[Therapeutic effects of moxibustion on neonatal mice with hypoxia-ischemia brain injury].

Objective: To investigate the effects of moxibustion on the behavioral performance, brain morphological structure of mice with hypoxia-ischemia brain injury and to explore its mechanisms. Methods: One hundred and six ICR mice were randomly divided into three groups, sham group (n=23), model group (n=46) and moxibustion-treated group (n=37). Neonatal hypoxic-ischemia brain injury was induced by ligation of common carotid artery followed by hypoxia (8% oxygen, 100 min), and pups in the moxibustion-treated group were administered suspended moxibustion on the Dazhui points (GV14) at a height of approximately 2 cm over a hairless area of the skin once a day for 4 days (i.e. at 2, 24, 48 and 72 hours after hypoxia-ischemia procedure). Behavioral tests were used to evaluate behavioral performance. HE staining was used to observe brain morphological structure. Western blot was used to detect the expression of SOD2 protein, and spectrophotometry was used to determine the content of MDA in the ipsilateral brain. Results: Mouse pups in sham group showed that the behavioral performance was normal, the brain tissue cells were densely and neatly arranged, the expression of SOD2 and the level of MDA in the brain tissues were normal. Compared with sham group, mouse pups in the HI model group exhibited a significant longer latency to complete the righting reflex, geotaxis reflex, cliff avoidance (P＜0.05) and a marked shorter latency to complete the grip test (P＜0.05); and the HI model group had dramatic brain morphological changes showing missing regions, decreased expression of SOD2 protein (P＜0.05) and increased level of MDA in the brain. Compared with HI model group, mouse pups in the moxibustion-treated group exhibited a significant shorter latency to complete the righting reflex, geotaxis reflex, cliff avoidance test (P＜0.05) and a marked longer latency to complete the grip test (P＜0.05); and the moxibustion-treated group had less brain morphological changes, increased expression of SOD2 protein (P＜0.05) and decreased level of MDA in the brain (P＜0.05) . Conclusion: Moxibustion could improve behavioral performance and attenuate hypoxia-ischemia brain injury, which might be related to increasing the expression of SOD2 protein and decreasing the content of MDA, thus enhancing the anti-oxidative ability.

[Effect of moxibustion on learning and memory in neonatal hypoxic-ischemia encephalopathy mice].

OBJECTIVE: To observe the effect of moxibustion on the expression of phosphorylated calcium/calmodulin-dependent protein kinase Ⅱα(pCaMKⅡα) and neuronal nuclei (NeuN) and the ability of learning and memory in the neonatal mice model of hypoxic-ischemia encephalopathy(HIE), so as to explore its mechanism underlying improvement of learning and memory. METHODS: ICR mice (aged 7 days) were randomly divided into sham operation, model and moxibustion groups. HIE model was induced by ligation of the right common carotid artery combined with hypoxia in a closed transparent chamber. Mice in the moxibustion group were treated with gentle moxibustion at "Dazhui"(GV14) for 35 min，once daily for 3 consecutive days. The pathological changes of brain tissues were observed with the naked eyes and under microscope after H.E. staining, respectively. The expressions of pCaMKⅡα and NeuN in the ischemic penumbra were examined by immunofluorescent staining, and the learning and memory ablility was tested with Morris maze. RESULTS: No infarcts were found in the brain tissue of the mice in the sham operation group. Compared with the sham operation group, mice in the model group had infarcts and the expression of pCaMKⅡα and NeuN in the ischemic penumbra was significantly reduced (P<0.01), and the latency to find a platform was significantly prolonged in Morris maze test (P<0.01). After moxibustion, in comparison with the model group showed that, small areas of infarction were seen in the right hemisphere of the moxibustion group, and the expressions of pCaMKⅡα, NeuN increased significantly (P<0.01), and the latency to find a platform was significantly shortened (P<0.01). CONCLUSION: Moxibustion can improve the ability of learning and memory in the neonatal mice with HIE, which might be related to alleviating brain injury and increasing the expression of pCaMKⅡα in neurons of ischemic brain tissues.

