Effect of Tongxinluo Treated Conditioned Medium of Cardiac Microvascular Endothelial Cells on Rats' Embryo Cardiomyocyte Apoptosis / 中国循环杂志

Objective: To explore the effect of impaired cardiac microvascular endothelial cells on rat's embryo cardiomyocyte apoptosis and the impact of Tongxinluo in such process. Methods: 3 kinds conditioned medium of cardiac microvascular endothelial cells were prepared. ① normal rat's cardiac microvascular endothelial cell (RCMECs) conditioned medium, ②homocysteine induced impaired RCMECs conditioned medium and ③ Tongxinluo treated RCMECs conditioned medium. Rat's embryo H9c2 cardiomyocytes were divided into 4 groups:① Control group, cells were cultured in DMEM without serum, ② N-CM group, cells were cultured in RCMECs conditioned medium, ③ H-CM group, cells were cultured in homocysteine induced impaired RCMECs conditioned medium and ④ T-CM group, cells were cultured in Tongxinluo treated RCMECs conditioned medium. Cell mitochondrial membrane potential (MMP) was detected by JC-1 agent, ROS level was examined by flow cytometry, cardiomyocytes apoptosis was measured by live cell imaging system, expression levels of cytochrome C (Cyt-C), cysteinyl aspartate specific proteinase-3 (Caspase-3), Bcl-2 associated X protein (Bax) and B-cell leukemia/lymphoma 2 protein (Bcl-2) were detected by Western blot analysis. Results: MMP, expression levels of ROS, Cyt-C and cardiomyocyte apoptosis were similar between Control group and N-CM group, P>0.05. Compared with N-CM group, H-CM group had decreased MMP, increased ROS and cardiomyocyte apoptosis, up-regulated expressions of Cyt-C, Caspase-3 and Bax, all P<0.01. Compared with H-CM group, T-CM group had increased MMP, decreased ROS and cardiomyocyte apoptosis, down regulated expressions of Cyt-C, Caspase-3 and Bax, all P<0.05; while up-regulated expression of Bcl-2, P<0.01. Conclusion: Conditioned medium of impaired cardiac microvascular endothelial cells may induce cardiomyocyte apoptosis and such process could be inhibited by Tongxinluo.

Effect of Tongxinluo on Apoptosis of Rat Cardiac Microvascular Endothelial Cells / 中国中西医结合杂志

<p><b>OBJECTIVE</b>To observe the protective effects of Tongxinluo (TXL) on apoptosis of rat cardiac microvascular endothelial cells (RCMECs) resulting from homocysteine (Hcy) induced endoplasmic reticulum stress (ERS), and to determine the signaling pathway behind its protection.</p><p><b>METHODS</b>Primary cultured RCMECs were isolated from neonatal rats using tissue explant method. The morphology of RCMECs was observed using inverted microscope, identified and differentiated by CD31 immunofluorescence method. Selected were well growing 2nd-4th generations of RCMECs. The optimal action time was determined by detecting the expression of glucose regulated protein 78 (GRP78) using immunofluorescence method. In the next experiment RCMECs were divided into 5 groups, i.e., the blank control group, the Hcy induced group (Hcy 10 mmol/L, 10 h), the Hcy + TXL group (Hcy 10 mmol/L + TXL 400 µg/mL), the Hcy +LY294002 group (Hcy 10 mmol/L + LY294002 5 µmol/L, LY294002 as the inhibitor of PI3K), the Hcy + LY294002 + TXL group (Hcy 10 mmol/L + LY294002 5 µmol/L + TXL 400 µg/mL). The apoptosis rate of RCMECs was detected by flow cytometry. mRNA and protein expressions of GRP78, C/ EBP homologous protein (CHOP), and cysteinyl aspartate specific proteinase-12 (caspase12) were detected by real-time reverse transcription PCR (RT-PCR) and Western blot respectively. Expression levels of phosphorylation of phosphatidylinositol 3-kinase (P-PI3K), total phosphatidylinositol 3-kinase (T- P13K) , phosphorylation of kinase B (P-Akt) , and total kinase B (T-Akt) were detected by Western blot.</p><p><b>RESULTS</b>Ten hours Hcy action time was determined. Compared with the blank control group, the apoptosis rate was increased (22.77%), mRNA and protein expressions of GRP78, CHOP, and Caspase-12 were increased, protein expressions of P-PI3K and P-Akt,ratios of P-PI3K/T-PI3K and P-Akt/T-Akt were decreased in the Hcy induced group (P < 0.05, P < 0.01). Compared with the Hcy induced group, the apoptosis rate was decreased (10.17%), mRNA and protein expressions of GRP78, CHOP, and Caspase-12 were decreased, and expression levels of P-PI3K, P-Akt, P-PI3K/T-PI3K, and P-Akt/T-Akt were increased in the Hcy + TXL group (P < 0.05, P < 0.01). Compared with the Hcy + TXL group, the apoptosis rate was increased (17.9%), mRNA and protein expressions of GRP78, CHOP, and Caspase-12 were increased, expression levels of P-PI3K and P-Akt, ratios of P-PI3K/T-PI3K and P-Akt/T-Akt were decreased in the Hcy + TXL + LY294002 group (P < 0.05, P < 0.01).</p><p><b>CONCLUSION</b>TXL could inhibit the apoptosis of RCMECs resulting from Hcy-induced ERS and its mechanism might be associated with activating PI3K/Akt signaling pathway.</p>

