| BackgroundHypertension is one of the familiar risk factors for the human health. The changes of arterial structural and functional remodeling are the adaptive reaction to the disorders of hemodynamics, neurohumor and local endocrinic factors in hypertension. Furthermore, they are the major pathological base of target organ damage, such as heart, brain and kidney, in the development of hypertension. Therefore, it is important for prevention and treatment of hypertension and target organ damage to reverse the vascular remodeling to aim directly at its developed mechanisms.The renin-angiotensin system (RAS) plays a major role in the regulation of blood pressure. Angiotensinâ…¡(Angâ…¡) is one of the most important factors in RAS. Some studies showed that Angâ…¡had many biological functions, such as improving synthesis of the nucleic acid, regulating gene expression and stimulating vascular hyperplasia and thickening, besides adjusting the vascular tonicity and the aldosterone secreting. It is not clear that the mechanisms of Angâ…¡taking part in the process of hypertensive vascular remodeling.Basic fibroblast growth factor (bFGF) is a growth factor belonging to polypeptide family with effects of vascular activity. Recently, bFGF was proved to take part in the process of hypertensive vascular remodeling through inducing the proliferation and the migration of vascular smooth muscle cells (VSMCs) and improving the synthesis of extracellular matrix (ECM). Osteopontin (OPN) is an arginine-glycine-aspartate (RGD) -containing extracellular matrix phosphoprotein involved in cell's attachment and migration. Recently, OPN was considered to play a key role in the development of vascular remodeling in hypertension. Qindan Capsule (QC), a prepared compound of traditional Chinese medicine, has been used as an anti-hypertensive agent for years in clinic. In the present study, the changes of the pathology and the ultrastructure of arterial media were observed in spontaneous hypertensive rats (SHR), and the variation of bFGF and OPN mRNA and protein expression was detected by real-time PCR and Western blot. The possible mechanism of QC in reversing vascular remodeling in hypertension was investigated, which could provide scientific evidence for the treatment of hypertensive vascular damage in clinic. Aim1. To investigate the anti-hypertensive effects of QC through observing the changes of endothelin-1 (ET-1), calcitonin gene-related peptide (CGRP) and Ang II in plasma and Ang II in local vascular tissues.2. To investigate the possible mechanism of QC in reversing vascular remodeling in hypertension through observing the changes of the morphology and the ultrastructure and detecting the variation of bFGF and OPN mRNA and protein expression.Methods40 male (14 weeks old) SHRs and 8 male (14 weeks old) Wistar Kyoto(WKY) rats were randomly divided into 6 groups (n=8): the WKY control group, the SHR control group, the Niuhuang Jiangya Capsule (SHR+NJC) group, the Captopril (SHR+CAP) group, the high dosage QC group (SHR+QCH), and the low dosage QC group (SHR+QCL). The SHR+NJC group was treated with Niuhuang Jiangya Capsule for 200 mg/kg per day, equivalent to 15 folds of the dosage for human adult used in clinical practice; the SHR+CAP group was treated with Captopril for 15 mg/kg per day, equivalent to 20 folds; the SHR+QCH group was treated with high dosage (750 mg/kg per day) of QC, equivalent to 15 folds; and the SHR+QCL group was treated with low dosage (150 mg/kg per day) of QC, equivalent to 15 folds. The WKY group and the SHR 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) Determination of the levels of ET-1, CGRP, and Angâ…¡in plasma and contents of Angâ…¡in tissues of mesenteric artery; (3) Observation of the pathological structure in aortic wall; (4) Measurement of the morphological index of aortic wall: the medial thicken (MT), the luminal diameter (LD), and the ratio of MT to LD; (5) Determination of the volume fraction of collagen (VFC) in the medial; (6) Ultrastructural observation of the VSMCs in the media; (7) Analysis of bFGF and OPN mRNA expression by real-time PCR; (8) Analysis of bFGF and OPN protein expression by Western blot.Results1. Comparison of SBPThe SBP of rats in the SHR group with 14 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. After 12 weeks, the SBP of rats in the SHR group with 26 weeks old was significantly higher than those in the WKY group (P<0.01). From the second week of treatment, the SBP was lower in the SHR+NJC group, the SHR+CAP group, the SHR+QCH group, and the SHR+QCL group than that in the SHR group (all P<0.01). After six weeks of treatment, the SBP in the SHR+QCH group