Zuanfang Li

Millimeter wave treatment promotes chondrocyte proliferation via G1/S cell cycle transition

Millimeter waves, high-frequency electromagnetic waves, can effectively alleviate the clinical symptoms in osteoarthritis patients, as a non-pharmaceutical and non-invasive physical therapy regimen. However, the molecular mechanisms of the therapeutic effects of millimeter wave treatment are not well understood. In the present study, the effect of millimeter waves on the G1/S cell cycle progression in chondrocytes and the underlying mechanism was investigated. Chondrocytes isolated from the knee of SD rats were cultured and identified using toluidine blue staining. The second generation chondrocytes were collected and stimulated with or without millimeter waves for 48 h. Chondrocyte viability was analyzed using the MTT assay. The cell cycle distribution of chondrocytes was analyzed by flow cytometry. mRNA and protein expression levels of cyclin D1, cyclin-dependent kinases 4 and 6 (CDK4 and CDK6) and p21 were detected using real-time PCR and western blotting, respectively. Millimeter wave stimulation was found to significantly enhance chondrocyte viability. Moreover, the percentage of chondrocytes in the G0/G1 phase was significantly decreased, whereas that in the S phase was significantly increased. In addition, following millimeter wave treatment, cyclin D1, CDK4 and CDK6 expression was significantly upregulated, whereas p21 expression was significantly downregulated. The results indicate that millimeter wave treatment promotes chondrocyte proliferation via cell cycle progression.

Millimeter Wave Treatment Inhibits Apoptosis of Chondrocytes via Regulation Dynamic Equilibrium of Intracellular Free Ca2

The molecular mechanisms of TNF-α-induced apoptosis of chondrocyte and the role of Ca2+ mediating the effects of MW on TNF-α-induced apoptosis of chondrocytes remained unclear. In this study, we investigated the molecular mechanism underlying inhibiting TNF-α-induced chondrocytes apoptosis of MW. MTT assay, DAPI, and flow cytometry demonstrated that MW significantly increased cell activity and inhibited chromatin condensation accompanying the loss of plasma membrane asymmetry and the collapse of mitochondrial membrane potential. Our results also indicated that MW reduced the elevation of [Ca2+]i in chondrocytes by LSCM. Moreover, MW suppressed the protein levels of calpain, Bax, cytochrome c, and caspase-3, while the expressions of Bcl-2, collagen II, and aggrecan were increased. Our evidences indicated that MW treatment inhibited the apoptosis of chondrocytes through depression of [Ca2+]i. It also inhibited calpain activation, which mediated Bax cleavage and cytochrome c release and initiated the apoptotic execution phase. In addition, MW treatment increased the expression of collagen II and aggrecan of chondrocytes.

Neuroprotective effects of Gua Lou Gui Zhi decoction against glutamate-induced apoptosis in BV-2 cells.

Gua Lou Gui Zhi decoction (GLGZD), a traditional Chinese medicine consisting of different herbal medicines, has been used for centuries in the treatment of muscular spasticity following stroke, epilepsy or spinal cord injury. However, the precise mechanisms involved remain poorly understood. In the present study, we investigated the neuroprotective effects of GLGZD on glutamate-induced apoptosis in cultured BV-2 cells, as well as the underlying mechanisms. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was applied to assess the viability of the cells. An Annexin V/propidium iodide (PI) assay was utilized to analyze cellular apoptosis. Mitochondrial membrane potential (MMP) was evaluated by flow cytometry and laser scanning confocal microscopy. The gene and protein expression of the apoptosis-related genes, Bcl-2 and Bax, was analyzed by RT-PCR and western blot analysis, respectively. Furthermore, the expression of cleaved caspase-3 protein was detected by immunofluorescence. Glutamate treatment induced the loss of BV-2 cell viability, which was associated with an increase in the apoptotic rate, as well as an increase in the Bax/Bcl-2 ratio and the extracellular levels of cleaved caspase-3. Treatment with GLGZD significantly reversed these phenotypes, with its maximum protective effects observed at the concentration of 1,000 µg/ml. These results indicate that GLGZD protects BV-2 cells from glutamate-induced cytotoxicity. These protective effects may be ascribed to its anti-apoptotic activities, in part, associated with the decrease in the Bax/Bcl-2 ratio and caspase-3 expression, as well as with the stability of high mitochondrial membrane potential.

Salidroside Reduces Cell Mobility via NF-κB and MAPK Signaling in LPS-Induced BV2 Microglial Cells

The unregulated activation of microglia following stroke results in the production of toxic factors that propagate secondary neuronal injury. Salidroside has been shown to exhibit protective effects against neuronal death induced by different insults. However, the molecular mechanisms responsible for the anti-inflammatory activity of salidroside have not been elucidated clearly in microglia. In the present study, we investigated the molecular mechanism underlying inhibiting LPS-stimulated BV2 microglial cell mobility of salidroside. The protective effect of salidroside was investigated in microglial BV2 cell, subjected to stretch injury. Moreover, transwell migration assay demonstrated that salidroside significantly reduced cell motility. Our results also indicated that salidroside suppressed LPS-induced chemokines production in a dose-dependent manner, without causing cytotoxicity in BV2 microglial cells. Moreover, salidroside suppressed LPS-induced activation of nuclear factor kappa B (NF-κB) by blocking degradation of IκBα and phosphorylation of MAPK (p38, JNK, ERK1/2), which resulted in inhibition of chemokine expression. These results suggest that salidroside possesses a potent suppressive effect on cell migration of BV2 microglia and this compound may offer substantial therapeutic potential for treatment of ischemic strokes that are accompanied by microglial activation.

