| “Ying and Wei theory” is the core of the Vessel-Collateral Doctrine. Ying runs inside the vessel and Wei runs outside the vessels. Ying and Wei Convergence and transformation in collateral, which coincide with the substance exchange in the end of the microcirculation of Western medicine. Microvascular endothelial cells play an important role in that physiological function, and it is of great value to reveal the pathogenesis of the disease. Under the guidance of Ying and Wei “unblocking collaterals with subsequent convergence and substance-qi transformation” theory of the Vessel-Collateral Doctrine, based on the relationship between Ying and vascular endothelium, Wei and the vascular outer membrane and the systemic nerve-endocrine-immune network（NEI）, this study established the model of cardiac microvascular endothelial cells induced by homocysteine. To observe the effect of injuried microvascular endothelial cells conditioned medium on cardiomyocytes, and to explore the possible mechanism of the protective effect of Tongxinluo on reduced the injury of cardiomyocytes by protected the injured cardiac microvascular endothelial cells. This study to provide experimental data support for the theory of Ying and Wei “unblocking collaterals with subsequent convergence and substance-qi transformation”, and to open up a new way for the treatment of vascular diseases from the perspective of collateral integrity protection. Part Ⅰ Homocysteine induced injury of cardiac microvascular endothelial cells on cardiomyocytesObjective: To investigate whether the injured of cardiac microvascular endothelial cells conditioned medium can induce the injury of cardiomyocytes.Methods: This section Primitive extraction plant block method to separated rat cardiac microvascular endothelial cells and immunofluorescence method is used to identify these. Use 10 mmol/L Hcy to intervented rat cardiac microvascular endothelial cells for 24 hours that cardiac microvascular endothelial cells damaged model was established, and seted up normal group. In the two groups Take CCK-8 method to detected the cells viability of rat cardiac microvascular endothelial cells. The contents of ET-1, NO and LDH in two groups of rat cardiac microvascular endothelial cells supernatant were detected by visible spectrophotometry, nitrate reductase method and enzyme linked immunosorbent assay.The cardiac microvascular endothelial cells conditioned medium were extracted from normal cardiac microvascular endothelial cells（N-CM） and homocysteine injuried cardiac microvascular endothelial cells（H-CM）. The H9c2 cardiomyocytes were divided into normal group（Normal）, N-CM group and H-CM group. The morphological changes of the cells were observed by inverted microscope. CCK-8 method was used to detect the cells viability of H9c2 cardiomyocytes. Enzyme linked immunosorbent assay was used to detected the content of cTnT in the supernatant in each group of the H9c2 cardiomyocytes.Results: 1 Morphology and characterization of rat cardiac microvascular endothelial cells:The rat cardiac microvascular endothelial cells were cultured for 24 hours, shows spindle, a star or polygon. Then taken away the tissue block cultured to 96 hours, the cells could be seen like the sink synthetic paving stone. The CD31 immune fluorescent identification results showed that the extraction of rat cardiac microvascular endothelial cells purity greater than 95%. 2 The comparison of cell viability in two rat cardiac microvascular endothelial cells groups:Compared with Normal group, the cell viability of Hcy group was decreased（P<0.01）. 3 The comparison of LDH content in cell supernatant in the two rat cardiac microvascular endothelial cells groups:Compared with Normal group, the LDH content in cell supernatant of Hcy group was increased（P<0.01）. 