Synthesis and characterization of 9-O-alkyl substituted palmatine derivatives / 中国中药杂志

For the establishment of chemical library of protoberberines provided for the bio-activity screening, the target compounds were synthesized by thermal degradation and nucleophilic substitution reactions with the bio-active alkaloid, palmatine (1), as the raw material, and their structures were identified and conformed by 1H-NMR and MS spectra. Among them, 13 compounds were new.

The effect of baicalin on human brain microvascular endothelial cell under the glucose deprivation combined with hypoxia condition / 中国应用生理学杂志

<p><b>OBJECTIVE</b>To investigate the mechanisms of baicalin on anti-cerebral ischemic through observing the effect of baicalin on human brain microvascular endothelial cell under the glucose deprivation combined with hypoxia condition.</p><p><b>METHODS</b>Human brain microvascular endothelial cells (HBMVECs) cultured in vitro were divided into the following groups: normal group, model group, baicalin high dose group, baicalin middle dose group, baicalin low dose group, nimodipine group. The kits were used to detect the cell viability, leakage rate of lactate dehydrogenase (LDH), Na(+)-K(+)-ATPase, Ca(2+)-ATPase, the concentration of Ca2+ in each group, and apoptosis rates of each group were analyzed by flow cytometry (FCM).</p><p><b>RESULTS</b>Compared with the normal group, the cell viability, Na(+)-K(+)-ATPase and Ca(2+)-ATPase in model group decreased significantly (P < 0.01, P < 0.05). But the leakage rate of LDH, Ca2+ in cells and apoptosis rates increased remarkably (P < 0.01). Compared with the model group, the cell viability, Na(+)-K(+)-ATPase and Ca(2+)-ATPase increased obviously (P < 0.01, P < 0.05) in baicalin high dose group. But the leakage rate of LDH and Ca2+ in cells in baicalin high dose group decreased significantly comparing with that of model group (P < 0.05, P < 0.01). And the reduction of intracellular Ca2+ was superior to that of nimodipine group. Meanwhile, the apoptosis rates decreased significantly in both baicalin high and middle dose groups (P < 0.01).</p><p><b>CONCLUSION</b>Baicalin could improve the cell viability of HBMVECs under the glucose deprivation combined with hypoxia condition. And the mechanisms were related with improving the energy metabolism, inhibiting intracellular calcium overload and decreasing the apoptosis rate of cells further.</p>

Changes of HO/CO system in arterial tissues of rats with stagnacy of collateral-Qi in deficiency condition and treatment with Renshen, Shuangshen, and Tongxinluo / 第二军医大学学报

