[Tetramethylpyrazine regulates angiogenesis of endothelial cells in cerebral ischemic stroke injury via SIRT1/VEGFA signaling pathway].

This study aims to investigate whether tetramethylpyrazine(TMP) can stimulate angiogenesis in cerebral microvascular endothelial cells and alleviate cerebral ischemic stroke(CIS) and to explore the underlying mechanisms. In the animal study, adult Sprague-Dawley rats(n=15) were assigned into sham surgery(sham), middle cerebral artery occlusion/reperfusion(MCAO/R), and MCAO/R+TMP(intraperitoneal injection of 20 mg·kg~(-1)) groups. The neurological function was evaluated by the Z-Longa method. The cerebral infarction volume was detected by TTC staining. Enzyme-linked immunosorbent assay(ELISA) was employed to detect the expression of vascular endothelial growth factor(VEGF), angiopoietin(Ang), and platelet-derived growth factor(PDGF). Immunofluorescence staining was employed to detect Ki67 and the expression of vascular endothelial growth factor A(VEGFA) and slient information regulator 1(SIRT1). Western blot was employed to determine the expression levels of VEGFA, SIRT1, angiopoietin-2(Ang-2), and platelet-derived growth factor B(PDGFB). In the cell study, mouse brain-derived endothelial cells(Bend.3) were cultured, and the optimal concentration of TMP was determined. Then, VEGF, Ang, and PDGF were detected by ELISA after the addition of cabozantinib. Western blot was employed to measure the expression of VEGFA, Ang-2, and PDGFB. Immunofluorescence staining was used to detect CD31, CD34, and Ki67, and the proliferation, migration, and tube formation ability of Bend.3 cells were observed in vitro. Western blot and immunofluorescence staining were performed to measure the expression of SIRT1 and VEGFA after addition of the SIRT1-specific inhibitor selisistat(EX-527). The results showed that compared with the sham group, the MCAO/R group had severe neurological function damage, increased infarction volume, up-regulated expression of VEGF, VEGFA, Ang, Ang-2, PDGF, and PDGFB, and down-regulated expression of Ki67 and SIRT1(P<0.01). Compared with the MCAO/R group, the MCAO/R+TMP group presented alleviated neurological function damage, reduced infarction volume, and activated expression of VEGF, VEGFA, Ang, Ang-2, PDGF, PDGFB, Ki67, and SIRT1(P<0.01). The cell experiments showed that compared with the normal group, Bend.3 cells were activated by oxygen glucose deprivation/reoxygenation(OGD/R) treatment(P<0.05, P<0.01). Compared with the OGD/R group, the OGD/R+TMP group upregulated the expression levels of VEGF, VEGFA, Ang, Ang-2, PDGF, PDGFB, SIRT1, Ki67, CD31, and CD34, enhanced the angiogenic ability of Bend.3 cells without being inhibited by BMS or EX-527(P<0.05, P<0.01, P<0.001). The results suggest that TMP can activate the SIRT1/VEGFA signaling pathway to stimulate angiogenesis and alleviate CIS injury.

Inhibitory effects of lupane­type triterpenoid saponins from the leaves of Acanthopanax gracilistylus on lipopolysaccharide-induced TNF­α, IL­1ß and high­mobility group box 1 release in macrophages.

Acanthopanax gracilistylus (AGS) has long been used in traditional Chinese medicine for the treatment of various inflammatory diseases. 3­O­ß­D­glucopyranosyl 3α, 11α­dihydroxylup­20(29)­en­28­oic acid, acantrifoside A, acankoreoside D, acankoreoside B and acankoreoside A are major lupane­type triterpenoid saponins derived from AGS. In the present study, these five saponins were isolated from AGS by chromatography and their anti­inflammatory activities were investigated in lipopolysaccharide (LPS)­treated RAW264.7 macrophages. Cell viability was evaluated by MTT assay. Tumor necrosis factor (TNF)­α, interleukin (IL)­1ß and NF­κB p65 were measured by ELISA. The gene expression levels of TNF­α and IL­1ß was detected by reverse­transcription polymerase chain reaction. And high­mobility group box 1 (HMGB1) were analyzed by western blotting. The results demonstrated that these five saponins significantly suppressed LPS­induced expression of TNF­α and IL­1ß at the mRNA and protein level in RAW264.7 cells. Further analysis revealed that acankoreoside A and acankoreoside B were able to reduce the secretion of HMGB1 and NF­κB activity induced by LPS in RAW264.7 macrophages. Taken together, these results suggested that the anti­inflammatory activity of AGS­derived saponins may be associated with the downregulation of TNF­α and IL­1ß, and the 'late­phase' proinflammatory cytokine HMGB1, via negative regulation of the NF­κB pathway in RAW264.7 cells.

