Aging-Associated Thyroid Dysfunction Contributes to Oxidative Stress and Worsened Functional Outcomes Following Traumatic Brain Injury.

The incidence of traumatic brain injury (TBI) increases dramatically with advanced age and accumulating evidence indicates that age is one of the important predictors of an unfavorable prognosis after brain trauma. Unfortunately, thus far, evidence-based effective therapeutics for geriatric TBI is limited. By using middle-aged animals, we first confirm that there is an age-related change in TBI susceptibility manifested by increased inflammatory events, neuronal death and impaired functional outcomes in motor and cognitive behaviors. Since thyroid hormones function as endogenous regulators of oxidative stress, we postulate that age-related thyroid dysfunction could be a crucial pathology in the increased TBI severity. By surgically removing the thyroid glands, which recapitulates the age-related increase in TBI-susceptible phenotypes, we provide direct evidence showing that endogenous thyroid hormone-dependent compensatory regulation of antioxidant events modulates individual TBI susceptibility, which is abolished in aged or thyroidectomized individuals. The antioxidant capacity of melatonin is well-known, and we found acute melatonin treatment but not liothyronine (T3) supplementation improved the TBI-susceptible phenotypes of oxidative stress, excitotoxic neuronal loss and promotes functional recovery in the aged individuals with thyroid dysfunction. Our study suggests that monitoring thyroid function and acute administration of melatonin could be feasible therapeutics in the management of geriatric-TBI in clinic.

Multi-Targeting Andrographolide, a Novel NF-κB Inhibitor, as a Potential Therapeutic Agent for Stroke.

A key focus in the field of drug discovery has been motivated by the neuroprotection of natural compounds. Cerebral ischemia is a multifaceted pathological process with a series of mechanisms, and a perspective for the development of neuroprotectants from traditional herbal medicine or natural products is a promising treatment for this disease. Natural compounds with the effects of anti-oxidation, anti-inflammation, anti-apoptosis, and neurofunctional regulation exhibit therapeutic effects on experimental ischemic brain injury. Conferring to the pharmacological mechanisms underlying neuroprotection, a study found that androgapholide, a diterpene lactone compound, exhibits varying degrees of neuroprotective activities in both in vitro and in vivo experimental models of stroke. The neuroprotective mechanisms of andrographolide are suggested as: (I) increasing nuclear factor E2-related factor 2-heme oxygenase (Nrf2-HO-1) expression through p38-mitogen activated protein kinase (MAPK) regulation, (II) inducing cerebral endothelial cells (CEC) apoptosis and caspase-3 activation, (III) down regulating Bax, inducible nitric oxide synthase (iNOS), and (IV) inhibiting hydroxyl radical (OH-) formation, and activating transcription factor NF-κB signaling pathways. Recently, several researchers have also been trying to unveil the principal mechanisms involved in the neuroprotective effects of andrographolide. Therefore, this review aims to summarize an overview on the neuroprotective effects of andrographolide and exemplifies the essential mechanisms involved. This paper can provide information that andrographolide drug discovery may be a promising strategy for the development of a novel class of neuroprotective drug.

The neuroprotective effects of Tao-Ren-Cheng-Qi Tang against embolic stroke in rats.

BACKGROUND: Combinations of the traditional Chinese and Western medicines have been used to treat numerous diseases throughout the world, and there is a growing body of evidence showing that some of the herbs used in traditional Chinese medicine elicit significant pharmacological effects. The aim of this study was to demonstrate the neuroprotective effects of Tao-Ren-Cheng-Qi Tang (TRCQT) in combination with aspirin following middle cerebral artery occlusion (MCAO)-induced embolic stroke in rats. METHODS: A blood clot was embolized into the middle cerebral artery of rats to induce focal ischemic brain injury. After 24 h of MCAO occlusion, the rats were arbitrarily separated into five groups and subjected to different oral treatment processes with TRCQT and aspirin for 30 days before being evaluated in terms of their neurological behavior using a four-point system. The rats were sacrificed at 30 days after drug treatment and the infarct volumes were measured using a 2,3,5-triphenyltetrazolium chloride staining method. Tumor necrosis factor-α (TNF-α), c-Jun N-terminal kinases (JNK), activated caspase-3 and Bax were detected by western blot analysis. The apoptotic cells were identified by Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. ROS generation was also measured by electron spin resonance spectrometry. RESULTS: Rats treated with TRCQT alone or in combination with aspirin showed a significantly reduced infarct volume (P < 0.001) and improved neurological outcome compared with those treated with distilled water. Rats treated with TRCQT alone (P = 0.021) or in combination with aspirin (P = 0.02) also showed significantly reduced MCAO-induced expression levels of TNF-α and pJNK (P < 0.001) in their ischemic regions. Rats treated with TRCQT alone or in combination with aspirin showed decreased apoptosis by a reduction in the number of TUNEL positive cells, which inhibited the expression of activated caspase-3 (P = 0.038) and Bax (P = 0.004; P = 0.003). TRCQT also led to a significant concentration-dependent reduction in the formation of hydroxyl radicals (P < 0.001). CONCLUSIONS: TRCQT reduced brain infarct volume and improved neurological outcomes by reducing apoptosis, attenuating the expression of TNF-α and p-JNK, and reducing the formation of hydroxyl radicals in MCAO-induced embolic stroke of rats.

