data showed that the level of brain water content was significantly increased in TBI group. The increased water content was significantly attenuated in estradiol-treated (1mg/kg) TBI rats. Disruption of BBB after TBI was significantly inhibited just by estradiol treatment. Estrogen-treated animals showed a significant increase in ERα mRNA (18%) and protein (35%) levels in the brain tissue. Furthermore, in the brain-injured rats the levels of ERß mRNA were lower than those in control rats. Following estrogen treatment, the protein levels of ERß were closed to those in control group. In conclusion, the data demonstrate that estrogen treatment can protect brain against traumatic brain injury. Estrogen treatment increases ER mRNA and protein levels which were coincident with its protective effects. It seems that such phenomenon participates in the induction of neuroprotective effects of estrogen. This article is part of a Special Issue entitled 1618.

Inhibitory effect of Thymus caramanicus Jalas on hyperglycemia-induced apoptosis in in vitro and in vivo models of diabetic neuropathic pain.

ETHNOPHARMACOLOGICAL RELEVANCE: Since Thymus caramanicus Jalas is used as a folk medicine for the treatment of rheumatism, skin disorders, bacterial infections and diabetes and it contain antioxidant agents, we decided to investigate the possible effects of Thymus caramanicus Jalas (TCJ) extract on in vitro and in vivo models of diabetic neuropathy. MATERIALS AND METHODS: The high glucose-induced cell injury in Pheochromocytoma (PC12) cells and streptozotocin-induced diabetic rats were used. Tail-flick and rotarod treadmill assessments were used to determine nociceptive threshold and motor coordination. Cell viability was determined by MTT assay test. Western blotting was performed to measurement of apoptosis markers. RESULTS: The data showed that elevation of glucose consecutively increases functional cell injury and apoptosis. Furthermore, diabetic rats developed thermal hyperalgesia and motor deficit. Activated caspase 3, cytochrome c release and Bax/Bcl-2 ratio were significantly increased in high glucose-treated PC12 cells and in spinal cord of diabetic animals. TCJ extract (60 and 80 µg/ml) attenuates high glucose-induced PC12 cells damage and apoptosis. In diabetic animals, TCJ extract at daily doses of 100 and 150 mg/kg ameliorated hyperalgesia and suppressed spinal apoptosis. CONCLUSION: The data indicate that TCJ extract has neuroprotective effects against high glucose-induced neural damage. These protective effects are mediated, at least in part, through attenuation of neural apoptosis and suggest therapeutic potential of TCJ extract in amelioration of diabetic neuropathy.

Cisplatin toxicity reduced in human cultured renal tubular cells by oxygen pretreatment.

Cisplatin is an effective and widely used chemotherapy agent and its side effects, particularly nephrotoxicity, limit its usage and related platinum-based drugs. Cisplatin nephrotoxicity is mainly due to extremely increase in reactive oxygen species (ROS) generation leading to kidney tubular cell death. Preconditioning with oxidative stress has been demonstrated to stimulate the cellular adaptation to subsequent severe oxidative stress. Short term oxygen pre-exposure as a mild oxidative stress may enhance some endogenous defense mechanisms, so its effect on Cisplatin induced cell death was investigated in present research. We studied the effects of hyperoxic environment pre-exposure on Cisplatin toxicity in an in-vitro model of cultured human embryonic tubular epithelial cells (AD293). Viability of AD293 cells, as evaluated by MTT-assay, was affected by Cisplatin in a time (1-4 h) dependent model. Biochemical markers of cell apoptosis were evaluated using immunoblotting. Pretreatment with nearly pure oxygen (≥90%) for 2 h significantly reduced the level of cell damage. Activated caspase 3 and Bax/Bcl-2 ratio were significantly increased in Cisplatin-treated cells. Oxygen pretreatment inhibited caspase 3 activation and decreased Bax/Bcl-2 ratio. Oxygen pre-treatment itself not showed any cytotoxicity in exposure times up to 3 h. Our data indicate that hyperoxic preconditioning reduces Cisplatin toxicity in cultured human tubular epithelial cells. The exact mechanism of protection is unclear, though enhancement of some endogenous defense mechanisms and subsequently scavenging of free oxygen radicals may play an important role.

Satureja khuzestanica attenuates apoptosis in hyperglycemic PC12 cells and spinal cord of diabetic rats.