4 The comparison of NO and ET-1 content in cell supernatant in the two rat cardiac microvascular endothelial cells groups:Compared with Normal group, the NO content was decreased and the ET-1 content was increased in cell supernatant of Hcy group（all P<0.01）. 5 H9c2 cardiomyocytes Morphological changes in different groups:In Normal group H9c2 cardiomyocytes were short fusiform or irregular triangle, with clear edge and the nucleus is oval centered. Compared with Normal group, the morphology of the cells in the N-CM group without significant change. Compared with N-CM group, the cells nucleus concentration, edge blur and more celsl fell off, a clusters of cells decreased significantly, the injury was serious. 6 The comparison of cell viability in different H9c2 cardiomyocytes groups:Compared with Normal group, N-CM group had no significant changed in cell viability（P>0.05）. Compared with N-CM group, the cell viability was decreased in H-CM group（P<0.01）. 7 The comparison of c Tn T content in cell supernatant in different H9c2 cardiomyocytes groups:Compared with Normal group, the content of c Tn T in cell supernatant was no significant changed in N-CM group（P>0.05）. Compared with N-CM group, the content of c Tn T in cell supernatant was increased in H-CM group（P<0.01）.Part Ⅱ Protective effect of Tongxinluo on cardiac microvascular endothelial cells induced by homocysteineObjective: To investigate the protective effect and mechanism of Tongxinluo on cardiac microvascular endothelial cells.Methods:The cardiac microvascular endothelial cells cells were divided into 5 groups: normal group（Normal）, homocysteine group（Hcy）, Tongxinluo low concentration group（TXL-L）, Tongxinluo middle concentration group（TXL-M）, and Tongxinluo high concentration group（TXL-H）. CCK-8 assay was used to detect the cardiac microvascular endothelial cells cell viability; hydroxylamine method, TBA method and enzyme linked immunoassay were detection of the culture supernatant of SOD, MDA, IL-6 and ICAM-1 content; flow cytometry was used to detected the cells in ROS and apoptosis rate; Real-Time fluorescent quantitative Reverse Transcription PCR（RT-PCR） was used to detected ET-1 m RNA expression; Western Blot was used to detected the cells e NOS and NF-κB protein expression.The cardiac microvascular endothelial cells were divided into 7 groups: normal group（Normal）, Hcy intervented 5 hours group（Hcy 5 h）, Hcy intervented 5 hours+TXL group（Hcy 5 h+TXL）, Hcy intervented 10 hours group（Hcy 10 h）, Hcy intervented 10 hours+TXL group（Hcy 10 h+TXL）, Hcy intervented 20 hours group（Hcy 20 h） and Hcy intervented 20 hours+TXL group（Hcy 20 h+TXL）. Used high-throughput high content cells analysis system to detected the GRP78 protein immunofluorescence strength. The cardiac microvascular endothelial cells were divided into 5 groups: normal group（Normal）, Hcy group, Hcy+TXL group, Hcy+LY294002 group（LY294002 as the inhibitor of PI3K） and Hcy+LY294002+TXL group. Use flow cytometry to detected the cells apoptosis rate; RT-PCR and Western blot assay for detected the GRP78, CHOP, Caspase-12 m RNA and protein expression; Western blot was used to detected the expression of p-PI3 k, t-PI3 K, p-Akt and t-Akt protein expression.Results: 1 The comparison of cell viability in different rat cardiac microvascular endothelial cells groups: Compared with Normal group, the cell viability was decreased in Hcy group（P<0.01）. Compared with Hcy group, the cell viability was increased in TXL-H group（P<0.01）. 