Antrodia camphorata Potentiates Neuroprotection against Cerebral Ischemia in Rats via Downregulation of iNOS/HO-1/Bax and Activated Caspase-3 and Inhibition of Hydroxyl Radical Formation.

Antrodia camphorata (A. camphorata) is a fungus generally used in Chinese folk medicine for treatment of viral hepatitis and cancer. Our previous study found A. camphorata has neuroprotective properties and could reduce stroke injury in cerebral ischemia animal models. In this study, we sought to investigate the molecular mechanisms of neuroprotective effects of A. camphorata in middle cerebral artery occlusion (MCAO) rats. A selective occlusion of the middle cerebral artery (MCA) with whole blood clots was used to induce ischemic stroke in rats and they were orally treated with A. camphorata (0.25 and 0.75 g/kg/day) alone or combined with aspirin (5 mg/kg/day). To provide insight into the functions of A. camphorata mediated neuroprotection, the expression of Bax, inducible nitric oxide synthase (iNOS), haem oxygenase-1 (HO-1), and activated caspase-3 was determined by Western blot assay. Treatment of aspirin alone significantly reduced the expressions of HO-1 (P < 0.001), iNOS (P < 0.001), and Bax (P < 0.01) in ischemic regions. The reduction of these expressions was more potentiated when rats treated by aspirin combined with A. camphorata (0.75 g/kg/day). Combination treatment also reduced apoptosis as measured by a significant reduction in active caspase-3 expression in the ischemic brain compared to MCAO group (P < 0.01). Moreover, treatment of A. camphorata significantly (P < 0.05) reduced fenton reaction-induced hydroxyl radical (OH(•)) formation at a dose of 40 mg/mL. Taken together, A. camphorata has shown neuroprotective effects in embolic rats, and the molecular mechanisms may correlate with the downregulation of Bax, iNOS, HO-1, and activated caspase-3 and the inhibition of OH(•) signals.

Hinokitiol Negatively Regulates Immune Responses through Cell Cycle Arrest in Concanavalin A-Activated Lymphocytes.

Autoimmune diseases are a group of chronic inflammatory diseases that arise from inappropriate inflammatory responses. Hinokitiol, isolated from the wood of Chamaecyparis taiwanensis, engages in multiple biological activities. Although hinokitiol has been reported to inhibit inflammation, its immunological regulation in lymphocytes remains incomplete. Thus, we determined the effects of hinokitiol on concanavalin A- (ConA-) stimulated T lymphocytes from the spleens of mice. In the present study, the MTT assay revealed that hinokitiol (1-5 µM) alone did not affect cell viability of lymphocytes, but at the concentration of 5 µM it could reduce ConA-stimulated T lymphocyte proliferation. Moreover, propidium iodide (PI) staining revealed that hinokitiol arrested cell cycle of T lymphocytes at the G0/G1 phase. Hinokitiol also reduced interferon gamma (IFN-γ) secretion from ConA-activated T lymphocytes, as detected by an ELISA assay. In addition, hinokitiol also downregulated cyclin D3, E2F1, and Cdk4 expression and upregulated p21 expression. These results revealed that hinokitiol may regulate immune responses. In conclusion, we for the first time demonstrated that hinokitiol upregulates p21 expression and attenuates IFN-γ secretion in ConA-stimulated T lymphocytes, thereby arresting cell cycle at the G0/G1 phase. In addition, our findings also indicated that hinokitiol may provide benefits to treating patients with autoimmune diseases.

Chinese medicines and bioactive compounds for treatment of stroke.

