The Protective Effects Of Tongluo Drugs On Endothelial Dysfunction Induced By Over-fatigue

Objective:The pressure from social living and psychology has important effects to vascular disease, even resulting in over-fatigue and cardiovascular events. The Collaterals disease theory in Traditional Chinese Medicine (TCM) supposed that collateral-qi in a deficiency and stagnancy condition caused by over-fatigue is the early stages of vascular disease from dysfunction to organic disease and was the trigger item of "Mailuo-Vascular System Disease", which existed in it at all time. The different phase including self-adaptive, self-regulating, self-steady state dysfunction caused by over-fatigue was closely related to endothelial dysfunction. Endothelial dysfunction, throughout every process of vascular disease, was the common pathological basis in occurrence and development of vascular disease. In this study, the over-fatigue experimental animal model was developed to explore the damage of endothelium caused by lipid peroxidation and phospholipaseA2 activation and to observe the preventive effects of Tongluo drugs. It would be helpful for further probing the internal relationship between psychosocial factors and cardiovascular disease.Methods:1 The effect of Tongluo drug on the structure and function of vascular endothelial cell in over-fatigue rat aortic. Seventy-five male Wistar rats were randomly divided into the following four groups: (1) Control group, (2) Fatigue group, (3) Renshen group (1.2g powder/kg·d), (4) TXL large dose group (1.2g powder/kg·d), (5)TXL small dose group(0.6g powder/kg·d). To evaluate the fatigue status with the methods of general observation, the behavior score, climbing time. The light microscope and transmission electron microscope (TEM) were used to observe the aorta morphology. The immunohistology chemistry was adopted to analyze the the expression of endothelin-1 (ET-1) and endothelial nitric oxide synthase (eNOS). And the level of endothelin (ET) and nitro oxygen (NO) in blood were determined to token the function of EC.2 The effects of Tongluo drugs on the SOD, MDA in serum and LOX-1 receptor expression in aorta of over fatigue rats.Sixty male Wistar rats were randomly divided into the following four groups: (1) Control group, (2) Fatigue group, (3) Renshen group(1.2g powder/kg?d), (4) TXL group(1.2g powder/kg?d). The method of developing experimental animal model was the same as part 1, at the end of the experiment, blood samples and aortic tissues were collected. The activity of serum SOD was detected by xanthine oxidase method. The content of serum MDA was detected by colorimetry. Aortic tissue LOX-1 receptor mRNA and protein expression were detected by RT-PCR and western blot Methods. To observed the effects of Tongluo drugs on lipid peroxidation and ox-LDL receptor expression induced by over-fatigue.3 The effects of Tongluo on the expression of phospholipase A2 and GPR4 receptor in overfatigue rats'aorta.The experimental animal groups and the modeling methods were the same as the second part. At the end of the experiment, blood samples and aortic tissues were collected. The lp-PLA2 levels in serum were detected by enzyme-linked immunosorbent assay. The expressions of aortic tissue PLA2 were detected by immunohistochemistry. Aortic tissue GPR4 receptor mRNA and protein expression were detected by RT-PCR and western blot methods. Linear correlation was analyzed between serum lp-PLA2 and ET and MDA. The results indicated that Tongluo drugs can improve the abnormal metabolism in membrane phospholipid and lipoprotein phospholipid composition induced by the activation of phospholipase A2 who played an important role on endothelial dysfunction induced by over-fatigue.Results:1 The effect of Tongluo drug on the structure and function of vascular endothelial cell in over-fatigue rat aortic. 1.1 The changes of general condition and behavior in rats of different groups.Compared with control group, with the time prolonging, the symptom in model group rats appeared, including tiredness and laziness and sluggishness and weight loss, skin and pelage withered and dim, reduced shoulder extrados, ears and tails pale and lusterless. Furthermore, the score of biology superficial syndrome was increased significantly than control group(P<0.01). The body weight decreased significantly at the end of the experiment (P<0.01). Climbing pole time was significantly shorter (P<0.01). So the fatigue status of rats was reflected by the behavior induced by weight-loading exhaustive swimming. Compared with the