Neuroprotective effects of a novel poly (ADP-ribose) polymerase-1 inhibitor, JPI-289, in hypoxic rat cortical neurons.

Excessive activation of poly (ADP-ribose) polymerase-1 (PARP-1) is known to develop neuronal apoptosis, necrosis and inflammation after ischaemic brain injury. Therefore, PARP-1 inhibition after ischaemic stroke has been attempted in successful animal studies. The purpose of present work was to develop a novel water soluble PARP-1 inhibitor (JPI-289) and explore its neuroprotective effect on ischaemic injury in an in vitro model. The half-life of JPI-289 after intravenous or oral administration in rats was relatively long (1.4-1.5 hours) with 65.6% bioavailability. The inhibitor strongly inhibited PARP-1 activity (IC50 =18.5 nmol/L) and cellular PAR formation (IC50 =10.7 nmol/L) in the nanomolar range. In rat cortical neuronal cells, JPI-289 did not affect cell viability up to 1 mmol/L as assayed by Trypan blue staining (TBS) and lactate dehydrogenase (LDH) assay. Treatment of JPI-289 for 2 hours after 2 hours of oxygen glucose deprived (OGD) rat cortical neuron attenuated PARP activity and restored ATP and NAD+ levels. Apoptosis-associated molecules such as apoptosis inducing factor (AIF), cytochrome C and cleaved caspase-3 were reduced after JPI-289 treatment in the OGD model. The present findings suggest that the novel PARP-1 inhibitor, JPI-289, is a potential neuroprotective agent which could be useful as a treatment for acute ischaemic stroke.

Newly developed glycogen synthase kinase-3 (GSK-3) inhibitors protect neuronal cells death in amyloid-beta induced cell model and in a transgenic mouse model of Alzheimer's disease.

Glycogen synthase kinase-3 (GSK-3) is emerging as a prominent therapeutic target of Alzheimer's disease (AD). A number of studies have been undertaken to develop GSK-3 inhibitors for clinical use. We report two novel GSK-3 inhibitors (C-7a and C-7b) showing good activity and pharmacokinetic (PK) profiles. IC50 of new GSK-3 inhibitors were in the range of 120-130 nM, and they effectively reduced the Aß-oligomers induced neuronal toxicity. Also, new GSK-3 inhibitors decreased the phosphorylated tau at pThr231, pSer396, pThr181, and pSer202, and inhibited the GSK-3 activity against Aß-oligomers induced neuronal cell toxicity. In B6;129-Psen1(tm1Mpm) Tg(APPSwe, tauP301L)1Lfa/Mmjax model of AD, oral administration of C-7a (20 mg/kg, 50 mg/kg) showed increased total arm entries and spontaneous alteration of Y-maze which was regarded as short-term memory. In particular, 50 mg/kg C-7a treated mice significantly decreased the level of phosphorylated tau (Ser396) in brain hippocampus. We suggest that new GSK-3 inhibitor (C-7a) is potential candidates for the treatment of AD.

Synthesis and evaluation of 8-amino-[1,2,4]triazolo[4,3-a]pyridin-3(2H)-one derivatives as glycogen synthase kinase-3 (GSK-3) inhibitors.

New potent glycogen synthase kinase-3 (GSK-3) inhibitors, 8-amino-[1,2,4]triazolo[4,3-a]pyridin-3(2H)-one derivatives, were designed by modeling, synthesized and evaluated in vitro. Compound 17c showed good potency in enzyme and cell-based assays (IC50=111 nM, EC50=1.78 µM). Moreover, it has demonstrated desirable water solubility, PK profile, and moderate brain penetration.

Anti-arthritic effects of Ephedra sinica STAPF herb-acupuncture: inhibition of lipopolysaccharide-induced inflammation and adjuvant-induced polyarthritis.