2 The comparison of e NOS protein expression in different rat cardiac microvascular endothelial cells groups:3 The comparison of ET-1 mRNA expression in different rat cardiac microvascular endothelial cells groups: Compared with Normal group, the ET-1 m RNA expression was increased in group Hcy（P<0.01）. Compared with Hcy group, the ET-1 m RNA expression was decreased in TXL-L group, TXL-M group and TXL-H group（P<0.01）.4 The Comparison of SOD activity and MDA content in cell supernatant in different rat cardiac microvascular endothelial cells groups:Compared with Normal group, the SOD activity was reduced and theMDA content was increased in cell supernatants in Hcy group（all P<0.01）.Compared with Hcy group, the SOD activity was increased in cell supernatantin TXL-L group, TXL-M group and TXL-H group（P<0.01）, the MDAcontent was decreased in cell supernatant in TXL-M group and TXL-H group（P<0.01）. Compared with TXL-L group, the SOD activity in cell supernatantwas increased in TXL-H group（P<0.05）, the MDA content was decreased incell supernatant in TXL-M group and TXL-H group（P<0.01）. Compared withTXL-M group, the MDA content was decreasedin cell supernatant in TXL-Hgroup（P<0.01）.5 The comparison of ROS fluorescence intensity in different rat cardiacmicrovascular endothelial cells groups:Compared with Normal group, the ROS fluorescence intensity was increased in Hcy group（P<0.05）. Compared with Hcy group, the ROS fluorescence intensity was decreased in TXL-H group（P<0.05）.6 The comparison of IL-6 and ICAM-1 content in cell supernatant in differentrat cardiac microvascular endothelial cells groups:Compared with Normal group, the IL-6 and ICAM-1 content were increased in cell supernatant in Hcy group（P<0.01）. Compared with Hcy group, IL-6 and ICAM-1 content in cell supernatant were decreased in TXL-M group and TXL-H group（P<0.01）. Compared with TXL-M group, the IL-6 and ICAM-1 content were decreased in TXL-H group（P<0.05）.7 The comparison of NF-κB protein expression in different rat cardiacmicrovascular endothelial cells groups:Compared with Normal group, the NF-κB protein expression was increased in Hcy group（P<0.01）. Compared with Hcy group, the NF-κB protein expression was decreased in TXL-L group, TXL-M group and TXL-H group（P<0.01）. Compared with TXL-L group, the NF-κB protein expression was decreased in TXL-M group and TXL-H groups（P<0.01）. Compared with TXL-M group, the NF-κB protein expression was decreased in TXL-H group（P<0.05）.8 The comparison of GRP78 protein expression in different rat cardiamicrovascular endothelial cells groups:Compared with Normol group, the GRP78 protein expression had no significant change in Hcy 5 h group（P>0.05）, the GRP78 protein expression were increased in Hcy 10 h group and Hcy 20 h group（P<0.01）. Compared with Hcy 10 h, the GRP78 protein expression was decreased in Hcy 10 h+TXL group（P<0.01）. Compared with Hcy 20 h, the GRP78 protein expression was decreased in Hcy 20 h+TXL group（P<0.01）.9 The comparison of p-PI3 K, t-PI3 K, p-Akt and t-Akt protein expression in different rat cardiac microvascular endothelial cells groups:Compared with Normal group, p-PI3 K, p-PI3K/t-PI3 K, p-Akt and p-Akt/t-Akt protein expression were decreased（P<0.05 or P<0.01）, t-PI3 K and t-Akt were no significant changed in Hcy group（P>0.05）; p-PI3 K, p-PI3K/t-PI3 K, p-Akt and p-Akt/t-Akt protein expression were decreased（P<0.01）, t-PI3 K and t-Akt protein expression were no significant changed in Hcy+LY294002 group and Hcy+LY294002+TXL group（P>0.05）. Compared with Hcy group, p-PI3 K, p-PI3K/t-PI3 K, p-Akt and p-Akt/t-Akt proteinexpression were increased（P<0.05 or P<0.01）, t-PI3 K and t-Akt protein expression were no significant changed in Hcy+TXL group（P>0.05）.10 The Comparison of cell apoptosis rate in different rat cardiac microvascular endothelial cells groups:Compared with Normal group, the cell apoptosis rate was increased in Hcy group（P<0.01）. Compared with Hcy group, the apoptosis rate were decreased in Hcy+TXL group and Hcy+LY294002+TXL group（all P<0.01）, the cell apoptosis rate was no significant changed in Hcy+LY294002 group（P>0.05）. Compared with Hcy+TXL group, the cell apoptosis rate was increased in Hcy+LY294002+TXL group（P<0.01）.11 The Comparison of GRP78, CHOP and Caspase-12 m RNA expression in different rat cardiac microvascular endothelial cells groups:Compared with Normal group, GRP78, CHOP and Caspase-12 mRNA and protein expression were increased in Hcy group（all P<0.01）; Compared