[Mechanisms of moxibustion preconditioning underlying improving learning-memory ability by regulating polarization of microglia via TLR4/NF-κB signaling pathway in AD rats].

OBJECTIVE: To observe the effect of moxibustion preconditioning on learning-memory ability, Toll like receptor 4(TLR4)/nuclear factor-kappa B(NF-κB) signal pathway related proteins and microglia in rats with Alzheimer's disease (AD), so as to explore its possible mechanisms underlying improvement of AD. METHODS: Male SD rats were randomly divided into normal, sham operation, AD model and pre-moxibustion groups, with 9 rats in each group. Moxibustion was applied to "Baihui"(GV20), "Shenshu"(BL23) and "Zusanli"(ST36) for 15 min, once daily, 6 days as a course of treatment for 3 courses. At the end of moxibustion, the AD model was established by injection of Aß25-35 aggregation solution into the bilateral hippocampus. The sham operation group was only injected with the same amount of 0.9% Nacl solution. The spatial learning-memory ability of rats was detected by Morris water maze test, the ultrastructure of hippocampal neurons was observed by transmission electron microscope (TEM). The histopathological changes of hippocampus tissue were observed by HE staining, and the protein expression levels of TLR4 and NF-κB p65 in the hippocampus detected by Western blot, and the positive expressions of Iba-1, CD80 and CD206 in the hippocampal CA1 region were detected by immunofluorescence labeling. The contents of inflammatory factors IL-1ß, TNF-α and IL-10 in the hippocampus were measured by ELISA. RESULTS: Compared with the sham operation group, the escape latency was significantly increased (P<0.01), and the number of platform quadrant crossing times was decreased (P<0.01) in the model group. In comparison with the model group, the increased escape latency and the decreased platform quadrant crossing times were reversed in the pre-moxibustion group (P<0.01). TEM and light microscope observation showed loose arrangement of cells, enlarged cell space, degeneration, swelling and deformation of hippocampal neurons, rupture of membranes of a large number of cells, reduction of mitochondria, dilation of endoplasmic reticulum, and matrix vacuoles, uneven distribution of organelles and cytoplasm, and being difficult in distinguishing the nuclear cytoplasm in the model group, which was relatively milder in the pre-moxibustion group. The expression levels of hippocampal NF-κB p65 and TLR4, the mean immunofluorescence density of Iba-1 and CD80, as well as the contents of IL-1ß and TNF-α in hippocampal CA1 region were significantly increased in the model group than those in the sham operation group (P<0.01), and obviously decreased in the pre-moxibustion group than those in the model group (P<0.05, P<0.01). Whereas the expression of CD206 and the content of IL-10 were evidently decreased in the model group than those in the sham operation group (P<0.01), and strikingly increased in the pre-moxibustion group than those in the model group (P<0.01). No significant differences were found between the sham operation group and the normal group in all the indexes mention above (P>0.05). CONCLUSION: Pre-moxibustion at GV20, BL23 and ST36 can improve learning-memory ability in AD rats, which may be associated with its functions in promoting the polarization of microglia from M1 to M2 and reducing the neuroinflammatory response by way of TLR4/NF-κB signaling pathway.

[Involvement of miR-126-3p via mTOR/HIF-1α signaling pathway in effect of electroacupuncture on angiogenesis in rats with cerebral ischemia].

