Traditional Chinese medicine suppresses left ventricular hypertrophy by targeting extracellular signal-regulated kinases signaling pathway in spontaneously hypertensive rats

Chinese herbal medicine Bu-Shen-Jiang-Ya decoction (BSJYD) is reported to be beneficial for hypertension. Over expression of extracellular signal regulated kinases (ERK) pathway plays an important role in left ventricular hypertrophy (LVH). This study aimed to observe effects of BSJYD on LVH in spontaneously hypertensive rats (SHRs) and explore its possible mechanism on regulation of ERK pathway. Sixty 12-week-old SHRs were randomly allocated into 5 groups: BSJYD high dose group, middle dose group, low dose group, captopril group, and control group. Besides, a control group of Wistar-Kyoto rats was established. All rats were treated for 8 weeks. Systolic blood pressure (SBP), heart rate (HR), pathology, and left ventricular mass index (LVMI) were measured. Western blotting and Real-time PCR were used to assess the expressions of BDNF, Ras, ERK1/2, and c-fox levels. SBP and HR were significantly decreased compared with the control group and LVMI was markedly improved by BSJYD treatment in a dose-dependent manner. BSJYD inhibited the expression of BDNF, Ras, ERK1/2, and c-fox mRNA in LVH. In conclusion, BSJYD suppressed hypertension-induced cardiac hypertrophy by inhibiting the expression of ERK pathway. These changes in gene expression may be a possible mechanism by which BSJYD provides myocardial protection from hypertension.

Scientific RepoRts | 7:42965 | DOI: 10.1038/srep42965 Histological characteristics. Eight weeks after the treatment, the cardiac structure was measured through histological examinations. A significant difference was identified in the left ventricular apical biopsy between the C group and the Ca group (Fig. 2, HE staining 200x magnification). The single ventricular myocytes in the Bh group and the Ca group were larger than those in the C group.

Effect of BSJYD on the proteins expression of ERK signaling by WB analysis. Western blotting
analysis was performed to examine the proteins expression of ERK signaling pathway, including BDNF, Ras, ERK1/2, and c-fox in left ventricular myocardium among the six groups (n = 12 per group). Figure 3 (a) showed the expression of these proteins in the left ventricular areas. As shown in Fig. 3(b), the expression of BDNF was significantly increased in the C group, the Ca group, the Bh group, the Bm group, and the Bl group, when compared with that of the WKY group (P < 0.01). However, it was significantly decreased in the Bh group and the Ca group when compared with the C group (P < 0.01). However, no significant difference between the Ca group and Bh group on the expression of BDNF was identified (P > 0.05). As shown in Fig. 3(c), the expression of Ras was significantly increased in the C group, the Ca group, the Bh group, the Bm group, and the Bl group when compared with the WKY group (P < 0.01). However, when compared with that of the C group, the expression level of Ras was reduced in the Ca group, the Bh group, the Bm group, and the Bl group (P < 0.05). As compared to the Ca group, Ras was increased in the Bl group (P < 0.05); but no statistically significant differences were observed in the Bh group and the Bm group in terms of their expression of Ras (P > 0.05). As shown in Fig. 3(d), the expression of ERK1/2 was significantly reduced in the Ca group, the Bh group, the Bm group, the Bl group, and the WKY group when compared to the C group (P < 0.01). As compared to the Ca group, it was significantly increased in the Bl group (P < 0.05); however, no statistically significant difference was identified in the Bh group and the Bm group (P > 0.05). As shown in Fig. 3(e), c-fox was significantly increased in the C group, the Ca group, the Bh group, the Bm group, and the Bl group compared with the WKY group (P < 0.05). However, the expression of c-fox was decreased in the Ca group, the Bh group, the Bm group, the Bl group, and the WKY group when compared with the C group (P < 0.05). It was increased in the Bh group (P < 0.05), significantly increased in the Bm group and the Bl group compared to the Ca group (P < 0.01).
Effect of BSJYD on the mRNA expression of ERK signaling by Real-time PCR assay. Real-time PCR was also performed to examine the mRNA expression of BDNF, Ras, ERK1/2, and c-fox in left ventricular myocardium. As shown in Fig. 4(a), the mRNA expression of BDNF in the C group, the Ca group, the Bh group, the Bm group, and the Bl group were significantly increased when compared to the WKY group (P < 0.01). As compared with the C group, the expression of BDNF mRNA in the Ca group, the Bh group, and the Bm group were significantly decreased (P < 0.01), while there is no difference in the Bl group (P > 0.05). As compared with the Ca group, the expression of BDNF mRNA in the Bh group, the Bm group, and the Bl group were significantly increased (P < 0.05). As shown in Fig. 4(b), the mRNA expression of Ras in the C group, the Bh group, the Bm group, the Bl group, and the Ca group were significantly increased when compared to the WKY group (P < 0.05).
As compared with the C group, the expression of Ras mRNA in the Ca group, the Bh group, and the Bm group were significantly decreased (P < 0.01), while there is no difference in the Bl group (P > 0.05). As compared with the Ca group, the expression of Ras mRNA in the Bl group was significantly increased (P < 0.01), while there were no difference in the Bh group and the Bm group (P > 0.05). As shown in Fig. 4(c), the mRNA expression of ERK1/2 in the C group, the Bh group, the Bm group, and the Bl group were significantly increased (P < 0.01); while there is no difference in the Ca group when compared to the WKY group (P > 0.05). The expression of ERK1/2 mRNA in the Ca group, the Bh group, and the Bm group were significantly decreased (P < 0.01), while there is no difference in the Bl group (P > 0.05) when compared to the C group. As compared to the Ca group, the expression of ERK1/2 mRNA in the Bl group was significantly increased (P < 0.01), while there were no difference in the Bh group and the Bm group (P > 0.05). As shown in Fig. 4(d), the mRNA expression of c-fox in the C group, the Ca group, the Bh group, the Bm group, and the Bl group were significantly increased when compared to the WKY group (P < 0.01). The expression of c-fox mRNA in the Ca group, the Bh group, the Bm group, and the Bl group were significantly decreased when compared with the C group (P < 0.01). As compared with the Ca group, c-fox mRNA in the Bh group, the Bm group, and the Bl group were significantly increased (P < 0.05).