fatigue group, Ginseng and Tongxinluo can improve the general situation of rats, the scores of biology superficial syndrome were significantly decreased (all P<0.01). The body weights were increased, but no significant difference. The last day exhaustive swimming time of Ginseng group was significantly longer than the model group (P<0.05). Climbing pole time was longer than the model group, but no significant difference. The last exhaustive swimming times of TXL large and small doses groups were significantly prolonged than the model group (P<0.05, P<0.01), climbing pole times were significantly longer (P<0.05, P<0.01).1.2 The morphology of aorta endothelium1.2.1 The morphology of light microscopeThe light microscope analysis showed that vascular wall layers were clear, the structure of internal elastic membrane was smooth, endothelial cells were integrity and continuous, and the middle and outer membrane were normal in control group. Edema could be observed in endothelial cell, internal elastic membrane was rupture, edema and hyperplasia could also be observed in the smooth muscle layer, and inflammatory cell infiltration could be seen in fatigue groups. Minor edema could be observed in endothelial cells and smooth muscle layer in Ginseng group. Minor edema could be observed in endothelial cells, vascular wall layers were clear, the internal elastic membrane and elastic fibers in the middle membrane were visible in TXL large dose group. Minor edema could be observed in endothelial cells and smooth muscle layer, vascular wall layers were clear in Tongxinluo small dose group.1.2.2 The morphology of TEMThe Electron microscope analysis showed that the rules of vascular endothelial cells were tightly, basement membrane intacted, nucleus and organelles were existing, mitochondria structure was normal in control group. The majority of mitochondrial cristae and membrane were fusion, vague, varying degrees of degranulation could be found in the rough surfaced endoplasmic reticulum, the counts of pinocytotic vesicles could be found decreased, part of the nucleus gap was expansion in fatigue groups. Part of mitochondrial cristae and membrane were fusion, vague, part of varying degrees of degranulation could be found in the rough surfaced endoplasmic reticulum, microvilli, pinocytotic vesicles could be found decreased in Ginseng group. A little part of mitochondrial cristae and membrane were fusion, vague, mild degranulation could be seen in rough endoplasmic reticulum, the counts of microvilli and pinocytotic vesicles were decreased in TXL large dose group. Part of mitochondrial cristae and membrane were fusion, vague, part of varying degrees of degranulation could be found in the rough surfaced endoplasmic reticulum, microvilli, pinocytotic vesicles could be found minor decreased in TXL small dose group.1.3 The changes of correlated index of VED1.3.1 The changes of NO and ETCompared with control group, the serum NO level of fatigue group was significantly decreased (P<0.05), plasma ET was significantly increased (P<0.01). Compared with fatigue group, the serum NO levels of the Ginseng, TXL large and small doses groups were significantly increased (all P<0.01), plasma ET levels were significantly decreased(all P<0.01), and the level of TXL-large dose intervention group had the most significant improvement of endothelial function.1.3.2 The expression of ET-1 and eNOS in aorta tissueET-1 expression: The immunohistology chemistry analysis showed that weak positive staining could be observed on aortic endothelial cells in control group. Strong positive staining could be observed under endothelium in fatigue group, and the remaining floors saw weak positive or negative staining. Weak positive staining could be observed on aortic endothelium in Ginseng and TXL large and small dose groups.eNOS expression: The immunohistology chemistry analysis showed that positive staining on artery layers in control group. Weak positive staining could be observed on arterial layers in fatigue group. Positive staining could be observed on endothelial cells in Ginseng group. Strong positive staining could be observed on endothelium in TXL large and low doses group, weak staining could be observed on the remaining layers. 