OBJECTIVE: To observe the effect of electroacupuncture (EA) on miRNA-126-3p and mammalian target of rapamycin (mTOR)/hypoxia-inducible factor-1α (HIF-1α) signaling pathway in rats with cerebral ischemia (CI), so as to explore the underlying mechanism of EA on angiogenesis. METHODS: Male SD rats were randomly divided into control group, model group, EA group and EA+inhibitor group (inhibitor group), which were further divided into 3, 7 and 14 d subgroups, with 12 rats in each sub-group. The CI model was established by occlusion of the middle cerebral artery. EA (2 Hz/20 Hz, 0.5 mA) was applied to "Dazhui" (GV14), "Baihui" (GV20) for 20 min, once daily for 14 days at most. Rats of the inhibitor group were given an intraperitoneally injection of mTOR inhibitor (0.1 mg/mL, 0.3 mg/kg) before daily EA. The neurological function was evaluated by modified neurological severity score (mNSS). The ultrastructure of cortical neurons and microvascular endothelial cells in ischemic penumbra was observed by transmission electron microscope, and the microvessel density (MVD) of cortical endothelium in ischemic penumbra was detected by immunohistochemistry. Western blot and quantitative real-time PCR were used to detect the protein and mRNA expression of mTOR, HIF-1α and the expression of miR-126-3p in the cortex of ischemic penumbra, respectively. RESULTS: After modeling, compared with the control group at the same time point, the mNSS of the model group was increased (P<0.01), and decreased over time (P<0.01). The cortical neurons and brain microvascular endothelial cells in the ischemic penumbra were edema, and the cell structure was damaged obviously in the model group.The MVD value and the expressions of mTOR、HIF-1α proteins and mRNAs were increased (P<0.01), while the expression of miR-126-3p decreased (P<0.01) in the model group relative to the control group. Compared with the model group at the same time point, the mNSS of both intervention groups was significantly reduced (P<0.01, P<0.05), the neuron and cerebral microvascular structure improved to varying degrees, and the MVD value, the expressions of mTOR and HIF-1α protein and mRNA, and the expression of miR-126-3p of the two treatment groups were increased (P<0.01, P<0.05) at all time points (excep MVD at day 7 in the inhibitor group). Compared with the EA group at the same time point, MVD, the expressions of mTOR, HIF-1α proteins and mRNAs and miR-126-3p in the inhibitor group were all decreased (P<0.05,P<0.01). Compared with the group itself at 4 hours after modeling and day 3 and day 7, the mNSS was decreased at day 14 （P<0.01） in the model, EA and inhibitor groups. Compared with the group itself at day 3, the MVD value and the expression of mTOR protein were increased at day 7 and day 14 in the model, EA and inhibitor groups (P<0.01, P<0.05). Compared with the group itself at day 3 and day 7, the expression of mTOR mRNA and miR-126-3p were up-regulated at day 14 in the model and EA groups (P<0.01, P<0.05).Compared with the group itself at day 3, the mRNA expressions of mTOR and HIF-1α were increased at day 7 and day 14 (P<0.01, P<0.05) in the inhibitor group. CONCLUSION: EA at GV14 and GV20 can alleviate neurological deficit and improve angiogenesis in rats with CI, which may be related with its effect in up-regulating the expression of mTOR and HIF-1α, improving activation of miR-126-3p in the cortex of ischemic penumbra.

[Electroacupuncture combined with rehabilitation training improves regional cerebral blood flow and reduces infarct volume by promoting expression of angiogenesis-related factors in acute cerebral ischemia rats].

OBJECTIVE: To observe the effect of electroacupuncture (EA) combined with rehabilitation training on regional cerebral blood flow (rCBF) and angiogenesis in rats with acute cerebral ischemia (ACI), so as to explore its mechanisms underlying improvement of ACI. METHODS: A total of 135 male SD rats were divided into 5 groups: sham-operation (sham), model, EA, rehabilitation training and EA+rehabilitation training (combined treatment) groups (n=27 rats in each group). The ACI model was established by occlusion of the middle cerebral artery with thread embolus. EA (2 Hz/20 Hz, 3-5 V) was applied to "Baihui" (GV20), "Shuigou" (GV26) and bilateral "Neiguan" (PC6) for 20 min, once daily for 14 days. The rehabilitation training including hair-brushing in an enriched environment (10 min), round wooden-stick turning (10 min), grid-board climbing (10 min), and treadmill running (30 min/d) was condacted once daily for 14 days. The rCBF was measured by Doppler ultrasound. The cerebral infarct volume (CIV) was measured after 2, 3, 5-triphenyltetrazolium chloride (TTC) staining. The expression of CD34+ in the ischemic penumbra region of brain tissue was detected by immunohistochemistry, and the expressions of angiogenesis-related factors as vascular endothelial growth factor (VEGF), vascular endothelial growth factor receptor 2 (VEGFR2) and basic fibroblast growth factor (bFGF) proteins in the ischemic brain tissue were detected by Western blot. RESULTS: Following modeling, the rCBF levels at the 5 min, 3rd, 7th and 14th day were significantly decreased in the model group relevant to the sham group (P<0.01). After the intervention, the rCBF levels were significantly increased on day 3, 7 and 14 in the combined treatment group and on day 7 and 14 in both the EA and rehabilitation training groups in comparison with the model group (P<0.01). The CIV was obvious in the model group in comparison with the sham group (P<0.01), but was markedly smaller in the EA, rehabilitation training and combined treatment groups on day 3,7 and 14 than in the model group (P<0.01). The number of CD34+ positive cells, and the expression levels of VEGF, VEGFR2, and bFGF proteins in ischemic brain tissues were significantly higher on day 3, 7 and 14 in the model group than in the sham group (P<0.01, P<0.05), and were further up-regulated considerably at the 3 time-points in the 3 treatment groups (P<0.01, P<0.05). The therapeutic effect of EA+rehabilitation training was significantly superior to that of simple EA and simple rehabilitation training in up-regulating rCBF, CD34+ positive cell number, and expression levels of VEGF, VEGFR2 and bFGF, and in down-regulating the CIV on day 3,7 and 14 (P<0.05, P<0.01). No significant differences were found between the EA and rehabilitation groups in the above-mentioned 6 indexes (P>0.05). CONCLUSION: EA combined with rehabilitation training can reduce the infarct volume and increase rCBF in ACI rats, which is probably associated with its effects in promoting the expression of angiogenesis-related factors of ischemic brain tissues. The effect of EA combined with rehabilitation training is markedly better than that of EA and rehabilitation training alone.