Inhibitory effects of 3α-hydroxy-lup-20(29)-en-23, 28-dioic acid on lipopolysaccharide-induced TNF-α, IL-1ß, and the high mobility group box 1 release in macrophages.

We investigated the anti-inflammatory effects of 3α-hydroxy-lup-20(29)-en-23, 28-dioic acid (HLEDA)-a lupane-type triterpene isolated from leaves of Acanthopanax gracilistylus W. W.Smith (AGS), as well as the underlying molecular mechanisms in lipopolysaccharide (LPS)-induced RAW264.7 cells. Our results demonstrated that HLEDA concentration-dependently reduced the production of nitric oxide (NO), significantly suppressed LPS-induced expression of TNF-α and IL-1ß at the mRNA and protein levels in RAW264.7 cells. Further analysis revealed that HLEDA could reduce the secretion of High Mobility Group Box 1 (HMGB1). Additionally, the results showed that HLEDA efficiently decreased nuclear factor-kappaB (NF-κB) activation by inhibiting the degradation and phosphorylation of IκBα. These results suggest that HLEDA exerts anti-inflammatory properties in LPS-induced macrophages, possibly through inhibition of the NF-κB signaling pathway, which mediates the expression of pro-inflammatory cytokines. These results warrant further studies that would concern candidate therapy for diseases, such as fulminant hepatitis and rheumatology of triterpenoids in AGS.

[Effect of Acupuncture plus Different Frequency Shock-wave Interventions on Pain Reactions and Motor Function in Knee Osteoarthritis Patients].

OBJECTIVE: To observe the clinical effect of acupuncture plus shock-wave (SW) intervention for osteoarthritis (WA), so as to explore its practicability in clinical practice. METHODS: A total of 120 cases of knee OA patients were randomly divided into 4 groups, namely acupuncture (acupunct) + LFSW, acupunct + MFSW, acupunct + HFSW and routine acupunct groups, with 30 cases in each group. Xuehai (SP 10) , Liangqiu (ST 34), Yanglingquan (GB 34), Xiyan (ST 35) and Ashi-point were punctured with filiform needles which were manipulated with uniform reinforcing-reducing techniques for 15-20 min, once every other day for 7 times. In addition, these acupoints were also respectively stimulated with shock waves(10 Hz, 14 Hz and 18 Hz, pressure: 1-4 bar) delivered from a DolorClastEMS therapeutic apparatus for 600 times in 3 acupunct+ SW groups. The patients' pain response changes of the knee-joint were assessed by using visual analog scale (VAS) and the motility was evaluated by using a 0-3 grade scale. RESULTS After 7 times of treatment, the patients' VAS scores and motility scores were significantly decreased in the acupunct+ LFSW, acupunct+ MFSW, acupunct+ HFSW and routine acupunct groups compared with their own basic values before treatment (P < 0.01), and the therapeutic effect of the acupunct+ MFSW group was significantly superior to those of the other 3 groups in reducing both VAS and motility scores (P < 0.05). Correspondingly, the Deqi sensation score of the acupunct+ MFSW group was markedly higher than those of the other 3 groups (P < 0.05). CONCLUSION: Shock wave acu-puncture treatment is effective in relieving OA patients' knee-joint pain and functional activity, and the therapeutic effect of acu- punct + 14 Hz-SW is better, which is closely with Deqi-sensation.

[Study on inter-individual variability of cerebral response to acupuncture with fMRI].

OBJECTIVE: Effects of inter-individual variability on fMRI of acupuncture were observed and the possible influencing factors were further analyzed. METHODS: Twenty-six healthy volunteers were selected. And acupuncture was applied at Zusanli (ST 36) on the left side with even manipulation. The same experimental designation and data collecting reference were adopted to collect functional data. Then, the same data processing method was applied for analyzing individual data. Data which did not confirm with data analyzing qualification were rejected. The 26 individual data which met the requirement were taken randomly for 5 times according to the principle of random group division. Five groups named with A, B, C, D and E were thus generated with 11 samples in each. Images were processed with the AFNI software for every group, and the activated brain areas were revealed. RESULTS: Activated areas in the brain were observed in all the 5 groups, and the results vary a lot among different groups. Decreased signals of activated brain areas were observed in group C, while increased signals were seen in group D. Partial increasing and partial decreasing signals appeared in the other 3 groups. Compared with other groups, group D demonstrated totally different activated areas. The rate of difference among different groups is 46.7%-100.0%, and most of the differences were over half of the activated areas. CONCLUSION: Under the pre-requisites of strict control of experimental designation, acupuncture method, data collecting and processing, great differences have been found in the activated areas of the brain. It indicates that obvious individual differences existes in the activated areas of the brain with acupuncture. And the difference may greatly influence the researching result of fMRI as well as conclusions of those results.

