Modified Saengmaeksan, an herbal formula containing six herbs, improves hypertension through RhoA/Rho kinase-mediated vasorelaxation

Figures

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  Fig. 1. Effect of mSMS water and EtOH extracts on vascular contraction. Isolated rat aorta rings were pre-contracted with U46619 and then treated with mSMS water extract and ethanol extract. The contractile response was measured using the organ bath method. (a-c) The relaxation responses were expressed as the percentage to maximal contractions in response to U46619 (10 nmol/L). mSMS was added cumulatively (0.1–1.0 mg/ml) to elicit relaxation in aortic rings precontracted with U46619. (d, e) Pretreatment effect of mSMS on U46619-induced contraction was determined. Developed tension is expressed as a percentage of the contraction to KCl (50 mmol/L). Data are expressed as means of 5 experiments with vertical bars showing SEM. *P < 0.05 vs. vehicle.

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  Fig. 2. Effect of mSMS water and EtOH extracts on Rho/Rho kinase pathway. (a) RhoA activation was assessed by G-LISA after treatment with U46619 (10 nmol/L). Modified Saengmaeksan had been pretreated for 30 min. Absorbance at the control (OD of around 0.4) was expressed as 1 arbitrary unit. (b) Phosphorylation levels of MLC20, MYPT1, and CPI17 were measured by western blot. (c) Relative ratios of phosphorylation against the total protein forms of MCL20, MYPT1, and CPI17 were measured. Data are expressed as means of 4 experiments with vertical bars showing SEM. (d) Effect of mSMS on PDBu-induced contraction in PKC-inhibited conditons. PDBu (0.1 µmol/L) was added to elicit tension in the presence or absence of Ro31–8220, mSMS (0.6 or 1.0 mg/ml) for 30 min in the aortic rings with endothelium denuded. Developed tension was expressed as a percentage of the maximum contraction to KCl (50 mmol/L). (e) CPI17 phosphorylation at Thr38 was assessed by Western blots after PDBu treatment. ## P < 0.01 vs. vehicle; * P < 0.05, ** P < 0.01 vs. U46619 alone (a-c) or PDBu alone (d, e).

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  Fig. 3. Effect of mSMS extracts on blood pressure in l-NAME-induced hypertensive mice. (a) Male C57BL6 mice or (b) female ICR mice were administered l-NAME for 3 weeks to induce hypertension. mSMS water extract or ethanol extract (500 mg/kg/day) was orally administrated simultaneously. Blood pressure (SBP and DBP) was measured once a week using tail-cuff method. Bar graph show the blood pressure at Week 3 as mean ± SEM. ##P < 0.01, ###P < 0.001 vs. control group; * P < 0.05, ** P < 0.01 vs. l-NAME hypertension group.

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  Fig. 4. Effect of mSMS extracts on blood pressure in SHR hypertensive rats. SHR rats were orally administrated with mSMS ethanol extract at 200, 400, and 800 mg/kg/day. Blood pressure (SBP and DBP) was measured once a week using tail-cuff method for 4 weeks. Losartan (20 mg/kg/day) and amlodipine (10 mg/kg/day) were used as positive controls. WKY rats were fed PBS as a control group. Bar graph show the blood pressure at Week 4 as mean ± SEM. ####P < 0.0001 vs. WKY control group; *P < 0.05, **P < 0.01 vs. SHR hypertension group.

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  Fig. 5. Effect of mSMS extract on histological changes and oxidative stress in aorta of SHR rats. (a) Representative images of aorta sections stained with H&E, showing the structural integrity and morphology of the aortic wall. Bar graph represent the thickness of aorta. (b) Representative images of aorta sections stained with DCF-DA to assess ROS levels. Increased fluorescence indicates higher levels of oxidative stress in the aortic tissue. Bar graph represent the relative fluorescence intensities. ###P < 0.001 vs. WKY control group; *P < 0.05, **P < 0.01, ***P < 0.001 vs. SHR hypertension group.

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  Fig. 6. Effect of mSMS extract on histological changes and AT1R expression in kidney of SHR rats. (a) Representative images of kidney sections stained with H&E. Bar graph quantifies the kidney injury score, highlighting the degree of renal damage. (b) IF staining of kidney sections for AT1R. Bar graph presents the quantification of AT1R fluorescence intensity. ##P < 0.01, ###P < 0.001 vs. WKY control group; *P < 0.05, ***P < 0.001 vs. SHR hypertension group.