Stroke is an important cause of mortality and morbidity worldwide but effective therapeutic strategy for the prevention of brain injury in patients with cerebral ischemia is lacking. Although tissue plasminogen activator has been used to treat stroke patients, this therapeutic strategy is confronted with ill side effects and is limited to patients within 3 h of a stroke. Due to the complexity of the events and the disappointing results from single agent trials, the combination of thrombolytic therapy and effective neural protection therapy may be an alternative strategy for patients with cerebral ischemia. Chinese medicine (CM) is the most widely practiced form of herbalism worldwide, as it is a sophisticated system of medical theory and practice that is specifically different from Western medicine. Most traditional therapeutic formulations consist of a combination of several drugs. The combination of multiple drugs is thought to maximize therapeutic efficacy by facilitating synergistic actions and preventing possible adverse effects while at the same time marking at multiple targets. CM has been labeled in ancient medicine systems as a treatment for various diseases associated with stroke. This review summarizes various CMs, bioactive compounds and their effects on cerebral ischemia.

Potential advantages of Chinese medicine Taohong Siwu Decoction () combined with tissue-plasminogen activator for alleviating middle cerebral artery occlusion-induced embolic stroke in rats.

OBJECTIVE: To investigate whether combination treatment with Taohong Siwu Decoction (, TSD) and recombinant tissue-type plasminogen activator (rt-PA) potentiate in reducing infarct volume and alleviate thromboembolic stroke in an in vivo rat model. METHOD: Adult male Wistar rats were subjected to embolic middle cerebral artery occlusion (MCAO) and treated with rt-PA (4 and 8 mg/kg) alone (n=5), TSD [0.7 g/(kg·day)] alone (n=5), combination of rt-PA and TSD, 24 h after stroke. Rats were sacrificed at 14 days after treatment and lesion volumes were measured. To investigate the underlying mechanism of neuroprotective effect of the combination treatment, cleaved caspase-3, tumor necrosis factor alpha (TNF-α), hypoxia-inducible factor (HIF)-1α, and inducible nitric oxide synthase (iNOS) immunostaining were performed. RESULTS: Combination treatment significantly reduced infarct volume of cerebral ischemic regions compared with treatment of rt-PA and TSD alone and that of the saline control group (P<0.01). A combined treatment of rt-PA (4 mg/kg) with TSD [0.7 g/(kg·day)] significantly increased cerebral blood flow in a time (100 and 120 min) dependent manner (P<0.05). Interestingly, despite treatment of rt-PA (4 mg/kg) alone significantly reduced the expressions of HIF-1α, TNF-α, and iNOS in ischemic regions, reduction of these expressions were more potentiated when combined with TSD (P<0.05). Combination treatment also reduced apoptosis as measured by a significant reduction in active caspase-3 expression in the ischemic brain compared with the MCAO group (P<0.01). CONCLUSIONS: A combination of low-dose rt-PA and TSD after embolic stroke reduced infarct volume, improved cerebral blood flow and provided neuroprotection and these effects were associated with reduction of apoptosis and attenuation of HIF-1α, TNF-α and iNOS expression. These results provide a positive contribution to better understand the therapeutic value of the combination of TSD with rt-PA in ischemic stroke and may support further clinical evaluation.

Extract of Antrodia camphorata exerts neuroprotection against embolic stroke in rats without causing the risk of hemorrhagic incidence.

In this study, the neuroprotective effect of an extract of Antrodia camphorata (A. camphorata), a fungus commonly used in Chinese folk medicine for treatment of viral hepatitis and cancer, alone or in combination with aspirin was investigated in a rat embolic stroke model. An ischemic stroke was induced in rats by a selective occlusion of the middle cerebral artery (MCA) with whole blood clots and then orally treated with A. camphorata (0.25 and 0.75 g/kg/day) alone and combined with aspirin (5 mg/kg/day). Sixty days later, the brains were removed, sectioned, and stained with triphenyltetrazolium chloride and analysed by a commercial image processing software program. Brain infarct volume, neurobehavioral score, cerebral blood perfusion, and subarachnoid and intracerebral hemorrhage incidence were perceived. In addition, potential bleeding side effect of the combinative therapy was assessed by measuring hemoglobin (Hb) content during intracerebral hemorrhage and gastric bleeding, prothrombin time (PT), and occlusion time (OT) after oral administration. Posttreatment with high dose A. camphorata significantly reduced infarct volume and improved neurobehavioral score (P < 0.05). Since A. camphorata alone or with aspirin did not alter the Hb level, this treatment is safe and does not cause hemorrhagic incident. Remarkably, the combination of A. camphorata and aspirin did not show a significant effect on the bleeding time, PT and OT increase suggesting that A. camphorata may have the neuroprotective effect without the prolongation of bleeding time or coagulation time. From these observations, we suggest that combinative therapy of A. camphorata and aspirin might offer enhanced neuroprotective efficacies without increasing side effects.