Enzyme-assisted extraction of flavonoids from Ginkgo biloba leaves: improvement effect of flavonol transglycosylation catalyzed by Penicillium decumbens cellulase.

We report a novel enzyme-involved approach to improve the extraction of flavonoids from Ginkgo biloba, in which the enzyme is employed not only for cell wall degradation, but also for increasing the solubility of target compounds in the ethanol-water extractant. Penicillium decumbens cellulase, a commercial cell wall-degrading enzyme with high transglycosylation activity, was found to offer far better performance in the extraction than Trichoderma reesei cellulase and Aspergillus niger pectinase under the presence of maltose as the glycosyl donor. TLC, HPLC and MS analysis indicated that P. decumbens cellulase could transglycosylate flavonol aglycones into more polar glucosides, the higher solubility of which led to improved extraction. The influence of glycosyl donor, pH, solvent and temperature on the enzymatic transglycosylation was investigated. For three predominant flavonoids in G. biloba, the transglycosylation showed similar optimal conditions, which were therefore used for the enzyme-assisted extraction. The extraction yield turned to be 28.3mg/g of dw, 31% higher than that under the pre-optimized conditions, and 102% higher than that under the conditions without enzymes. The utilization of enzymatic bifunctionality described here, naming enzymatic modification of target compounds and facilitation of cell wall degradation, provides a novel approach for the extraction of natural compounds from plants.

[Discussion on the national standard, the Name and Location of Acupoints].

Location of a part of acupoints in the national standard, The Name and Location of Acupoints, are studied. In combination with anatomy, record of ancient literature and teaching experience, the location of Naohui (TE 13), Chengshan (BL 57), Fengshi (GB 31), Zhongdu (GB 32), Toulinqi (GB 15), Yinbao (LR 9) and Shaoshang (LU 11) in the national standard are analyzed and the relative location methods are raised.

[Research of aeration with bio-film technology to treat urban landscape water].

Research of the aeration with bio-film technology was carried out to treat scenic water of a sanatorium in Beijing. The aim of the research was improving the water habitat by increasing the transparency and reducing the concentration of N and P. The equipments were set in a 5,000 m2 water area, which combined the plug flow jet aerator with the elastic biological filler. The research indicated that the transparency increased from 25 cm to 120 cm by the technology. The removal efficiencies of NH4(+)-N, NO3(-)-N and TP were 86.6% , 90% and 73.3%, but there was only 22.4% for TN. The concentration of DO increased from 4.3 mg/L to 7 mg/L. In a word, the aeration with bio-film technology was an effective measure to improve the water habitat by increasing the transparency.

[Effects of acupuncture at Neiguan (PC 6) on human brain functional imaging in different functional states].

OBJECTIVE: To study on effects of acupuncture at a same point at physiological or pathological state on brain functional imaging. METHODS: Electroacupuncture was given at Neiguan (PC 6) of the healthy senile persons (normal group) and the patients of Alzheimer's disease (AD group), and they were scanned on a 1.5 T MRI scanner and the data of functional magnetic resonance imaging (fMRI) were analyzed by Statistical Parametric Mapping (SPM99). The functional imaging of different brain regions were obtained from both the healthy group and the AD group. RESULTS: Both the frontal lobe and the temporal lobe were activated in varying degrees by electroacupuncture at Neiguan (PC 6) in the healthy group. However, the frontal lobe, the temporal lobe and the cingulate gyrus as well as the cerebellum were activated in varying degrees and other brain regions were not activated by electroacupuncture at Neiguan (PC 6) in the AD group. CONCLUSION: Acupuncture at Neiguan (PC 6) can activate functions of different brain regions in the healthy group and the AD group, indicating that acupuncture at Neiguan (PC 6) has different effects on functional magnetic resonance imaging in different functional states.