Anti-inflammatory and anti-arthritic effects of water distillates of Ephedra sinica STAPF (ES), in herb-acupuncture, on the inflammatory responses of arthritis was investigated using phorbol 12-myristate 13-acetate (PMA)/lipopolysaccharide (LPS)-induced human macrophage and adjuvant-induced arthritic rat. The luciferase reporter vectors driven by the tumor necrosis factor (TNF)-alpha and cyclooxygenase-2 promoters were transiently transfected into U937 cells, which were then differentiated and stimulated by PMA and LPS, respectively, to develop an in vitro anti-inflammation assay system. The luciferase activities, observed in the activated U937 cells, were significantly inhibited by ES herb-acupuncture, compared to those of PD98509 and berberine. To evaluate ES herb-acupuncture as a novel anti-arthritic therapy, a polyarthritic rat model was developed using heat-killed Mycobacterium tuberculosis, and 50 mul of ES distillate was subcutaneously injected into the ST36 acupoint on each knee joint. While the articular indexes of arthritic rats were evidently decreased by ES herb-acupuncture, their body weights did not regain their initial levels. This may be due to the accelerating effects of ES on weight-loss and fat consumption. The mRNA expressions of TNF-alpha and interleukin (IL)-6 genes, which were closely stimulated in the arthritic rat joints, were found to be restored to the normal levels through the ES treatment. In the case of IL-1beta, the recovery was not significant but substantial. The anti-arthritic effect of ES herb-acupuncture was not found in the ES-treated/non-acupoint group. In conclusion, the ES herb-acupuncture into the ST36 acupoint was found to be effective in alleviating the inflammatory response and thus arthritic symptoms in adjuvant-induced arthritic rats.

Gene expression profiling in hypothalamus of immobilization-stressed mouse using cDNA microarray.

To investigate the effects of repeated immobilization-stress challenge on HPA axis, genomic transcriptome in the hypothalamus of immobilization-stressed mouse was analyzed by using cDNA microarray. With the 1.5-fold cutoff of arbitrary criteria, the expression levels of 108 genes out of 6016 genes were significantly modulated in the hypothalamus by the stress. Energy metabolism-, lipid metabolism-, and apoptosis- and signal transduction-related genes were activated while DNA repair-, protein biosynthesis-, and structure integrity-related genes were down-regulated in the hypothalamus. Eighteen genes among them were selected for RT-PCR analysis to confirm the change of their expression levels on agarose gels. Besides, dozens of novel genes, which have not been previously reported, were screened to be modulated by the immobilization stress through the transcriptome analysis. These genes are related to apoptosis, tumor-suppression, DNA-binding and protein folding, and thus may be used as potential targets for the development of therapeutics of chronic stress or depressant.

Therapeutic effects of Hominis placenta injection into an acupuncture point on the inflammatory responses in subchondral bone region of adjuvant-induced polyarthritic rat.

Rheumatoid arthritis (RA) is characterized by chronic inflammation of the synovial membrane in the joint, which leads to the progressive destruction of articular cartilage, ligament and bone. Several cytokines such as tumor necrosis factor alpha (TNF-alpha), interleukin 1beta (IL-1beta), and interleukin 6 (IL-6) have been implicated in the pathological mechanisms of synovial tissue proliferation, joint destruction and programmed cell death in rheumatoid joint. In the Korean traditional medicine, Hominis placenta (HP) as an herbal component of herb-acupuncture has been widely used to treat chronic inflammatory diseases such as RA. To study the therapeutic effects of HP injection into the ST36 acupoint (HP herb-acupuncture) on the inflammatory responses of a subchondral region of rheumatoid joint, the polyarthritis-induced Sprague-Dawley (SD) rat was developed as a rheumatoid arthritis model by the intradermal injection of dried cells of Mycobacterium tuberculosis emulsified in squalene to the base of tail. After the onset stage (11 d after adjuvant injection) of polyarthritis, a fixed volume of HP extract was daily injected to Zusanli (ST36) acupoint on the rat's leg for 2 weeks. The body weight, paw volume of the knee joint and articular index were exploited as an assessment method addressing arthritic symptoms, and the expression profiles of TNF-alpha, IL-1beta and IL-6 at the subchondral bone of the joint were analyzed using an immunohistochemistry. After the treatment of arthritic rats with HP, the body weights and paw volumes of arthritic rats were almost restored to the levels of normal rats whereas the evaluation by the articular index was not remarkable. The TNF-alpha, IL-1beta and IL-6 positive cells in the immunohistological sections of subchondral bone region of the joint significantly decreased in HP-treated (ST36 acupoint) arthritic group as compared with those in non-treated or HP-treated (non-acupoint) ones, which was coincident with the behavioral studies. In conclusion, the HP herb-acupuncture was found to be effective to alleviate the arthritic symptoms in adjuvant-induced arthritis rats as regards the body weight, joint appearance and the expression profiles of inflammatory cytokines.