Amarogentin, a secoiridoid glycoside, abrogates platelet activation through PLC γ 2-PKC and MAPK pathways.

Amarogentin, an active principle of Gentiana lutea, possess antitumorigenic, antidiabetic, and antioxidative properties. Activation of platelets is associated with intravascular thrombosis and cardiovascular diseases. The present study examined the effects of amarogentin on platelet activation. Amarogentin treatment (15~60 µM) inhibited platelet aggregation induced by collagen, but not thrombin, arachidonic acid, and U46619. Amarogentin inhibited collagen-induced phosphorylation of phospholipase C (PLC) γ2, protein kinase C (PKC), and mitogen-activated protein kinases (MAPKs). It also inhibits in vivo thrombus formation in mice. In addition, neither the guanylate cyclase inhibitor ODQ nor the adenylate cyclase inhibitor SQ22536 affected the amarogentin-mediated inhibition of platelet aggregation, which suggests that amarogentin does not regulate the levels of cyclic AMP and cyclic GMP. In conclusion, amarogentin prevents platelet activation through the inhibition of PLC γ2-PKC cascade and MAPK pathway. Our findings suggest that amarogentin may offer therapeutic potential for preventing or treating thromboembolic disorders.

Hinokitiol, a natural tropolone derivative, offers neuroprotection from thromboembolic stroke in vivo.

Hinokitiol ( ß -thujaplicin), a tropolone-related compound found in the heartwood cupressaceous plants, is widely used in hair tonics, tooth pastes, cosmetics, and food as an antimicrobial agent. Increasing evidence has confirmed that hinokitiol exhibits anticancer activity in a variety of cancers through inhibition of cell proliferation. In the present study, we have investigated the neuroprotective effect and mechanisms of hinokitiol in rats against middle cerebral artery occlusion (MCAO)-induced thromboembolic stroke. Treatment with hinokitiol (0.2 and 0.5 mg/kg; intraperitoneally) 30 min before MCAO dose dependently attenuated cerebral ischemia and improved neurobehavioral deficits in cerebral ischemic rats. Intraperitoneal administration of hinokitiol significantly reduced infarct size compared to that in control rats. MCAO-induced focal cerebral ischemia was associated with increased expressions of hypoxia-inducible factor (HIF)-1 α , inducible nitric oxide synthase (iNOS), tumor necrosis factor (TNF)- α , and active caspase-3 in ischemic regions. However, these expressions were obviously inhibited by hinokitiol (0.2 and 0.5 mg/kg) treatment. This study demonstrates for the first time that in addition to being originally considered as an agent against microbes and variety of cancers, hinokitiol possesses potent neuroprotective activity. This activity is mediated, at least in part, by inhibition of inflammatory responses (i.e., HIF-1 α , iNOS expression) and apoptosis (i.e., TNF- α , active caspase-3), resulting in a reduction of infarct volume and improvement in neurobehavior in rats with cerebral ischemia. Therefore, the therapeutic potential of hinokitiol may lead to novel role for treatment or prevention of ischemia/reperfusion injury-related disorders.

Mechanisms of andrographolide-induced platelet apoptosis in human platelets: regulatory roles of the extrinsic apoptotic pathway.

Andrographolide, a novel nuclear factor-κB (NF-κB) inhibitor, is isolated from the leaves of Andrographis paniculata. Platelet activation is relevant to a variety of coronary heart diseases. Our recent studies revealed that andrographolide possesses potent antiplatelet activity by inhibition of the p38 MAPK/(●) HO-NF-κB-ERK2 cascade. Although platelets are anucleated cells, apoptotic machinery apparatus recently has been found to regulate platelet activation and limit platelet lifespan. Therefore, we further investigated the regulatory effects of andrographolide on platelet apoptotic events. In this study, apoptotic signaling events for caspase-3, -8, and Bid were time (10-60 min)- and dose (25-100 µΜ)-dependently activated by andrographolide in human platelets. Andrographolide could also disrupt mitrochondrial membrane potential. In addition, caspase-8 inhibitor (z-IETD-fmk, 50 µΜ) was found to reverse andrographolide-induced caspase-8 activation, whereas the antagonistic anti-Fas receptor (ZB4, 500 ng/mL) and anti-tumor necrosis factor-R1 (H398, 10 µg/mL) monoclonal antibodies did not. In conclusion, this study for the first time demonstrated that andrographolide might limit platelet lifespan by initiating the caspase-8-dependent extrinsic apoptotic pathway, in spite of no direct evidence that death receptors are involved in this process proved. Overall, the various medicinal properties of andrographolide suggest its potential value in treating patients with thromboembolic disorders.