Several studies have indicated the involvement of oxidative stress and high glucose-induced cell death in the development of diabetic neuropathy. Satureja khuzestanica has been recommended in the literature as a remedy for the treatment of diabetes, and also contains antioxidant agents. Here, we investigated the possible neuroprotective effects of Satureja khuzestanica extract (SKE) on in vitro and in vivo models of diabetic neuropathy pain. High-glucose-induced damage to pheochromocytoma (PC12) cells and in streptozotocin-induced diabetic rats was studied. Tail-flick and rotarod treadmill tests were used to access nociceptive threshold and motor coordination, respectively. Cell viability was determined by MTT assay. Activated caspase 3 and Bax/Bcl-2 ratio-biochemical markers of apoptosis-were evaluated using immunoblotting. We found that elevating the glucose in the medium (to 4× normal) increased cell toxicity and caspase-3 activation in PC12 cells. Incubation with SKE (200 and 250 µg/ml) decreased cell damage. Furthermore, the diabetic rats developed neuropathy, which was evident from thermal hyperalgesia and motor deficit. Administering SKE at a daily dose of between 50 and 200 mg/kg to the diabetic animals for 3 weeks ameliorated hyperglycemia, weight loss, hyperalgesia, and motor deficit, inhibited caspase 3 activation, and decreased the Bax/Bcl-2 ratio. The results suggest that SKE exerts neuroprotective effects against hyperglycemia-induced cellular damage. The mechanisms of these effects may be related to (at least in part) the prevention of neural apoptosis, and the results suggest that Satureja has the therapeutic potential to attenuate side effects of diabetes, such as neuropathy.

Olive (Olea europaea L.) leaf extract attenuates early diabetic neuropathic pain through prevention of high glucose-induced apoptosis: in vitro and in vivo studies.

AIM OF STUDY: Since the leaves of olive have been recommended in the literature as a remedy for the treatment of diabetes and they also contain antioxidant agents, we decided to investigate the possible effects of olive leaf extract (OLE) on in vitro and in vivo models of diabetic pain neuropathy. MATERIALS AND METHODS: The high glucose-induced cell damage in naive and NGF-treated Pheochromocytoma (PC12) cells and streptozotocin-induced diabetic rats were used. Tail-flick test was used to access nociceptive threshold. Cell viability was determined by MTT assay. Biochemical markers of neural apoptosis were evaluated using immunoblotting. RESULTS: We found that elevation of glucose (4 times of normal) sequentially increases functional cell damage and caspase-3 activation in NGF-treated PC12 cells. Incubation of cells with OLE (200, 400 and 600 µg/ml) decreased cell damage. Furthermore, the diabetic rats developed neuropathic pain which was evident from decreased tail-flick latency (thermal hyperalgesia). Activated caspase 3 and Bax/Bcl2 ratio were significantly increased in spinal cord of diabetic animals. OLE treatment (300 and 500 mg/kg per day) ameliorated hyperalgesia, inhibited caspase 3 activation and decreased Bax/Bcl2 ratio. Furthermore, OLE exhibited potent DPPH free radical scavenging capacity. CONCLUSION: The results suggest that olive leaf extract inhibits high glucose-induced neural damage and suppresses diabetes-induced thermal hyperalgesia. The mechanisms of these effects may be due, at least in part, to reduce neuronal apoptosis and suggest therapeutic potential of olive leaf extract in attenuation of diabetic neuropathic pain.

Establishment, characterization, and drug sensitivity of a new Ewing sarcoma cell line (SS-ES-1).

Ewing sarcoma (ES) is one of the most malignant tumors in children and young adults. We present here a new ES cell line, SS-ES-1, established from the left thoracic tumor of a 16-year-old female patient. The SS-ES-1 cells retained genotype, morphology, and growth rate for over 150 passages. Immunocytochemical staining showed the strong immunoreactivity for cytokeratin, epithelial membrane antigen, neurofilament, CD99, P53, Ki-67, platelet-derived growth factor receptor-ß, estrogen receptor-α (ER-α), and Bcl-2, but no reactivity for glial fibrillary acidic protein, epidermal growth factor receptor, and HER-2/neu. The presence of the type 1 EWS/FLI-1 fusion transcripts was confirmed by reverse transcriptase-polymerase chain reaction. On the basis of the MTT assay results, GW2974, a dual inhibitor of epidermal growth factor receptor and HER-2/neu, exhibited only a weak cytotoxic response in SS-ES-1 cells. In contrast, tyrphostin A9, a specific inhibitor of platelet-derived growth factor receptor, had a high cytotoxic effect against these cells. Surprisingly, it was found that SS-ES-1 cells displayed a high sensitivity to 4OH-tamoxifen. In conclusion, the SS-ES-1 cell line shows unique cellular properties, which makes it a useful model for studying various aspects of the biology of ES. In addition, the results suggest that ER can be a good therapeutic target for ER + ES.