Tongqiao Huoxuetang Improves Neurological Deficits in CIRI Rats by Regulating Glu-Gln Circulation to Reduce Glutamate Excitotoxicity of Astrocytes / 中国实验方剂学杂志

Objective:To observe and compare the protective effects of Tongqiao Huoxue decoction （TQHX） prepared by three methods against cerebral ischemia-reperfusion injury （CIRI）， and to explore its mechanism through the glutamate （Glu） metabolic pathway in astrocytes. Method:The male SD rats of SPF grade were subjected to CIRI model induction by the modified middle cerebral artery occlusion method. The model rats were randomly divided into a model group， a sham operation group， and water-decocted， wine-decocted， and alcohol-extracted TQHX （6.3 g·kg^-1·d^-1） groups. The rats were treated correspondingly for 7 days. Those in the sham operation group and the model group were treated with an equal volume of normal saline by gavage. After the final treatment， the neurological function of rats was assessed by the modified neurological severity score （mNSS）. Hematoxylin-eosin （HE） staining was used to observe the morphological changes of ischemic brain tissues in rats. High-performance liquid chromatography （HPLC） was used to detect glutamate （Glu） in ischemic brain tissues. The expression of glutamate transporter-1 （GLT-1） and glial fibrillary acidic protein （GFAP） and co-expression of glutamine synthetase （GS） and GFAP in ischemic brain tissues were detected by immunofluorescence assay. Western blot was used to detect the protein expression of GFAP， GLT-1， and GS. Result:Compared with the sham operation group， the model group showed increased mNSS （<italic>P</italic><0.01）， large necrosis of cerebral cortex in ischemic brain tissues with disordered cell arrangement， obscure boundary， intracellular edema， and inflammatory infiltration， elevated Glu in ischemic brain tissues （<italic>P</italic><0.01）， declining GLT-1-GFAP co-expression and GS-GFAP co-expression （<italic>P</italic><0.01）， up-regulated expression of GFAP protein， and reduced protein expression of GLT-1 and GS（<italic>P<</italic>0.05，<italic>P<</italic>0.01）. Compared with the model group， the TQHX groups showed decreased mNSS （<italic>P<</italic>0.01）， relieved injury in the cerebral cortex and hippocampal nerve cells in ischemic brain tissues， reduced Glu expression（<italic>P<</italic>0.05，<italic>P<</italic>0.01）， elevated co-expression of GLT-1 and GFAP （<italic>P<</italic>0.05，<italic>P<</italic>0.01）， and up-regulated protein expression of GFAP and GLT-1（<italic>P<</italic>0.05，<italic>P<</italic>0.01）. The co-expression of GS and GFAP （<italic>P<</italic>0.05，<italic>P<</italic>0.01）and the expression of GS （<italic>P<</italic>0.01）were increased in the wine-decocted and alcohol-extracted TQHX groups. Compared with the water-decocted TQHX group， the alcohol-extracted group showed increased GLT-1-GFAP and GS-GFAP co-expression（<italic>P<</italic>0.05）； the wine-decocted and alcohol-extracted TQHX groups exhibited elevated GS protein expression （<italic>P<</italic>0.05）； the alcohol-extracted TQHX group displayed declining Glu content （<italic>P</italic><0.01） and increased protein expression of GFAP and GLT-1 （<italic>P<</italic>0.05， <italic>P<</italic>0.01）. Compared with the wine-decocted TQHX group， the alcohol-extracted TQHX group showed increased protein expression of GFAP and GLT-1（<italic>P<</italic>0.05，<italic>P<</italic>0.01）. Conclusion:TQHX prepared by three methods can improve neurological deficits in CIRI rats. The effect is presumedly achieved by promoting the further activation of astrocytes， increasing the expression of GLT-1 and GS， promoting the clearance of Glu accumulated in the synaptic cleft by astrocytes through the Glu-glutamine （Gln） circulation， and reducing the excitotoxicity of Glu. The alcohol-extracted TQHX group was superior to the water-decocted and wine-decocted TQHX groups in reducing the content of Glu in ischemic brain tissues， promoting the activation of astrocytes， and enhancing the protein expression of GLT-1 and GS.