Traditional Chinese medicine, Xue-Fu-Zhu-Yu decoction, potentiates tissue plasminogen activator against thromboembolic stroke in rats.

AIM OF THIS STUDY: The Xue-Fu-Zhu-Yu decoction (XFZYD) is a well-known traditional Chinese medicine for treating cardiovascular diseases. The therapeutic effects of this XFZYD have been well documented especially in treating of atherosclerosis and hyperlipidemia. Since this decoction can induce endothelial progenitor cell angiogenesis, it can provide experimental evidence for the treatment of ischemic diseases. Patients who are admitted to the hospital with acute ischemic stroke are initially considered candidates for the recombinant tissue plasminogen activator (rt-PA). However, rt-PA therapy is still lesser than ideal due to its major side effect of hemorrhaging. Therefore, medical research has been devoted to finding an alternative and/or complementary therapy for ischemic stroke. In the present study, we evaluated the protective effect of the combination of XFZYD with or without rt-PA in a rat model of thromboembolic stroke. MATERIALS AND METHODS: A cerebral thromboembolic stroke animal model and immunoblotting analysis were used to assess the effects of XFZYD and rt-PA. RESULTS: Treatment with rt-PA (8 mg/kg) or XFZYD (1.5 and 3.0 g/kg/day) alone showed slight reductions in the infarct volume compared to solvent-treated rats. However, XFZYD (1.5 and 3.0 g/kg/day) obviously potentiated rt-PA-mediated reduction in the infarct volume in cerebral ischemic regions. In addition, treatment with rt-PA significantly reduced both tumor necrosis factor (TNF)-α and inducible nitric oxide synthase (iNOS) but not hypoxia-inducible factor (HIF)-1 α or active caspase-3 expressions in ischemic regions, whereas treatment with XFZYD (3.0 g/kg/day) significantly reduced all of these protein expressions in ischemic regions. Moreover, treatment with XFZYD (1.5 and 3.0 g/kg/day) obviously potentiated rt-PA-mediated reductions in TNF-α, iNOS, HIF-1 α, and active caspase-3 expressions. CONCLUSIONS: Results of this study suggest that XFZYD potentiated rt-PA-mediated neuroprotection against thromboembolic stroke in rats. This neuroprotection is probably mediated by the inhibition of HIF-1 α and TNF-α, followed by the inhibition of inflammatory responses (i.e., iNOS) and apoptosis (active caspase-3). These results provide a better understanding of the scientific validation of the therapeutic value of the combination of XFZYD with rt-PA in ischemic stroke.

Neuroprotection by the Traditional Chinese Medicine, Tao-Hong-Si-Wu-Tang, against Middle Cerebral Artery Occlusion-Induced Cerebral Ischemia in Rats.

Tao-Hong-Si-Wu-Tang (THSWT) is a famous traditional Chinese medicine (TMC). In the present study, oral administration of THSWT (0.7 and 1.4 g kg(-1)day(-1)) for 14 days before MCAO dose-dependently attenuated focal cerebral ischemia in rats. MCAO-induced focal cerebral ischemia was associated with increases in hypoxia-inducible factor (HIF)-1α, inducible nitric oxide synthase (iNOS), tumor necrosis factor (TNF)-α, and active caspase-3 expressions in ischemic regions. These expressions were obviously inhibited by 0.7 g kg(-1)day(-1) THSWT treatment. In addition, THSWT inhibited platelet aggregation stimulated by collagen in washed platelets. In an in vivo study, THSWT (16 g kg(-1)) significantly prolonged platelet plug formation in mice. However, THSWT (20 and 40 µg mL(-1)) did not significantly reduce the electron spin resonance (ESR) signal intensity of hydroxyl radical (OH(•)) formation. In conclusion, the most important findings of this study demonstrate for the first time that THSWT possesses potent neuroprotective activity against MCAO-induced focal cerebral ischemia in vivo. This effect may be mediated, at least in part, by the inhibition of both HIF-1α and TNF-α activation, followed by the inhibition of inflammatory responses (i.e., iNOS expression), apoptosis formation (active caspase-3), and platelet activation, resulting in a reduction in the infarct volume in ischemia-reperfusion brain injury.