was decreased significantly compared with that in the SHR+QCL group (P<0.01 or P<0.05). After twelve weeks of treatment, the SBP in each group was decreased in different degree compared with the SHR group (all P<0.01). The SBP in the SHR+QCH group, the SHR+NJC group, and the SHR+CAP group was lower than that in the SHR+QCL group (P<0.01 or P<0.05), but no significant difference was shown in comparison among these three groups (P>0.05).2. Comparison of levels of ET-1, CGRP and Angll in plasma(1) The plasma ET-1 level in the SHR group was higher than that in the WKY group (P<0.01). The levels of ET-1 in each treatment groups were decreased in different degree compared with those in the SHR group, and among those treatment groups, the SHR+QCH group had the lowest level of ET-1 in plasma (P<0.05). It was no significant difference in the SHR+NJC group and the SHR+CAP group compared with the SHR group (P>0.05). The ET-1 level in the SHR+QCH was lower than that in the SHR+CAP group (P<0.05). No significant difference was shown between the SHR+QCH group and the SHR+QCL group (P>0.05).(2) The plasma CGRP level in the SHR group was lower than that in the WKY group (P<0.01). It was no significant difference shown in the SHR+NJC group and the SHR+CAP group compared with the SHR group (P>0.05). The plasma CGRP level in the SHR+QCH group was higher than those in the SHR group (P<0.05), and a significant difference was shown in the SHR+QCH group compared with the SHR+NJC and the SHR+CAP groups (all P<0.05). The plasma CGRP level in the SHR+QCL group was higher than that in the SHR group (P<0.01). No significant difference was shown in comparison between the SHR+QCH group and the SHR+QCL group (P>0.05).(3) No significant difference of plasma Angâ…¡level was shown in comparison among these six groups (all P>0.05).3. Comparison of levels of Angll in tissues of mesenteric arteryThe tissue Angll level in the SHR group was higher than that in the WKY group (P<0.01). No significant difference was shown in comparison between the SHR+NJC group and the SHR group (P>0.05). The tissue Angll levels was lower in the SHR+CAP, the SHR+QCH and the SHR+QCL groups than those in the SHR group and the SHR+NJC group (all P<0.01), but no significant difference was shown in comparison among these three groups (P>0.05).4. Observation of sections of aortic wall stained with HEThe structure of the intima, the media and the adventitia of aortic wall was very clear and was no thickening in the WKY rats. The VSMCs in media were shown a Fusiform shape. The nucleus were stained in blue and arranged regularly as concentric circles. The aortic walls were shown obviously thickened in the SHR group. The arrangement of its VSMCs was disordered, and a large amount of nucleus was shown in the unit area. The aortic walls were shown slightly thickened in the SHR+NJC, the SHR+CAP, the SHR+QCH and the SHR+QCL groups. The arrangement of their VSMCs was slightly disordered, and the amount of nucleus shown in the unit area in these four groups was smaller than that in the SHR group.5. Comparison of morphological index of aortic wall(1) MT was increased significantly in the SHR group compared with the WKY group (P<0.01). The SHR+NJC group was shown a lower MT than the SHR group (P<0.05). MT was decreased significantly in the SHR+CAP, the SHR+QCH and the SHR+QCL groups compared with that in the SHR group (all P<0.01). No significant difference was shown in comparison among these three groups above (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 decreased slightly in the SHR+NJC group, but no significant difference was shown compared with the SHR group (P>0.05). The ratio of MT/LD was significantly lower in the SHR+CAP, the SHR+QCH and the SHR+QCL groups than the SHR group (all P<0.05), but no significant difference was shown in comparison among these three groups above (all P>0.05).6. Observation of sections of aortic wall stained with MassonThe Masson stain showed that the VSMCs were stained in red and the collagen were stained in green. It was shown in the WKY group that the collagen was well distributed and the medial elastic fibers were arranged in order. The collagen of aortic wall in the SHR group was increased obviously. The medial elastic fibers in the SHR group were shown spastic and even ruptured, and the VSMCs were hyperplastic significantly. The volume of collagen in the SHR+NJC group was not shown the difference with the SHR group. But the volume of collagen was decreased in the SHR+CAP, the SHR+QCH, the SHR+QCL groups compared with that in the SHR group.7. Comparison of the volume fraction of collagen (VFC) The VFC of aortic media in the SHR group was