Antihypertensive and Antifibrosis Effects of Acupuncture at PC6 Acupoints in Spontaneously Hypertensive Rats and the Underlying Mechanisms

Figures

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  FIGURE 1. Correlation analysis in WKY rats and SHRs. Systolic blood pressure (SBP) was measured by the tail-cuff method after 8 weeks of EA treatment. The left ventricles were harvested for various analyses. The relationship of hydroxyproline (Hyp) content with SBP, left ventricular mass index (LVMI) with SBP, and LVMI with hydroxyproline content is shown in (A), (B), and (C), respectively (n = 6 each group).

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  FIGURE 2. Effect of EA treatment on cardiac interstitial fibrosis in SHR. The tissue was taken after 8 weeks of EA treatment from SHR or at the same time course from WKY, and stained by Van Gieson staining. (A) Representative micrographs of cardiac collagen in the interstitial space of the left ventricle, which were ameliorated by EA treatment (bar = 25 μm). (B) Quantitative analysis of collagen volume fraction (CVF) by Van Gieson staining in myocardial tissues. ***P < 0.001 vs. WKY; ΔP < 0.05 vs. SHR (n = 3 each group). (C) The hydroxyproline content (Hyp, μg/mg) was estimated after 8 weeks of EA treatment. ***P < 0.001 vs. WKY; ΔΔP < 0.01 vs. SHR (n = 6 each group).

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  FIGURE 3. Effect of EA treatment on Collagen I and Collagen III deposition in the left ventricle myocardium. Spontaneously hypertensive rats (SHRs) were treated with EA for 8 weeks. (A) The SHR and WKY rats’ left ventricles were harvested for fluorescent immunohistochemical staining with Collagen I (red), Collagen III (green), and DAPI (blue). (a–d) Interstitial fibrosis in SHR myocardium documented by accumulation of fibrillary collagen I and collagen III. (a1–d1) The magnified photos from the areas of the boxes in (a–d), respectively, showing the Collagen I and Collagen III in detail. (a2–a3,b2–b3,c2–c3,d2–d3) represent the tryptase (a2–d2) and phalloidin (a3–d3) distribution in (a1–d1), respectively. Scale bar for (a–d) is shown in (a) (bar = 200 μm), for (a1–a3,b1–b3,c1–c3) shown in (a1) (bar = 50 μm). Western blotting analysis of Collagen I (B) and Collagen III (C) protein in left ventricular myocardium after 8 weeks of EA treatment. ***P < 0.001 vs. WKY; ΔΔΔ P < 0.001 vs. SHR (n = 3 each group).

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  FIGURE 4. Effect of EA treatment on TGF-β1 deposition in the left ventricle myocardium. Spontaneously hypertensive rats (SHRs) were treated with EA for 8 weeks. (A) The SHR and WKY rats’ left ventricles were harvested for fluorescent immunohistochemical staining and histochemical staining with TGF-β1 (red), phalloidin (PHA, green), and DAPI (blue). (a–d) Fibrosis of the myocardium of SHRs and WKY rats as revealed by TGF-β1 (red) accumulation. (a1–d1) The magnified photos from the areas of the boxes in (a–d), respectively, showing the TGF-β1 and myocardial structure in detail. (a2-a3,b2-b3,c2-c3,d2-d3) represent the TGF-β1 (a2–d2) and phalloidin (a3–d3) distribution in (a1–d1), respectively. Scale bar for (a–d) is shown in (a) (bar = 200 μm), for (a1-a3,b1-b3,c1-c3) shown in (a1) (bar = 50 μm). Western blotting analysis of TGF-β1 (B) protein and real-time PCR analysis of TGF-β1 (C) mRNA in left ventricular myocardium after 8 weeks of EA treatment. ***P < 0.001 vs. WKY; Δ P < 0.05, Δ Δ P < 0.01 vs. SHR (n = 3 each group). (D) The concentration of TNF-αin myocardium measured by ELISA after 8 weeks of EA treatment (n = 6 each group).

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  FIGURE 5. Effect of EA treatment on CTGF deposition in the left ventricle myocardium. Spontaneously hypertensive rats (SHRs) were treated with EA for 8 weeks. (A) The SHR and WKY rats’ left ventricles were harvested for fluorescent immunohistochemical staining and histochemical staining with CTGF (red), phalloidin (PHA, green), and DAPI (blue). (a–d) Fibrosis of the myocardium of SHR and WKY rats as revealed by TGF-β1 (red) accumulation. (a1–d1) The magnified photos from the areas of the boxes in (a–d), respectively, showing the CTGF and myocardial structure in detail. (a2-a3,b2-b3,c2-c3,d2-d3) represent the CTGF (a2–d2) and phalloidin (a3–d3) distribution in (a1–d1), respectively. Scale bar for (a–d) is shown in (a) (bar = 200 μm), for (a1-a3,b1-b3,c1-c3) shown in (a1) (bar = 50 μm). Western blotting analysis of CTGF (B) protein and real-time PCR analysis of CTGF (C) mRNA in left ventricular myocardium after 8 weeks of EA treatment. **P < 0.01, ***P < 0.001 vs. WKY; Δ P < 0.05, Δ Δ P < 0.01 vs. SHR (n = 3 each group).

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  FIGURE 6. The roles of TNF-α, MMP-2, and MMP-9 in the cardiac protective effects of EA on SHR. The concentration of TNF-αin serum (A) measured by ELISA and heart tissue (B) measured by real-time PCR after 8 weeks of EA treatment. Western blotting analysis of MMP-2 (C) and MMP-9 (D) protein in left ventricular myocardium after 8 weeks of EA treatment. ***P < 0.001 vs. WKY; Δ Δ P < 0.01, ΔΔΔ P < 0.001 vs. SHR (n = 6 each group).

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  FIGURE 7. The transition of fibroblasts to myofibroblasts is an early event in hypertension-induced myocardial fibrosis. Cardiac fibroblasts can produce a number of active substances (such as Ang II, TGF-β1, CTGF, TNFα), which change the collagen deposition due to both a stiffening of the ECM synthesis and an altered balance of MMPs and their inhibitors (TIMP). The altered molecular and cellular events of the cardiac myocytes collectively result in progressive cardiac dysfunction.