Suppressive effects of Gua Lou Gui Zhi decoction on MCAO-induced NO and PGE2 production are dependent on the MAPK and NF-κB signaling pathways

The present study aimed to investigate the inhibitory effects, and underlying mechanisms, of Gua Lou Gui Zhi decoction (GLGZD) in a rat model of neuroinflammation. Sprague-Dawley rats were treated with GLGZD following middle cerebral artery occlusion (MCAO). Neurological function and infarct volume were evaluated to confirm successful generation of the rat model. Subsequently, brain tissues and blood samples were collected for further analysis. Nitric oxide (NO) and prostaglandin E2 (PGE2) were evaluated in peripheral blood samples using the Griess reagent assay and an ELISA, respectively. The relative expression levels of inducible nitric oxide synthase (iNOS) and cylooxygenase-2 (COX-2) were detected by quantitative polymerase chain reaction and immunohistochemistry. The associated pathways, including nuclear factor-κB (NF-κB) and mitogen-activated protein kinases (MAPK) signaling pathways, were detected by electrophoretic mobility shift assay and western blotting. The results demonstrated that treatment with GLGZD significantly inhibited MCAO-induced inflammation; GLGZD suppressed the production of NO and PGE2, and the expression of iNOS and COX-2, by inhibiting NF-κB activation and MAPK phosphorylation. These findings suggest that GLGZD, a potential agent for post-stroke treatment, may exert anti-inflammatory effects, thus providing neuroprotection.

Anti-inflammatory effects of Gualou Guizhi decoction in transient focal cerebral ischemic brains. [Corrected].

The aim of the present study was to explore the neuroprotective effects of Gualou Guizhi decoction (GLGZD) in a rat model of middle cerebral artery occlusion (MCAO). Sprague-Dawley rats were divided into three groups: Sham (no MCAO), MCAO (MCAO with no GLGZD treatment) and GLGZD (MCAO with GLGZD treatment). Rats in the MCAO and GLGZD groups were subjected to permanent occlusion of the left middle cerebral artery. Neurological function and infarct volume were measured. Microglial activation and inflammatory cell accumulation were measured using immunohistochemistry. mRNA and protein expression of inflammatory mediators were examined using reverse transcription-quantitative polymerase chain reaction and an enzyme-linked immunosorbent assay. The expression of proteins associated with the nuclear factor κ-B (NF-κB) inflammation signaling pathway was analyzed using western blotting. The results of the present study suggested that infarct size was significantly reduced and neurological behavior function was improved in rats with MCAO treated with GLGZD compared with rats in the MCAO group. Amoeboid microglial expansion and inflammatory cell migration were observed in the infarcted areas of rats in the GLGZD group and were not identified in those of the MCAO group. Target mRNA and protein levels, and inflammatory cell infiltration were significantly reduced in the GLGZD group compared with the MCAO model group. Notably, GLGZD treatment induced neuroprotective effects, reducing inflammation and inhibiting NF-κB signaling compared with the MCAO group. Therefore, GLGZD may exhibit anti-inflammatory effects against ischemia-reperfusion brain injury and may be a therapeutic target for ischemic stroke.

Gua Lou Gui Zhi decoction attenuates post‑stroke spasticity via the modulation of GABAB receptors

The aim of the present study was to investigate the mechanisms underlying the neuroprotective and antispastic effects of Gua Lou Gui Zhi decoction (GLGZD) in a rat model of middle cerebral artery occlusion (MCAO). The MCAO rats were treated with GLGZD (14.3 g/kg body weight) once a day for a period of seven days. Neurological deficit scores and screen tests were analyzed every other day. Following treatment with GLGZD for 7 days, the ischemic infarct volume of the rat brains was measured using 2,3,5‑triphenyl tetrazolium chloride staining. Reverse transcription‑polymerase chain reaction was performed in order to determine the mRNA expression levels of γ‑amino butyric acid B (γ‑GABAB) receptor (R) in the cortical infarct region. Furthermore, the protein expression levels of GABAB R were detected in the cortical infarct region by western blot analysis. Following 7 days, treatment with GLGZD significantly ameliorated the neurological defects and cerebral infarction in the MCAO rats. In addition, treatment with GLGZD ameliorated motor performance in the MCAO rats, as determined by screen tests. Furthermore, GLGZD was able to upregulate the mRNA and protein expression levels of GABAB1 R and GABAB2 R in the ischemic cerebral cortex. The results of the present study suggested that GLGZD may exert neuroprotective and antispastic effects in a cerebral ischemia model, through upregulating the expression of GABAB R.