with Hcy group, GRP78, CHOP and Caspase-12 m RNA and protein expression were decreased in Hcy+TXL group and Hcy+LY294002+TXL group（all P<0.01）, GRP78, CHOP and Caspase-12 m RNA and protein expression were no significant changed in Hcy+LY294002 group（P>0.05）. Compared with Hcy+TXL group, GRP78, CHOP and Caspase-12 m RNA and protein expression were increased in Hcy+LY294002+TXL group（P<0.01）.Part Ⅲ Protective effect of Tongxinluo on the conditioned medium of injuried cardiac microvascular endothelial cells on cardiomyocytesObjective: To clarify the indirect protective effect of Tongxinluo on cardiomyocytes through improving the secretion function of cardiac microvascular endothelial cells.Methods: The H9c2 cardiomyocytes were divided into 4 groups: normalgroup（Normal）, normal rat cardiac microvascular endothelial cellsconditioned medium group（N-CM）, homocysteine induced rat cardiacmicrovascular endothelial cells conditioned medium group（H-CM） and ratcardiac microvascular endothelial cells conditioned medium protectived byTongxinluo group（T-CM）. CCK-8 assay was used to detected thecardiomyocytes cell viability; enzyme linked immunoassay was to detected the cells supernatant c Tn T content; JC-1 fluorescence probe was to detected the cells mitochondrial membrane potentiall; flow cytometry was to detected the cells ROS fluorescence intensity; Western Blot was applied to detected the cells Cytochrome C, calreticulin（CRT）, Caspase-3, Bax and Bcl-2 protein expression; cell immune fluorescent staining method was used to detected the CRT protein expression; live cell imaging system dynamic observated the apoptosis of cardiomyocytes in each group.Results:1 The comparison of cell viability in different H9c2 cardiomyocytes groups:Compared with Normal group, the cell viability was no significant changed in N-CM group（P>0.05）. Compared with N-CM group, the cell survival viability was decreased in H-CM（P<0.01）. Compared with H-CM group, the cell viability was increased in T-CM（P<0.01）.2 The comparison of c Tn T content in cell supernatant in different H9c2 cardiomyocytes groups: Compared with Normal group, the content of c Tn T in cell supernatant was no significant changed in N-CM group（P>0.05）. Compared with N-CM group, the content of c Tn T in cell supernatant was increased in H-CM group（P<0.01）.Compared with H-CM group, the content of c Tn T in cell supernatant was decreased in T-CM group（P<0.01）.3 The comparison of mitochondrial membrane potential（MMP） in different H9c2 cardiomyocytes groups:Compared with Normal group, the MMP had no significant changed inN-CM group（P>0.05）. Compared with N-CM group, the MMP was decreasedin H-CM group（P<0.01）. Compared with H-CM group, the MMP wasincreased in T-CM group（P<0.01）.4 The comparison of ROS fluorescence intensity in different H9c2 cardiomyocytes groups:Compared with Normal, the level of ROS was no significant changed in N-CM group（P>0.05）. Compared with N-CM group, the level of ROS wasincreased in H-CM group and the fluorescence intensity was increased（P<0.01）. Compared with H-CM group, the level of ROS was decreased in T-CM group（P<0.05）.5 The comparison of CRT protein expression in different H9c2 cardiomyocytes groups: Compared with Normal group, the CRT protein expression had no significant changed in N-CM group. Compared with N-CM group, the CRT protein expression was increased and the cells shrinkaged in H-CM group. Compared with H-CM group, the CRT protein expression was increased and the cells shrinkaged significantly reduced in T-CM group.6 The comparison of CRT and Cyt C protein expression in different H9c2 cardiomyocytes groups: Compared with Normal group, the CRT and Cyt C protein expression had no significant changed in N-CM group（P>0.05）. Compared with N-CM group, the CRT and Cyt C protein expression were increased in H-CM group（P<0.01）. Compared with H-CM group, the CRT and Cyt C protein expression were decreased in T-CM group（P<0.05 or P<0.01）. 