Objective: To investigate the vascular protective mechanism of different levels of herbs through detecting activity of HO-1/CO system in arterial tissues of rats with stagnancy of collateral-Qi in deficiency condition. Methods: Healthy male Wistar rats of cleanness level were randomly divided into control group, stagnancy of collateral-Qi in a deficiency condition group (SCQDC group), Renshen group, Shuangshen group, and Tongxinluo group. The ultrastructure of endothelium cells in the artery was observed. RT-PCR, Western blot, and immunohistochemical approaches were used to detect JNK, c-Jun mRNA and albumen expression in the arterial tissues. Meanwhile, the content of CO in the arterial tissue was also detected. Results: In SCQDC group, the counts of pinocytotic cells and microvili on the free surface of vascular endothelial cells were significantly decreased; most crests of mitochondria fused with membrane, some even disappeared; serious degranulation was observed in the rough surfaced endoplasmic reticulum. Immunohistochemistry showed that the HO-1 positive signals in the arterial histiocyte weakened significantly compared with the control group. The HO-1 expression was increased in the 3 treatment groups. Compared with control group, SCQDC group had decreased HO-1 mRNA and albumen expression(P<0.01,P<0.001). The 3 different kinds of Herbs to dredge collaterals increased the HO-1 mRNA and albumen expression to different degrees (P<0.001). The increase of albumen in the Tongxinluo group was more than those in the Shuangshen group and the Renshen group(P<0.001). Tongxinluo also significantly increased the CO content. Conclusion: Herbs to dredge collaterals can induce CO production by ameliorating or recovering the HO System, which may be one of the mechanisms for protecting the vascular tissues.

Effect of serum from overfatigue rats on JNK/c-Jun/HO-1 pathway in human umbilical vein endothelial cells and the intervening effect of Tongxinluo superfine powder / 中国结合医学杂志

<p><b>OBJECTIVE</b>To cultivate human umbilical vein endothelial cells (HUVECs) in the serum of overfatigue rats with the intervention of Tongxinluo superfine powder (TXLSP). By examining the variation of the activity of JNK/c-Jun/HO-1 pathway, the possible mechanisms of vascular endothelial dysfunction under overfatigue conditions and the intervening effect of TXLSP were explored.</p><p><b>METHODS</b>The HUVECs were randomly divided into the normal control group, the model group, the SP600125 (a specific antagonist of JNK) group, the TXLSP group and the TXLSP + SP600125 group. The content of carboyhemoglobin (COHb) and the leak rate of lactic dehydrogenase (LDH) in different groups were measured. The mRNA and protein expression of JNK, c-Jun, HO-1 and the phosphorylation level of c-Jun (P-c-Jun) were detected using Western blot and PCR methods.</p><p><b>RESULTS</b>Compared with the normal control group, the COHb level in supernatant was increased significantly in the model group, and the expression of HO-1, JNK, c-Jun mRNA and corresponding proteins and P-c-Jun were also increased remarkably. The increases in these parameters were significantly decreased by SP600125. TXLSP showed remarkable up-regulation on the expression of JNK, c-Jun, P-c-Jun and HO-1 mRNA and their protein expression. Compared with the SP600125 group, the expressions of JNK, c-Jun, P-c-Jun and HO-1 mRNA and its protein in the TXLSP+SP600125 group were significantly increased at different time points (P<0.05, P<0.01).</p><p><b>CONCLUSIONS</b>The vascular endothelial dysfunction under overfatigue conditions is related to the activity of the JNK/c-Jun/HO-1 pathway. One of the mechanisms of TXLSP in improving the vascular endothelial function is to adjust the activity of the JNK/c-Jun/HO-1 pathway at gene and protein levels.</p>

The endothelium injuries caused by homocysteine and treatmental effects of Tongxinluo powder / 中国应用生理学杂志

<p><b>AIM</b>To observe the effect of homocysteine (HCY) on the function of endothelium cell, and to discuss the possible mechanisms that Tongxinluo super powder affected.</p><p><b>METHODS</b>Healthy male Wistar rats were divided into randomly the control group, the model group, the Tongxinluo group. The effect of Ach on isolated rat thoracic aorta in vitro was examined, the microcirculation was observed by microcirculation meter, the activity of SOD and GSH-PX and content of NO, MDA, ET, Ang II, TXA2, PGI2 was detected.</p><p><b>RESULTS</b>Compared with control group, the effect of Ach on isolated rat thoracic aorta in vitro weakened markablely (P < 0.01), the format and percentage that capillary dilated declined significantly (P < 0.05), after treatment with Tongxinluo powder, the effect of Ach on isolated rat thoracic aorta in vitro was improved obviously (p < 0.01), and the format and percentage that capillary dilated were increased compared with model group; comparing with the control group, the level of Ang II and ET, TXA2 in plasm increased obviously (P < 0.05, P < 0.01), while the content of PGI2 depressed manifestly (P < 0.05), at the same time, both content of NO and activity of SOD, (GSH-PX declined obviously (P < 0.001, P < 0.05). After treatment with Tongxinluo powder, the level of ET, AngII and TXA2 reduced significantly in different degree (P < 0.01), while the content of PGI2 appeared stepping up notably (P < 0.01), and both activity of SOD and NO level increased obviously (P < 0.01, P < 0.05).</p><p><b>CONCLUSION</b>(1) The high homocystein might cause the contracted and dilated function decreased, it might get involved in endothelium disfunction as a result of the massive free radicals production and diastolic-contract factors balance disorder induced by high homocystein. (2) Tongxinluo powder could improve the function of endothelium-dependment dilation induced by high homocystein, that associated with inhibitting the excessive production of free radicals, and improved function of endothelium.</p>