higher than that in the WKY group (P<0.01). No significant difference was shown in comparison between the SHR+NJC group and the SHR group (P>0.05). The VFC in the SHR+CAP group, the SHR+QCH group and the SHR+QCL group was lower than that in the SHR group (P<0.05 or P<0.01). There was a significant difference between the three groups mentioned above and the SHR+NJC group (P<0.05 or P<0.01).8. Ultrastructural observation of the VSMCs in the aortic mediaIn the WKY group, the VSMCs in the aortic media were shown the fusiform shape with the regular cell nucleus. Large numbers of dense area and dense bodies and a small quantity of mitochondria and endocytoplasmic reticula were observed in VSMCs of the WKY rats. The VSMCs in the SHR group were hypertrophic with the irregular nucleus. The number of dense area and dense bodies was decreased and the average volume of mitochondria and endocytoplasmic reticula were increased obviously, whilst mitochondria swelling and vacuolization were observed, which indicated that the phenotype of VSMC in SHR was changing. The VSMCs in the SHR+NJC group were hypertrophic too, and large numbers of swollen mitochondria and a small quantity of dense area and dense bodies were observed in the endochylema. However, the changes of mitochondria were ameliorated in the SHR+CAP group, the SHR+QCH group and the SHR+QCL group. The mitochondria with slight swelling, mild vacuolization and approximate normal numbers were observed in these three groups mentioned above compared with the SHR group.9. Comparison of expression of bFGF and OPN mRNA in aortic wall(1) Comparison of expression of bFGF mRNA: The expression of bFGF mRNA was higher in the SHR group than that in the WKY group (P<0.01). No significant difference was shown in comparison between the SHR+NJC group and the SHR group (P>0.05). The expression of bFGF mRNA was decreased significantly in the SHR+CAP group, the SHR+QCH group and the SHR+QCL group compared with that in the SHR group (all P<0.01). There was a significant difference between the three groups mentioned above and the SHR+NJC group (all P<0.01).(2) Comparison of expression of OPN mRNA: The expression of OPN mRNA was higher in the SHR group than that in the WKY group (P<0.01). No significant difference was shown in comparison between the SHR+NJC group and the SHR group (P>0.05). The expression of bFGF mRNA was decreased significantly in the SHR+CAP group, the SHR+QCH group and the SHR+QCL group compared with that in the SHR group (all P<0.01). And there was a significant difference between the three groups mentioned above and the SHR+NJC group (all P<0.01). The expression of bFGF mRNA was lower in the SHR+QCH group than that in the SHR+QCL group (P<0.05).10. Comparison of expression of bFGF and OPN protein in aortic wall(1) Comparison of expression of bFGF protein: The expression of bFGF protein was higher in the SHR group than that in the WKY group (P<0.01). The expression of bFGF protein was decreased in the four treatment groups than that in the SHR group (P<0.05 or P<0.01). And it was lower in the SHR+CAP group, the SHR+QCH group and the SHR+QCL group than that in the SHR+NJC group (P<0.05 or P<0.01).(2) Comparison of expression of OPN protein: The expression of OPN protein was higher in the SHR group than that in the WKY group (P<0.01). No significant difference was shown in comparison between the SHR+NJC group and the SHR group (P>0.05). The expression of OPN protein was lower in the SHR+CAP group, the SHR+QCH group and the SHR+QCL group than that in the SHR group and the SHR+NJC group (P<0.05 or P<0.01).Conclusion(1) A significant anti-hypertensive effect of QC on SHR was observed in our present study. QC could decrease the plasma ET-1 level and the local Angâ…¡level in the resistance vessels, increase the plasma CGRP level, and regulate the unbalanced net of vascular active peptide, which was possibly associated with the anti-hypertensive effect of QC.(2) A reversing effect of QC on the hypertensive vascular remodeling in SHR was observed in our present study. QC could improve the morphological index of aortic wall, and decrease VFC in the aortic media. The mechanisms were possibly associated with the suppressive effect of QC on the local Angll level and the expression of bFGF and OPN mRNA and protein.(3) QC could improve the changes of VSMCs phenotype in aortic media of SHR. QC could inhibit the transformation of VSMCs from systolic phenotype to synthetic one. The mechanisms were possibly associated with the inhibitive effect of QC on the promotion of Angâ…¡, bFGF and OPN to the transformation of VSMCs phenotype. |
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