[Effect of medicated thread moxibustion of Chinese Zhuang Minority medicine on colonic inflammatory impairment and Th17/IL-17F signaling in rats with ulcerative colitis].

OBJECTIVE: To observe the effect of medicated thread moxibustion of Zhuang Minority medicine on helper T cell 17 (Th17)/ Interleukin-17F(IL-17F) signaling pathway in ulcerative colitis (UC) rats, so as to explore its mechanisms underlying improvement of UC. METHODS: Forty male SD rats were randomly divided into normal control, model, medication and medicated thread moxibustion (MTM) groups, with 10 rats in each group. The UC model was induced by free drinking of 4% dextran sulfate sodium (DSS) for 10 d. After successful modeling, rats of the medication group were treated by gavage of salazosulfapyridine (SASP). Medicated thread moxibustion was applied to unilateral "Tianshu" (ST25) and "Qihai" (CV6) alternatively for rats of the MTM group, once daily for 14 d. The body mass, stool shape, and fecal occult test were recorded and conducted daily to perform disease activity index (DAI) score. H．E. staining was used to display pathological changes of the colon tissue. The Th17 cells and IL-17F and retinoic acid related orphan receptor γ t (ROR-γt) in the colon tissue were detected by flow cytometry, and enzyme-linked immunosorbent assay (ELISA), respectively, and the expression levels of RORγt and IL-17F mRNA in colon tissue were detected by quantitive real-time PCR. RESULTS: After modeling, the DAI score, colonic Th17 percentage, RORγt and IL-17F contents, and RORγt and IL-17F mRNA expression were significantly increased in the model group in contrast to the normal control group (P<0.01, P<0.05). Following the intervention, all the aforementioned indexes were reversed in both medication and MTM groups (P<0.01, P<0.05). No significant differences were found between the medication and MTM groups in the levels of the above mentioned indexes (P>0.05, except RORγt and IL-17F mRNA expression). H．E. staining showed disappearance of goblet cells, infiltration of a large number of inflammatory cells, exfoliation of the epithelial tissue and edema of colonic mucosal in rats of the model group, which was relatively milder in both medication and MTM groups. CONCLUSION: Medicated thread moxibustion of Zhuang Minority medicine can reduce the inflammatory damage of colon tissue in UC rats, which is associated with its effects in suppressing the expression of RORγt, production of Th17 cells, and secretion of pro-inflammatory factor IL-17F in colon tissue.

Comparison of the anti-apoptotic effects of 15- and 35-minute suspended moxibustion after focal cerebral ischemia/reperfusion injury.

Heat-sensitive suspended moxibustion has a neuroprotective effect against focal cerebral ischemia/reperfusion injury, but the underlying mechanisms remain unclear. The duration of heat-sensitive suspended moxibustion (usually from 30 minutes to 1 hour) is longer than traditional suspended moxibustion (usually 15 minutes). However, the effects of 15- and 35-minute suspended moxibustion in rats with cerebral ischemia/reperfusion injury are poorly understood. In this study, we performed 15- or 35-minute suspended moxibustion at acupoint Dazhui (GV14) in an adult rat model of focal cerebral ischemia/reperfusion injury. Infarct volume was evaluated with the 2,3,5-triphenyltetrazolium chloride assay. Histopathological changes and neuronal apoptosis at the injury site were assessed by hematoxylin-eosin staining and terminal deoxynucleotidyl transferase dUTP nick end labeling assay. Caspase-9 and caspase-3 expression at the injury site was detected using immunofluorescent staining. Bax and Bcl-2 expression at the injury site was assessed using western blot assay. In the 35-minute moxibustion group, infarct volume was decreased, neuronal apoptosis was reduced, caspase-9, caspase-3 and Bax expression was lower, and Bcl-2 expression was increased, compared with the 15-minute moxibustion group. Our findings show that 35-minute moxibustion has a greater anti-apoptotic effect than 15-minute moxibustion after focal cerebral ischemia/reperfusion injury.