7 The comparison of cell apoptosis in different H9c2 cardiomyocytes groups were observed with live cells imaging systemUse live cells imaging system dynamic observated H9c2 cardiomyocytes 15 hours. It was found that Normal group cells survived in good condition, the rounded nucleus had no shrinkaged, with clear edges, fluorescence intensity homogeneity and part of the cells appeared proliferation and very few cells occurred apoptosis（the nucleus were blue）. Compared with Normal group, in N-CM group there had no significant changed and part of the cells divisioned and proliferationed, very few cells apoptosised. Compared with N-CM group, in H-CM group within 5 hours had a handful of cell divisioned and proliferationed, at 5 hour began to appeared cell apoptosis, from 5 hours to 15 hours in the cells nuclear shrinkaged, hyperchromaticed, nuclear fragmentated, appeared apoptotic bodies and the cells apoptosised significantly.To be observed continuously for 15 hours the cells number were significantlyreduced. Compared with H-CM group, in T-CM group at 5 hours the cells didn’t appeare obvious cell apoptosis, from 5 hours to 15 hours part of the cells showed shrinkage, fragmentation, apoptotic bodies and cell apoptosis, but the number of apoptotic cells were significantly decreased.8 The comparison of Caspase-3, Bax and Bcl-2 protein expression in different H9c2 cardiomyocytes groups: Compared with Normal group, Caspase-3, Bax and bcl-2 protein expression had no significant changed in N-CM group（P>0.05）. Compared with N-CM group, Caspase-3 and Bax protein expression were increased and Bcl-2 protein expression was decreased in H-CM group（all P<0.01）. Compared with H-CM group, Caspase-3 and Bax protein expression was decreased and Bcl-2 protein expression was increased in T-CM group（all P<0.05 or P<0.01）.Conclusion:1 Under the guidance of Ying and Wei “unblocking collaterals with subsequent convergence and substance-qi transformation” theory of the Vessel-Collateral Doctrine, based on the relationship between Ying and vascular endothelium, Wei and the vascular outer membrane and the systemic nerve-endocrine-immune network（NEI）, and “collateral-microvascular” homoousia in anatomical morphology, this study to investigate the effect of homocysteine induced rat cardiac microvascular endothelial cells injury on cardiomyocytes and protective effects of Tongxinluo. This study to provide experimental data support for the theory of Ying and Wei “unblocking collaterals with subsequent convergence and substance-qi transformation”, and to open up a new way for the treatment of vascular diseases from the perspective of collateral integrity protection. 2 The experimental results showed that the conditioned medium of homocysteine induced cardiac microvascular endothelial cells injuried can decrease the viability of the cardiomyocytes, inhibited the function of mitochondria, and promoted the apoptosis of cardiomyocytes. Myocardial microvascular endothelial cell dysfunction may mediate cardiac muscle celldamage, that suggested of cardiac microvascular endothelial cells secretion dysfunction can mediated cardiomyocytes injuried. This was consistented with the course of the pathological damage caused by the Ying and Wei biochemical function of the “collateral-microvascular” at the end of the Vessel-Collateral.3 Tongxinluo can reduced the oxidative and inflammatory damage of cardiac microvascular endothelial cells, inhibited cells apoptosis, and improved the secretion function. The regulated effect of Tongxinluo on cardiac microvascular endothelial cells can further improve the viability of the damaged cardiomyocytes, improved the function of mitochondria, inhibited cells apoptosis, and then protect the cardiomyocytes.It was shown that Tongxinluo can inhibited the pathological injury of the viscera by protecting the “collateral- microvascular”. |
PDF Full Text Request