| Objective: Rheumatoid arthritis (RA) is a common chronic inflammatory autoimmune disease which involves peripheral joints mainly, but it can also affect other organs or tissues, such as heart, blood vessels, and so on. For the past few years, the morbidity and mortality of cardiovascular disease are increased in the context of rheumatoid arthritis. Evidence suggests that this increased cardiovascular disease can largely be attributed to atherosclerosis. Endothelial dysfunction is considered a first step for starting the process of atherosclerosis, so it associates with the generation and development of atherosclerosis closely. In this respect, currently, to further explore endothelial dysfunction and mechanisms for RA have been a focus in medical field. There is one of important goals to treat atherosclerosis that is protecting endothelial function which has consequential clinical significance. The aim of the study based on rat model of adjuvant-induced arthritis to investigate the effect of QingXinYin on endothelium-dependent relaxation of aortic rings, and on tumor necrosis factor-α(TNF-α), interleukin-1β(IL-1β), macrophage migration inhibitory factor (MIF) and adiponectin (APN) in AA rat′serum, the level of endothelin-1 (ET-1) and NT-proBNP in plasma of AA rat, the expression of ET-1 and MIF in vascular endothelium cell. Accordingly, we are to further probe the protective mechanisms of QingXinYin which includes anti-snake venom traditional Chinese medicine and afford experimental evidence for clinical work.Methods:Wistar rats whose weight from 150g to 180g were obtained from Medical University of Hebei, and the number of male and female was equal. Complete adjuvant was prepared by suspending heat-killed Mycobacterium butyricum (Difco, Detroit, MI) in mineral oil at 10 mg/ml. Rats were injected intradermally with 0.1ml of the adjuvant at the base of the tail to induce AA rat models. Simultaneously, eight of rats are injected equivalent saline. The day 19 post-adjuvant of injection, rats presenting secondary response are randomly divided into model group, MTX group, QXY group and MTX+QXY group. Each group includes 8 samples and of whose Arthritis Index (AI) is to exceed five. The rats injected saline as control group. After measuring the paws weight, thickness and volume of rats in each group, the treatment rats are given above-mentioned drugs for successive 21 days. After 21 days treatment, the animals from each group were anaesthetized in the morning with 4% hydral, cavitas abdominalis was opened and the serum and plasma are collected from abdominal aorta. Close behind, thorax was opened, and the descending thoracic aorta isolated carefully and cleaned of surrounding tissue. The isolated aortas were cut into 3.0-4.0mm long rings and placed in organ chambers. Norepinephrine was applied to achieve near-maximal contraction, and dose responses to various concentrations of acetylcholine were determined. Tissue sections were also stained with hematoxylin and eosin to observe histologic changes. The remanent isolated aortas were to determine the expression of ET-1 and MIF in vascular endothelium by immunohistochemistry. Semiquantitative analysis was carried out at equal pace. TNF-α, IL-1βand ET-1 are determined by radioimmunoassay. Meanwhile, NT-proBNP, APN and MIF are determined by enzyme-linked immunosorbent assay (ELISA). All procedures were performed in accordance with our institutional guidelines for animal research.The data was expressed as mean±SEM and median of the indicated number of samples studied. The data was assessed statistically using one-way analysis of variance (ANOVA) and nonparametric test. ANOVA was followed by LSD multiple range tests using SPSS statistical software (version 13.0) for comparison between different treatment groups. Pearson correlation analysis was adopted. Statistical significance was set at P < 0.05.Results:1. The effect of QingXinYin on general condition of AA rats Comparing with model group, other groups are improved in general condition. There was no significant difference in body weight among five groups before induction of rat AA. But after 18 days, the weight of model group, MTX group, QXY group and MTX+QXY group is lower than control group remarkably (P<0.01). However, there was no significant difference between QXY group and model group, MTX group and model group, QXY group and MTX group (P>0.05). The body weight of MTX+QXY group is notably higher than model group (P<0.01). We next examined the body weight gain of each group before and after treatment. The body weight gain of MTX group, QXY group and model group is lower than control group (P<0.01). The body weight gain of MTX group and MTX+QXY group is higher than model group. There was no significant difference between QXY group and model group (P>0.05). There was the same result between MTX group and QXY group. Although the body weight gain of MTX+QXY group was lower than control group, yet significant difference had not been shown.2. The effect of QingXinYin on paw swelling and AI of AA ratsAfter three weeks of treatment, the right paw swelling degree (%) of MTX group, QXY group and MTX+QXY group is lower than model group remarkably (P<0.05 and P<0.01). Compared with MTX and QXY group, the inhibition ratio of MTX+QXY group is higher (P<0.01). And it is shown that MTX group is lower than QXY group (P<0.01). But the average swelling degree (%) of left paws of model group, MTX group, QXY group and MTX+QXY group was 70.09±5.11, 59.02±4.90, 51.55±10.14 and 40.58±5.25, respectively. The statistical discovered that MTX group, QXY group and MTX+QXY group is obviously lower than model (P<0.01). The inhibition ratio of MTX+QXY group is higher than MTX and QXY group (P<0.01). And the average swelling degree (%) of left paws of QXY group is higher than MTX group.In addition to control group, the longer time was gone, the higher AI had been of each group. There was no significant difference among each group before giving treatment (P>0.05). At the end of experiment, however, AI of MTX group and MTX+QXY group is obviously lower than model group (P<0.05 