Neuroprotective Effects of a PSD-95 Inhibitor in Neonatal Hypoxic-Ischemic Brain Injury.

The postsynaptic density-95 inhibitor NA-1 uncouples NMDA glutamate receptors from downstream neurotoxic signaling pathways without affecting normal glutamate receptor function. NA-1 attenuates NMDA receptor-mediated neuronal cell death after stroke in multiple models and species. However, its efficacy in providing neuroprotection in models of neonatal hypoxic-ischemic brain injury has not yet been tested. In this study, a modified version of the Rice-Vannucci method for the induction of neonatal hypoxic-ischemic brain injury was performed on postnatal day 7 mouse pups. Animals received a single dose of NA-1 intraperitoneally either before or after right common carotid artery occlusion. All experiments were performed in a blinded manner. Infarct volumes were measured 1 and 7 days after the injury, while behavioral tests were conducted 1, 3, and 7 days after injury. Administration of NA-1 before right common carotid artery occlusion or immediately after ischemia significantly reduced infarct volume and improved neurobehavioral outcomes 1, 3, and 7 days post-injury. The neuroprotection and improvement in neurobehavioral outcomes conferred by NA-1 in this mouse neonatal hypoxic-ischemic injury model imply that NA-1 will be effective in reducing neonatal stroke damage and thus could potentially serve as a therapeutic drug for prevention or treatment of neonatal stroke.

Marine compound xyloketal B reduces neonatal hypoxic-ischemic brain injury.

Neonatal hypoxic-ischemic encephalopathy causes neurodegeneration and brain injury, leading to sensorimotor dysfunction. Xyloketal B is a novel marine compound isolated from a mangrove fungus Xylaria species (no. 2508) with unique antioxidant effects. In this study, we investigated the effects and mechanism of xyloketal B on oxygen-glucose deprivation-induced neuronal cell death in mouse primary cortical culture and on hypoxic-ischemic brain injury in neonatal mice in vivo. We found that xyloketal B reduced anoxia-induced neuronal cell death in vitro, as well as infarct volume in neonatal hypoxic-ischemic brain injury model in vivo. Furthermore, xyloketal B improved functional behavioral recovery of the animals following hypoxic-ischemic insult. In addition, xyloketal B significantly decreased calcium entry, reduced the number of TUNEL-positive cells, reduced the levels of cleaved caspase-3 and Bax proteins, and increased the level of Bcl-2 protein after the hypoxic-ischemic injury. Our findings indicate that xyloketal B is effective in models of hypoxia-ischemia and thus has potential as a treatment for hypoxic-ischemic brain injury.

[Effect of c-myb on hCG-induced testosterone secretion in isolated rat Leydig cells].

The present study was carried out to investigate the effect of antisense c-myb oligodeoxynucleotides (ODN) on hCG-induced testosterone secretion in isolated rat Leydig cells. The effects of cAMP, Ca(2+) and cycloheximide (CYX) on c-Myb protein expression and testosterone secretion were also observed. The results showed that antisense c-myb ODN inhibited hCG-induced testosterone secretion of isolated rat Leydig cells in a dose-dependent manner. At the same time, integral optical density immunostaining of Myb in Leydig cells was also remarkably reduced. Nonsense tat ODN had no effect on Leydig cells. Further experiments showed that dbcAMP (100 micromol/L) obviously increased hCG-induced testosterone secretion and integral optical density (IOD) immunostaining of Myb in Leydig cells. Verapamil (10 micromol/L), a Ca(2+) channel blocker, and cycloheximide (50 microg/ml), a protein synthesis inhibitor, reduced the immunostaining of c-Myb, and also lowered hCG-induced testosterone secretion in isolated rat Leydig cells. The results indicate that c-myb closely correlates with hCG-induced testosterone secretion, and that cAMP and Ca(2+)-dependent pathway participates in the expression of protooncogene.