Electroacupuncture ameliorate learning and memory by improving N-acetylaspartate and glutamate metabolism in APP/PS1 mice

ObjectiveTo explore the precise mechanism of electroacupuncture (EA) to delay cognitive impairment in Alzheimer disease.MethodsN-Acetylaspartate (NAA), glutamate (Glu) and myoinositol (mI) metabolism were measured by magnetic resonance spectroscopy, learning and memory of APP/PS1 mouse was evaluated by the Morris water maze test and the step-down avoidance test, neuron survival number and neuronal structure in the hippocampus were observed by Nissl staining, and BDNF and phosphorylated TrkB detected by Western blot.ResultsEA at DU20 acupuncture significantly improve learning and memory in behavioral tests, up-regulate NAA, Glu and mI metabolism, increase the surviving neurons in hippocampus, and promote the expression of BDNF and TrkB in the APP/PS1 transgenic mice.ConclusionThese findings suggested that EA is a potential therapeutic for ameliorate cognitive dysfunction, and it might be due to EA could improve NAA and Glu metabolism by upregulation of BDNF in APP/PS1 mice.

Electroacupuncture ameliorates cognitive impairment through inhibition of Ca2+-mediated neurotoxicity in a rat model of cerebral ischaemia–reperfusion injury

Background The hippocampus is vulnerable to severe damage after cerebral ischaemia–reperfusion (I/R) injury. This study aimed to explore the effect of electroacupuncture (EA) on cognitive impairment and its relationship with Ca2+neurotoxicity in a rat model of I/R injury induced by middle cerebral artery occlusion (MCAO). Methods 60 adult male Sprague-Dawley rats were randomly divided into three groups: control (sham surgery) group, untreated MCAO group and EA-treated MCAO+EA group. Rats in the MCAO and MCAO+EA groups underwent modelling of poststroke cognitive impairment by MCAO surgery. EA was performed for 30 min daily at GV20 and GV24 (1–20 Hz) for 1 week. The Morris water maze experiment was used to assess cognitive function. 2,3,5-triphenyl tetrazolium chloride staining was used to measure infarct volume. The intracellular Ca2+content in the Cornu Ammonis (CA)1 area of the hippocampus was assessed by laser confocal scanning microscopy. ELISA was performed to evaluate the concentration of glutamate (Glu) in the hippocampus, and the protein expression of two Glu receptors (N-methyl-D-aspartic acid receptor (NMDAR) 2A and NMDAR2B) were analysed by Western blotting. Results Compared with the untreated MCAO group, EA effectively ameliorated cognitive impairment (P=0.01) and shrunk the infarct volume (P=0.032). The content of intracellular Ca2+, Glu and NMDAR2B in the hippocampus was significantly raised by MCAO (P=0.031-0.043), while EA abrogated these effects. NMDAR2A was decreased by MCAO (P=0.015) but increased by EA (P=0.033). Conclusions EA had a beneficial effect on cognitive repair after cerebral I/R, and its mechanism of action likely involves a reduction of Ca2+influx via inhibition of Glu neurotoxicity and downregulation of NMDAR2B expression.

Ultrastructural change of the subchondral bone increases the severity of cartilage damage in osteoporotic osteoarthritis of the knee in rabbits

Osteoporotic osteoarthritis is a phenotype of osteoarthritis (OA) manifested as fragile and osteoporotic subchondral bone. However, the ultrastructural features of subchondral bone in osteoporosis OA have not been determined. The study was aimed to investigate the ultrastructural dynamic changes of subchondral bone in osteoporotic OA model and how the ultrastructural damage in the subchondral bone caused by osteoporosis deteriorated the cartilage damage in OA. Eighteen rabbits were equally randomized to three groups, including the control, the OA and the osteoporotic OA groups. The structural changes of cartilage were evaluated by HE and safranin-O fast green staining, the Mankins grading system was used to assess the stage of OA progression. And microstructural or ultrastructural changes in subchondral bone were assessed by micro-computed tomography or by scanning electron microscopy. According to the changes of cartilage histopathology, the OA group was in the early pathological stage of OA while the osteoporotic OA group was in the middle stage of OA based on Mankins grading system. In addition, the damage of cartilage surface, reduction in the number of chondrocytes and the matrix staining were more increased in the osteoporotic OA group compared to the OA group. Compared to the OA group, the subchondral bone in the microstructure and ultrastructure in the osteoporotic OA group showed more microfracture changes in trabecular bone with more destructions of the tree-like mesh. Moreover, the collagen fibers were random rough with a fewer amount of bone lacunae in subchondral cortical plate in the osteoporotic OA group compared to the OA group. These findings indicated that the subchondral bone ultrastructure in the osteoporotic OA model was characterized by the destruction of the network structure and collagen fibers. The subchondral bone ultrastructural damage caused by osteoporosis may change mechanical properties of the upper cartilage and aggravate OA cartilage. Therefore, early diagnosis and treatment of osteoporosis is of great significance to prevent early OA from further developing osteoporotic OA.