| Aim1. To investigate the anti-hypertensive effects of Qindan Capsule (QC) on spontaneous hypertensive rats (SHR), as well as to observe the changes of the morphology and the ultrastructure of the arteries.2. To investigate the possible mechanism of QC in reversing vascular adventitia remodeling in hypertension through detecting the variation of TGF-β1 and SM a-actin mRNA and protein expression.Methods32 male (40 weeks old) SHRs and 16 male (40 weeks old) Wistar Kyoto(WKY) rats were randomly divided into 6 groups (n=8 per group):the WKY control group, the WKY+QCH group, the SHR control group, the high dosage QC group (SHR+QCH), and the low dosage QC group (SHR+QCL), and the Losartan group (SHR+Losartan). The SHR+QCH group was treated with high dosage (750 mg/kg per day) of QC; the SHR+QCL group was treated with low dosage (150 mg/kg per day) of QC; the SHR+Losartan group was treated with losartan for 30 mg/kg per day; and the WKY+QCH group were treated with high dosage (750 mg/kg per day) of QC. The WKY control group and the SHR control group were treated with equal volume of normal saline instead. All the treatments were administered by gastrogavage once a day for 12 successive weeks.After the last administration, all rats were fasted with free access to water for 24 h, and anesthetized with intraperitoneal injection of 3% pentobarbital (30 mg/kg) which was followed by other experiments. The detection contents listed below:(1) Measurement of the systolic blood pressure (SBP); (2) Observation of the pathological structure in aortic wall, including the density of the nuclei in the adventitia (3) Measurement of the morphological index of aortic wall by laser scanning confocal microscope:the luminal diameter (LD), the medial thickness (MT), the adventitia thickness (AT), and the ratio of MT to AT; (4) Analysis of TGF-β1 and SM a-actin mRNA expression by real-time PCR, as well as protein expression by immuno-histochemical staining.Results1. Comparison of SBPThe SBP of rats in the SHR group with 40 weeks old was much higher than those in the WKY group (P＜0.01). The rats in the SHR group continuously developed further hypertension to a high pressure level. From the fourth week of treatment, the SBP was lower in the SHR+QCH group, the SHR+QCL group and the SHR+ Losartan group than that in the SHR control group (all P＜0.05). After seven weeks of treatment, the SBP in the SHR+QCH group was decreased significantly compared with that in the SHR+QCL group (P<0.05). After twelve weeks of treatment, the SBP in each group was decreased in different degree compared with the SHR control group (all P<0.05). Among all groups, the strongest decrease of SBP was shown in the SHR+ Losartan group, while the weakest decrease was shown in the SHR+QCL group. A statistically significant difference was shown between the SHR+ Losartan group and the SHR+QCL group. (P<0.05) (table 1).2. Observation of sections of thoracic aortic wall stained with HEThe structure of the intima, the media and the adventitia of thoracic aortic wall was very clear and was no thickening in the WKY rats. The thickness of adventitia was uniform, and the cells were arranged densely and regularly without proliferation. The thoracic aortic walls were shown obviously thickened in the SHR group, with active cell proliferation in the adventitia. Some sections showed that the adventitia was ruptured and even exfoliated. The medial elastic fibers were destroyed. The ratio of MT/LD/AT was decreased, while the density of the nuclei was obviously increased. After treatment, the pathologic alterations of the adventitia in the SHR+QCH, the SHR+QCL and the SHR+Losartan groups were ameliorated. But a significant difference was shown compared with the WKY group.(fig.l)3. Comparison of morphological index of thoracic aortic wall(1) MT was increased significantly in the SHR group compared with that in the WKY group (P＜0.01). The SHR+QCL group showed a lower MT than the SHR group (P＜0.05). MT was decreased significantly in the SHR+QCH and the SHR+Losartan groups compared with that in the SHR group (all P<0.01). However, no significant difference was shown in comparison among these three above groups (all P>0.05).(2) LD was decreased in different degree in the SHR group and other treatment groups compared with the WKY group, but no significant difference was shown in comparison among these six experimental groups (all P>0.05).(3) The ratio of MT/LD was significantly higher in the SHR group than the WKY group (P＜0.01). The ratio of MT/LD was significantly lower in the SHR+QCH, the SHR+QCL and the SHR+Losartan groups than the SHR group (all P<0.05), but no significant difference was shown in comparison among these three above groups (all P>0.05).(4) The adventitia thickness was increased significantly in the SHR group compared with that in the WKY group (P＜0.01). The adventitia thickness decreased significantly in the SHR+QCH, SHR+QCL and the SHR+Losartan groups compared with that in the SHR group (all P＜0.01). However, no significant difference was shown in comparison among these three above groups (all P>0.05).(5) The ratio of LD/MT/AT was significantly decereased in the SHR group than the WKY group (P＜0.01). The ratio of LD/MT/AT was not significantly lower in the SHR+QCH, the SHR+QCL and the SHR+Losartan groups than the SHR group (all P<0.05), and no significant difference was shown in comparison among these three above groups (all P>0.05).(table 2)4. Observation of sections of thoracic aortic wall stained with immunohistochemistryTGF-β1 was expressed in the cytoplasm, and stained in brown. Eight non-overlapping fields per tissue section, chosen at random, were scanned, and the optimal density (OD) values of TGF-β1 were calculated. Compared with the WKY group, the expression of TGF-(31 was significantly increased in the SHR group. After treatment, the expressions of TGF-P1 in the SHR+QCH, the SHR+QCL and the SHR+Losartan groups were all decreased compared with the SHR group. However, the levels of TGF-P1 in those above groups were still higher than the WKY group. (P< 0.05, Fig.2)SM a-actin was also expressed in the cytoplasm. However, the positive staining of SM a-actin could hardly be detected in the vascular adventitia in the WKY group. The expression of SM a-actin was obviously increased in the SHR group. After treatment, the expressions of SM a-actin in the SHR+QCH, the SHR+QCL and the SHR+Losartan groups were all decreased compared with the SHR group. However, the levels of SM a-actin in those above groups were still higher than the WKY group. (P< 0.05, Fig.3)5. Comparison of expression of TGF-β1 and SM a-actin mRNA in aortic wall(1) Comparison of expression of TGF-β1 mRNA:The expression of TGF-β1 mRNA was higher in the SHR group than that in the WKY group (P＜0.05). The expression of TGF-β1 mRNA was decreased significantly in the SHR+QCH group, the SHR+QCL group and the SHR+Losartan group compared with that in the SHR group (all P＜0.05, table 3).(2) Comparison of expression of SM a-actin mRNA:The expression of SM a-actin mRNA was higher in the SHR group than that in the WKY group (P<0.01). There was no significant difference in the SM a-actin mRNA expression in the SHR+QCH group, the SHR+QCL group and the SHR+Losartan group compared with that in the SHR group (all P>0.05, table 4).ConclusionA significant anti-hypertensive effect of QC on SHR was observed in our present study. QC could decrease the local TGF-β1 and SM a-actin level in the vascular adventitia, which was possibly associated with the anti-hypertensive effect of QC. Also, QC could reverse vascular adventitia remodeling in hypertension, the mechanisms were possibly associated with the suppressive effect of QC on the migration and activation of TGF-β1 and the phenotypic transition of SM a-actin. |
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