Distribution characteristics and source apportionment of polycyclic aromatic hydrocarbons (PAHs) in the Liao River drainage basin, northeast China.

The Liao River drainage basin, which is one of China's seven major rivers basins, is located in northeast China. This region is characterized by important industrial bases including steel factories and oil and chemical plants, all of which have the potential to contribute pollutants to the drainage basin. In this study, 16 polycyclic aromatic hydrocarbons (PAHs) in water and suspended particulate matter (SPM) in the major rivers of the Liao River drainage basin were identified and quantified by gas chromatography mass spectrometry (GC/MS). The total PAH concentrations ranged from 0.4 to 76.5 µg/g (dry weight) in SPM and 32.6 to 108 ng/L in surface water, respectively. Low-ring PAHs (including two- and three-ring PAHs) were dominant in all PAH samples, and the level of low-ring PAHs in surface water was higher than that in SPM. The proportion of two-ring PAHs was the highest, accounting for an average of 68.2 % of the total PAHs in surface water, while the level of three-ring PAHs was the highest in SPM, with an average of 66.3 %. When compared with other river systems, the concentrations of PAHs in the Liao River drainage basin were lower. Identification of the emission sources based on diagnostic ratios suggested petroleum and fossil fuel combustion were important contribution to PAHs in the study area.

Dissolved organic matter removal during coal slag additive soil aquifer treatment for secondary effluent recharging: Contribution of aerobic biodegradation.

Recycling wastewater treatment plant (WWTP) effluent at low cost via the soil aquifer treatment (SAT), which has been considered as a renewable approach in regenerating potable and non-potable water, is welcome in arid and semi-arid regions throughout the world. In this study, the effect of a coal slag additive on the bulk removal of the dissolved organic matter (DOM) in WWTP effluent during SAT operation was explored via the matrix configurations of both coal slag layer and natural soil layer. Azide inhibition and XAD-resins fractionation experiments indicated that the appropriate configuration designing of an upper soil layer (25 cm) and a mixture of soil/coal slag underneath would enhance the removal efficiency of adsorption and anaerobic biodegradation to the same level as that of aerobic biodegradation (31.7% vs 32.2%), while it was only 29.4% compared with the aerobic biodegradation during traditional 50 cm soil column operation. The added coal slag would preferentially adsorb the hydrophobic DOM, and those adsorbed organics could be partially biodegraded by the biomass within the SAT systems. Compared with the relatively lower dissolved organic carbon (DOC), ultraviolet light adsorption at 254 nm (UV-254) and trihalomethane formation potential (THMFP) removal rate of the original soil column (42.0%, 32.9%, and 28.0%, respectively), SSL2 and SSL4 columns would enhance the bulk removal efficiency to more than 60%. Moreover, a coal slag additive in the SAT columns could decline the aromatic components (fulvic-like organics and tryptophan-like proteins) significantly.