2 The effects of Tongluo drugs on.SOD, MDA in serum and LOX-1 receptor expression in aorta of ove -fatigue rats.2.1 The changes of serum SOD activity, MDA content in different groupsCompared with control group, the serum SOD activity of fatigue group was significantly decreased (P<0.01), the serum MDA level was significantly increased(P<0.01). Compared with fatigue group, the serum SOD activity of Ginseng and TXL group were significantly increased (P<0.01), the serum MDA levels were significantly decreased (P<0.01).2.2 The aorta LOX-1 receptor mRNA and protein expression of rats in different groupsCompared with control group, aorta LOX-1mRNA expression of fatigue group was significantly increased (P<0.01). Compared with fatigue group, Ginseng and TXL were able to significantly decreased expression of LOX-1mRNA, and both statistically significant (P <0.05, P <0.01).Compared with control group, aorta LOX-1 protein expression of fatigue group was significantly up-regulated. Compared with fatigue group, aorta LOX-1 protein expression of Ginseng and TXL groups were significantly reduced, and the more obvious down-regulation on TXL group.3 The effects of Tongluo drugs on phospholipase A2 and GPR4 receptor expression in aorta of over-fatigue rats. 3.1 The changes of serum lp-PLA2 level in different groupsCompared with control group, the serum lp-PLA2 level of fatigue group was significantly increased (P<0.01). Compared with fatigue group, the serum lp-PLA2 levels of Ginseng and TXL groups were significantly decreased (P<0.01). Linear correlation analysis showed: The serum lp-PLA2 was positively correlated with ET(r=0.678,P<0.01), the serum lp-PLA2 was positively correlated with MDA (r=0.743,P<0.01).3.2 The expression of rat aortic tissue PLA2 in different groups.The immunohistology chemistry analysis showed that weak positive staining could be observed on aortic endothelium, weak or negative staining could be observed on other layers in control group. Strong positive staining could be observed on endothelium and smooth muscle layer in fatigue group. Weak positive staining could be observed on aortic endothelium, negative staining could be observed on other layers in Ginseng and TXL groups.3.3 The GPR4 receptor mRNA and protein expression of rats aortic tissue in different groups.Compared with control group, the aortic GPR4mRNA expression of fatigue group was significantly increased (P<0.01). Compared with fatigue group, Ginseng and TXL were able to significantly decreased expression of GPR4mRNA, and both statistically significant (all P<0.01).Compared with control group, the aortic GPR4 protein expression of fatigue group was significantly increased. Compared with fatigue group, the GPR4 protein expression was significantly decreased in Ginseng and TXL drugs groups, and the more obvious down-regulation on TXL group.Conclusion:1 Under the background of the effects of over-fatigue and other psychosocial factors in vascular diseases, developing over-fatigue rat model by "restricted diet and weight-loading exhaustive swimming" method, to investigate the effects of over-fatigue on the structure and function of vascular endothelial cells. the results proved that psychosocial factors such as over-fatigue can lead to vascular endothelial cell injury and dysfunction. 2 Oxidative stress was enhanced by over-fatigue, resulting in a large number of free radicals. This process consumed SOD activity and promoted lipid peroxidation, to produce a large number of MDA. The endothelial specificity ox-LDL receptor LOX-1 was excess expression in the over-fatigue rats'aorta. It certificated that ox-LDL was recognized by specific receptor, and played a role in endothelial cells injured. Tongluo drugs could prevent endothelial cell injury from oxidative stress by reducing lipid peroxidation and inhibiting the over-expression of LOX-1 receptor.3 In over-fatigue rats, the large number of free radicals induced activation of phospholipase A2 to hydrolyze membrane phospholipids and phospholipids ingredient of ox-LDL. So some active substances as LPC and oxidized fatty acid which participated in vascular endothelial dysfunction were produced. Lp-PLA2, the key enzyme hydrolysis of ox-LDL, was significantly increased in the serum of over-fatigue rats. As the specific receptor for LPC to injure vascular endothelium, GPR4 receptor expression was significantly increased in over-fatigue rat aorta. And Tongluo drugs reduced the activity of phospholipase A2, inhibited the expression of GPR4 receptor, improved the membrane phospholipid and lipoprotein phospholipid ingredient metabolism, and maintained the integrity and normal function of endothelial cells. The protective effects of TXL were better than Ginseng.The experiment suggested that Tongluo drugs could improve vascular endothelial structure and function through reducing oxidative stress, improving phospholipid metabolism.