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. 2015 Nov;19(11):2607-16.
doi: 10.1111/jcmm.12645. Epub 2015 Jul 14.

Berberine via suppression of transient receptor potential vanilloid 4 channel improves vascular stiffness in mice

Jie Wang  1   2 , Tao Guo  1 , Qi-Sheng Peng  3 , Shou-Wei Yue  2 , Shuang-Xi Wang  1
Affiliations

Berberine via suppression of transient receptor potential vanilloid 4 channel improves vascular stiffness in mice

Jie Wang et al. J Cell Mol Med. 2015 Nov.

Abstract

Berberine, as an alkaloid found in many Chinese herbs, improves vascular functions in patients with cardiovascular diseases. We determined the effects of berberine in hypertension and vascular ageing, and elucidated the underlying mechanisms. In isolated aortas, berberine dose-dependently elicited aortic relaxation. In cultured cells, berberine induced the relaxation of vascular smooth muscle cells (VSMCs). Overexpression of transient receptor potential vanilloid 4 (TRPV4) channel by genetic approaches abolished the berberine-induced reduction in intracellular Ca(2+) concentration in VSMCs and attenuated berberine-elicited vessel dilation in mice aortas. In deoxycorticosterone acetate (DOCA)-induced hypertensive model, treatment of mice with berberine or RN-1734, a pharmacological inhibitor of TRPV4, significantly decreased systemic blood pressure (BP) in control mice or mice infected with an adenovirus vector. However, berberine-induced effects of lowering BP were reversed by overexpressing TRPV4 in mice by infecting with adenovirus. Furthermore, long-term administration of berberine decreased mean BP and pulse BP, increased artery response to vasodilator and reduced vascular collagen content in aged mice deficient in apolipoprotein E (Apoe-KO), but not in Apoe-KO old mice with lentivirus-mediated overexpression of TRPV4 channel. In conclusion, berberine induces direct vasorelaxation to lower BP and reduces vascular stiffness in aged mice through suppression of TRPV4.

Keywords: ageing; berberine; blood pressure; transient receptor potential vanilloid 4; vascular smooth muscle cells; vascular stiffness.