The protective function of puerarin to the injury of the lung and its mechanisms during diabetes / 中国应用生理学杂志

<p><b>AIM</b>To evaluate the roles of puerarin in alleviating the STZ-induced lung injury.</p><p><b>METHODS</b>DM model was established by streptozotocin (STZ) intraperitoneal injection to study the injury mechanisms of the lung. SD rats were divided randomly into control group (C group), diabetes group (DM group), diabetes + puerarin group (DM + Pur group). The blood glucose and weight were observed and recorded before and the 20 th, 40 th, 60 thd after administration of saline, STS, STZ+ Pur. Contents of NO and malondialdehyde (MDA) and activity of superoxide dismutase (SOD) were measured in lung tissues. Light microscope (LM), transmission electron microscope (TEM) and immunohistochemical analysis were also used.</p><p><b>RESULTS</b>(1) Compared with control group, the contents of NO and MDA were increased significantly (P < 0.01), while the activity of SOD reduced (P < 0.05). Compared with DM group, treatment with puerarin inhibited the increase of NO level (P < 0.01), and MDA content began to decline from 40 days after the model was established (P < 0.01), and inhibited the decrease of SOD activity induced by DM (P < 0.01). (2) LM and TEM results showed that alveolar and capillary basement membrane became thick, the number of tiny villus decreased markedly, the quantity of osmiophilic multilamellar body reduced remarkably, hyperplasia was shown in collgen fibre. Puerarin could alleviate above injuries induced by DM. (3) Immunohistochemical staining results showed that mild brown positive stain of NT could be seen in protoplasm of lung tissues. STZ administration induced the expression of NT in the protoplasm of cells, and led to stronger positive signals of NT than that of control group. Treatment with puerarin weakened the positive stain of NT.</p><p><b>CONCLUSION</b>(1) DM induced by STZ leads to a significant and sustained increase in blood glucose and obvious lung injury, which may be associated with the overproduction of free radicals. (2) The pathway of NO/ONOO- is one of the injury mechanisms of the lung tissues cells. (3) Puerarin suppresses the expression of NT and elevates the activity of SOD. Thereby, resulting in the reduces of the production of free radicals, which may be one of the mechanisms of its anti-oxidative-injuries.</p>

The effects of Tongxinluo Supermicro Powder on nuclear factor-kappaB, intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 expression in aorta of rabbits fed with high-lipid diet / 中华心血管病杂志

<p><b>OBJECTIVE</b>To observe the effects of Tongxinluo Supermicro Powder on the nuclear factor-kappaB (NF-kappaB), inter-cell adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) expression in aorta of rabbits fed with high-lipid diet.</p><p><b>METHODS</b>Healthy male New Zealand rabbits were randomly divided into 4 groups (n = 8 each): control group, model group, atorvastatin group (3 mg x kg(-1) x d(-1) per gavage), and Tongxinluo group (0.31 g x kg(-1) x d(-1) per gavage). At the end of 6 weeks, the expression of NF-kappaB, ICAM-1 and VCAM-1 were observed by immunochemistry methods, Western blotting and reverse transcription polymerase chain reaction (RT-PCR).</p><p><b>RESULT</b>The nuclear translocation of NF-kappaB in aortic endothelial cells and the gene expressions of NF-kappaB, ICAM-1 and VCAM-1 at protein and mRNA levels of the model group was significantly increased compared that in the control group (all P < 0.05), these effects could be significantly attenuated by atorvastatin and Tongxinluo Supermicro Powder (P < 0.01 vs. model group).</p><p><b>CONCLUSIONS</b>Similar as atorvastatin, Tongxinluo Supermicro Powder could relieve the process of atherosclerosis by decreasing the nuclear translocation of NF-kappaB and reducing the expression of ICAM-1, VCAM-1 in this model.</p>