Construction of a recombinant shRNA expression vector targeting EZH2 gene and its inhibitory effect on colon adenocarcinoma SW480 cells / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To construct a recombinant short hairpin RNA (shRNA) expression vector targeting EZH2 gene, and to determine its effect on the proliferation of colon adenocarcinoma SW480 cells.</p><p><b>METHODS</b>The DNA sequence with short hairpin structure was designed according to the EZH2 cDNA sequence and cloned into PGFP-V-RS vector to construct a recombinant expression vector silencing EZH2 gene. After identification, the shRNA-expressing vector was then transfected into SW480 cells. RT-PCR and Western blot were used to detect the inhibitory effect at both mRNA and protein levels. MTT was used to detect cell viability due to the alteration of EZH2 gene activity.</p><p><b>RESULTS</b>At 48 h after transfection, the expression of EZH2 mRNA in the gene silencing group and negative control group were 0.339 ± 0.013 and 1.968 ± 0.072, respectively. The expression of EZH2 protein in the gene silencing group and negative control group were 0.229 ± 0.008 and 1.168 ± 0.053, respectively. The expression of EZH2 in the gene silencing group was significantly lower than that in the negative control group (P < 0.01, P < 0.05). At 48 and 72 h after transfection, the inhibition rate of cell growth in the gene silencing group was 30.7% and 25.9%, respectively, indicating that the cell growth was significantly inhibited in comparison with that in the blank control group (P < 0.05).</p><p><b>CONCLUSIONS</b>A recombinant shRNA expression vector targeting EZH2 gene has been successfully constructed in this study, with a significant inhibitory effect on the proliferation of SW480 cells. This lays an experimental foundation for further exploring the mechanism underlying the action of EZH2 gene on tumor biology.</p>

Effects of acetoacetate extract of Radix Aconite on hepatic contents of LA, LDH, PA, Gn and ATPase activities in deficient cold model rats / 中国中西医结合杂志

<p><b>OBJECTIVE</b>To observe different effects of acetoacetate extract of Radix Aconite and Radix Aconite Decoction on the energy metabolism in deficient cold model rats.</p><p><b>METHODS</b>Wistar rats were randomly divided into the blank control group (n=10) and the deficient cold model group (n=30). The deficient cold rat model was established using decoction consisting of gypsum, Radix Gentianae, Cortex Phellodendri, and Rhizoma Anemarrhenae. The decoction was given to rats of the deficient cold model group by gastrogavage for 5 days. Then these rats were randomly divided into the acetoacetate extract of Radix Aconite group (n=10), the Radix Aconite Decoction group (n=10), and the model group (n=10). Rats in the model model group were administered with the decoction by gastrogavage. Rats in the other two groups were administered with the acetoacetate extract of Radix Aconite or Radix Aconite Decoction by gastrogavage for 5 days. The contents of lactic acid (LA), lactate dehydrogenase (LDH), pyruvate (PA), glycogen (Gn) and activities of Na(+) -K(+) -ATPase and Ca(2+) -Mg(2+) -ATPase in the hepatic tissue were detected.</p><p><b>RESULTS</b>Compared with the blank control group, the PA content, activities of Na(+)-K(+) -ATPase and Ca(2+) -Mg(2+) -ATPase decreased in the model group. Compared with the model group, the PA content increased in the other two groups. Compared with the control group, the contents of LDH and PA, and activities of Na(+) -K(+) -ATPase increased in the the acetoacetate extract of Radix Aconite group with statistical difference (P < 0.05).</p><p><b>CONCLUSIONS</b>The febricity of acetoacetate extract of Radix Aconite was slightly higher than that of Radix Aconite Decoction, seemingly generating more energy. But the final conclusions and concrete mechanisms of action need further studies.</p>