and P<0.01). There was no significant difference between MTX+QXY group and MTX group, MTX+QXY group and QXY group (P>0.05). QXY group was higher than MTX group (P<0.05). Although the AI of QXY group was lower than model group, yet significant difference had not been shown.3. The effect of QingXinYin on endothelium-dependent relaxation of aortic rings in response to acetylcholineWe found that the endothelium-dependent relaxation of the aortic ring was significantly depressed (P<0.01) in model group, MTX group, QXY group and MTX+QXY group compared with control rats. MTX group and QXY group were significantly higher than MTX+QXY group (P<0.01). MTX group was significantly increased compared with QXY group (P<0.05 or P<0.01).4. The effect of QingXinYin on TNF-α, IL-1β, MIF, APN in serum of AA rats4.1 Comparing the level of TNF-αand IL-1βin serum: model group, MTX group, QXY group and MTX+QXY group were significantly higher than control group (P<0.01). Whereas there was no significant difference between MTX+QXY group and control group (P>0.05). Compared with model rats, MTX group, QXY group and MTX+QXY group were significantly increased (P<0.01). The level of TNF-αand IL-1βin MTX group and QXY group lower than MTX+QXY group (P<0.01). MTX group was significantly depressed compared with QXY group (P<0.01).4.2 Comparing the level of MIF in serum: The level of MIF in model group, MTX group and QXY group were increased remarkably higher than control group (P<0.01). There was no significant difference between MTX+QXY group and control group (P>0.05). In contrast, MTX group, QXY group and MTX+QXY group lower than model group (P<0.01). The level of MIF in MTX group and QXY group is higher than MTX+QXY group (P<0.05 or P<0.01). Compared with MTX group, the level of MIF in QXY group was shown to be even higher (P<0.01). 4.3 Comparing the level of APN in serum: The date was demonstrating that level of APN in model group, MTX group, QXY group were significantly lower than control group (P<0.01). Meanwhile, the level of APN in QXY group and MTX+QXY group is higher than model group (P<0.05 and P<0.01). There was no significant difference in levels of APN between MTX group and model group (P>0.05). Compared with MTX+QXY group, MTX group's level is lower (P<0.01). QXY group and MTX+QXY group, MTX group and QXY group have no statistical significance.5. The effect of QingXinYin on ET-1, NT-proBNP in plasma of AA rats5.1 Comparing the level of ET-1 in plasma: The findings reveals that the level of ET-1 in model group, MTX group, QXY group and MTX+QXY group was significantly higher than control group (P<0.05). Control group, MTX group, QXY group and MTX+QXY group is lower than model group (P<0.01). Whereas the level of ET-1 in MTX group and QXY group is increased compared with MTX+QXY group (P<0.05). There was no significant difference in levels of ET-1 between QXY group and MTX group (P>0.05).5.2 Comparing the level of NT-proBNP in plasma: model group, MTX group, QXY group and MTX+QXY group were significantly higher than control group (P<0.01). MTX group, QXY group and MTX+QXY group is lower than model group (P<0.01). MTX group and QXY group is increased compared with MTX+QXY group (P<0.05 and P<0.01). There was no significant difference in levels of NT-proBNP between MTX group and QXY group (P>0.05).6. HE stain and the expression of MIF, ET-1 in vascular endothelium cell of AA rats6.1 Tissue sections were also stained with hematoxylin and eosin with the purpose of observing histologic changes (results shown in figures). The pathology of vascular endothelium was no changes in control group mice. Aortic tunica intima of model group is not smooth and glossy, the conjunction of endothelial cells destroyed, and same to swell up or desquamate. Moreover, here are some vacuole and edema of smooth muscle. The other groups, especially MTX+QXY group, have been presented to repair in different levels.6.2 We subsequently studied the levels of ET-1 and MIF expression in the isolated aortas.6.2.1 The levels of MIF expression in the isolated aortas: The level of MIF in model group, MTX group and QXY group were increased remarkably higher than control group (P<0.01). There was no significant difference between MTX+QXY group and control group (P>0.05). In contrast, MTX group, QXY group and MTX+QXY group lower than model group (P<0.05 and P<0.01). The level of MIF in MTX group and QXY group is higher than MTX+QXY group (P<0.05 and P<0.01). Compared with MTX group, the level of MIF in QXY group was shown to be even higher (P<0.05).6.2.2 The levels of ET-1 expression in the isolated aortas: We found that the level of ET-1 in model group, MTX group, QXY group and MTX+QXY group were significantly higher than control group (P<0.01). MTX group, QXY group and MTX+QXY group is lower than model group (P<0.01). Whereas the level of ET-1 in MTX group and QXY group is increased compared with MTX+QXY group (P<0.05). There was no significant difference in levels of ET-1 between MTX+QXY group and control group (P>0.05). The same findings are shown in QXY group and MTX group.Conclusions:1. Accumulating evidence suggests that AA rats are to be present correlation factors engendering endothelial function and the earliest stage of atherosclerosis.2. Our study shows that MIF and NT-proBNP seem to involve in pathology process of endothelial impairment. APN can be considered as one of the protective factors for endothelial impairment. So they are likely to be the forecasting agents for the earliest stage of atherosclerosis.3. QingXinYin can obviously decrease the paw swelling and the level of inflammatory cell factors, such as TNF-α, IL-1β. We identify QingXinYin combination with MTX can enhance the above-mentioned anti-inflammatory effect remarkably.4. QingXinYin is strikingly shown to protect vascular dysfunction of AA rats, of which have partial mechanisms as follows. QXY can not decrease the expression of ET-1, MIF in blood and vascular endothelium, the level of TNF-α, IL-1βin serum and NT-proBNP in plasma, but increase APN in serum, and so on.
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