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Figures

Figure 1
Figure 1
Berberine inhibits agonists-induced contraction and induces aortic relaxation in isolated mice aortas. (A–D) The contraction of isolated aortic ring in organ chamber was induced by PE (1 μM) or U46619 (30 nM). Berberine was added in to the organ bath as indicated dose when the contraction was stable. (A) The representative tracing of berberine-induced relaxation in PE-precontracted aorta ring. (B) Summary data for berberine-induced relaxation in A. (C) The representative tracing of berberine-induced relaxation in U46619-prechallenged aorta ring. (D) Summary data for berberine-induced relaxation in C. All quantitative results in B and D are expressed as mean ± SEM 6 mice were in each group. *P < 0.05 versus DMSO. (E and F) Isolated aortic ring was incubated with berberine (10 μM) or RN-1734 (10 μM) for 60 min. followed by stimulation with U46619 (30 nM) or PE (1 μM). The contraction of aortic ring was recorded. (E) The representative tracing of aortic ring contraction. (F) Summary data for the effects of berberine on agonists-induced contraction. Quantitative results are expressed as mean ± SEM 6 mice were in each group. *P < 0.05 versus DMSO.
Figure 2
Figure 2
Berberine reduces intracellular Ca2+ concentration and induces direct relaxation in cultured human vascular smooth muscle cells (VSMCs). (A) Cultured human VCMCs were incubated with berberine as indicated concentration (1–100 μM) for 60 min. and then stimulated with PE (1 μM) for 30 min. Total cell lysates were subjected to perform Western blot for detection of pMLC levels. The MW of pMLC (or tMLC) is about 20 kD, which matches the expected sizes. The blot is a representative blot obtained from three independent experiments. Quantitative results are expressed as mean ± SEM, *P < 0.05 versus Control, #P < 0.05 versus PE alone. (B) Cultured human VCMCs were incubated with berberine (10 μM) for 60 min. and then stimulated with PE (1 μM) for 30 min. The representative picture of cell morphology was taken by the microscope from three independent experiments. (C) Cultured human VCMCs were incubated with berberine (10 μM) for 30 min. and then stimulated with PE (1 μM) or U46619 (30 nM) for 30 min. Total cell lysates were subjected to perform Western blot for detection of pCaM levels. The MW of pCaM (or tCaM) is about 17 kD, which matches the expected sizes. The blot is a representative blot obtained from three independent experiments. Quantitative results are expressed as mean ± SEM, *P < 0.05 versus Control, #P < 0.05 versus PE or U46619 alone. (D) Cultured human VCMCs were incubated with berberine (10 μM) for 60 min. and then stimulated with PE (1 μM) for 30 min. Intracellular Ca2+ ([Ca2+]i) concentration was assayed by detecting Fluo-4-AM fluorescence. Relative [Ca2+]i concentration was calculated by the ratio of fluorescent intensity to control. The fluorescent intensity in control was set up as 1. The representative picture was presented from three independent experiments. (E) Summary data for [Ca2+]i concentration in D. Quantitative results are expressed as mean ± SEM, *P < 0.05 versus Control, #P < 0.05 versus PE alone.
Figure 3
Figure 3
Berberine via suppression of transient receptor potential vanilloid 4 (TRPV4) induces relaxation of vascular smooth muscle cells. Cultured human VCMCs infected with adenovirus containing vector or TRPV4 cDNA for 48 hrs were incubated with berberine (10 μM) for 60 min. followed by stimulation with PE (1 μM) for 30 min. (A) Intracellular Ca2+ ([Ca2+]i) concentration was assayed by detecting Fluo-4-AM fluorescence. Relative [Ca2+]i concentration was calculated by the ratio of fluorescent intensity to control. The fluorescent intensity in control was set up as 1. The representative picture was presented from three independent experiments. (B) Summary data for [Ca2+]i concentration in A. Quantitative results are expressed as mean ± SEM, *P < 0.05 versus Vector, #P < 0.05 versus Vector plus PE. NS indicates no significant difference. (C) Total cell lysates were subjected to perform Western blot for detection of pCaM, tCaM, pMLC and tMLC protein levels. The MWs of pMLC (or tMLC) and pCaM (or tCaM) match the expected sizes. The blot is a representative blot obtained from three independent experiments.
Figure 4
Figure 4
Overexpression of TRPV4 inhibits berberine-induced relaxation in mice aortas. Aortas from mice infected with adenovirus of containing vector or TRPV4 cDNA for 4 weeks were cut into rings and were mounted in organ chamber to detect vessel bioactivity. (A) Aortic ring was incubated with berberine (10 μM) for 60 min. followed by stimulation with PE (1 μM). The contraction of aortic ring was recorded by a computer. The representative tracing of aortic ring contraction was shown from six mice. (B) Summary data for the effects of berberine on aortic contraction in A. Quantitative results are expressed as mean ± SEM, N is 6 mice in each group. *P < 0.05 versus Vector alone. NS indicates no significant difference. (C) The contraction of aortic ring in organ chamber was induced by PE (1 μM) or U46619 (30 nM). Berberine was added into organ bath as indicated dose when the contraction was in the peak. The representative tracing of berberine-induced relaxation on aortic ring was shown from six mice. (D) Summary data for berberine-induced relaxation in PE-stimulated aorta in C. (E) Summary data for berberine-induced relaxation in U46619-stimulated aorta in C. Quantitative results are expressed as mean ± SEM 6 mice were in each group. *P < 0.05 versus Vector.
Figure 5
Figure 5
Administration of berberine via inhibition of TRPV4 reduces DOCA-induced hypertension in mice. (A and B) C57B16 mice at the age of 8–12 weeks old were fed with normal diet containing berberine (100 mg/kg/day) and RN-1734 (30 mg/kg/day) for 7 days prior to DOCA infusion. The hypertensive model was induced by DOCA (150 mg/kg) implantation. Blood pressures (BP) and heart rates were monitored by telemetry, as described in Materials and Methods. Systolic BP in A and diastolic BP in B were analysed. Quantitative results are expressed as mean ± SEM 10–15 mice in each group. *P < 0.05 versus Control mice. #P < 0.05 versus DOCA-implanted mice. (C and D) C57B16 mice at the age of 8-12 weeks were injected with adenovirus vector via tail vein. 2 weeks later, berberine (100 mg/kg/day) were provided in the diet followed by DOCA implantation for 35 days. Systolic BP in C and diastolic BP in D in adenovirus-vector-infected mice were analysed. Quantitative results are expressed as mean ± SEM 10–15 mice in each group. *P < 0.05 versus Mice infected with vector. #P < 0.05 versus DOCA-implanted mice infected with vector. (E and F) C57B16 mice were received infection of adenovirus containing TRPV4 cDNA by tail vein injection followed by administration of berberine (100 mg/kg/day) and DOCA (150 mg/kg) implantation. Systolic BP in E and diastolic BP in F in mice infected adenovirus containing TRPV4 cDNA were analysed. Quantitative results are expressed as mean ± SEM 10–15 mice in each group. *P < 0.05 versus Mice infected with TRPV4 cDNA.
Figure 6
Figure 6
Long-term administration of berberine via suppression of TRPV4 delays vascular stiffness in aged Apoe-KO mice. Male Apoe-KO mice at the age of 4 months old received lentivirus infection containing vector or TRPV4 cDNA by tail vein injection once every 2 months. Berberine (50 mg/kg/day) were provided in the high-fat diet starting from the end of 2nd month after lentivirus infection to the end of experiments. The total administrating time of berberine was 12 months. Blood pressures (BP) was monitored by telemetry, as described in Materials and methods. At the end of experiments, all mice were killed under anaesthesia. (A) Mean BP. (B) Pulse BP. (C) SNP-induced vessel relaxation was determined by organ chamber in descending aortas. (D) Phentolamine-induced vessel relaxation was assayed by organ chamber in descending aortas. (E) Picrosirius red staining in abdominal aorta was performed to detect collagens in vessel wall. (F) Summary data for the content of collagen in E. All quantitative results are expressed as mean ± SEM 10–15 mice in each group. *P < 0.05 versus Mice infected with vector. NS indicates no significant difference.
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