Discussion
Hypertension could be classified into the categories of "vertigo" and "headache" in TCM theory 28 . Pattern or syndrome is the basic unit and key concept of TCM 29 . It has been identified that TCM patterns are associated with clinical manifestations of hypertension and its related target organ damage 30 . According to the diagnosis method of syndrome differentiation, kidney yin deficiency syndrome is the critical type of hypertension, which included dizziness, headache, tinnitus, dysuria, weakness, fatigue, sexual dysfunction, lassitude in loins and legs, red tongue with less fur, and deep thready pulse 31,32 . A literature analysis of 13, 272 hypertensive patients revealed that kidney yin deficiency syndrome account for about 26.27% and the proportion of kidney yin deficiency syndrome increased with age 33,34 . Therefore, Chinese herbal medicine nourishing kidney yin deficiency have been widely utilized by TCM physicians in the treatment of hypertension, especially in elderly patients 35 . Recent researches have demonstrated that traditional Chinese medicinal herbs and formulas with kidney yin-tonifying effect display good clinical antihypertensive efficacy and its underlying multiple cardiovascular protective mechanisms might be relevant to inhibiting the activity of sympathetic nerve, blocking the renin-angiotensin system, improving endothelial function, preventing target organ damage, inhibiting inflammation, and improving insulin resistance as well as glucose and lipid metabolism 23,36,37 . According to the TCM basic theory, BSJYD, which is originated from a famous TCM classic herbal formula Liu Wei Dihuang pill, belongs to the category of Chinese herbal medicine with nourishing kidney yin deficiency effects. Antihypertensive therapy is the cornerstone of the treatment of hypertension 38 . Clinical trials have confirmed that antihypertensive therapy reduces the risk of cardiovascular disease outcomes, including incident stroke (by 35 to 40%), myocardial infarction (by 15 to 25%), and heart failure (by up to 64%) [39][40][41] . A recently published SPRINT study demonstrated that an intensive SBP control (less than 120 mmHg) might result in lower rates of fatal and nonfatal major cardiovascular events and death from any cause than standard SBP control (less than 140 mmHg) 42 . That is, strict BP control contributes to more cardiovascular protective effects. In our experiment, a significant reduction of SBP by BSJYD was identified when compared to the C group, and no significant difference between the Bh and C group was identified. Although difference between SHRs and hypertensive patients existed, the variation of BP in SHRs may partially reflect the antihypertensive effect of BSJYD. Biochemically, the pharmacological mechanism of BSJYD is closely related to the major active compounds including eucommia ulmoides lignans, gastrodin, polysaccharides of gastrodia rhizome, panax notoginseng saponins, and paeonol. Previously published studies also supported the BP-lowering effect of Chinese herbal medicine with nourishing kidney yin deficiency effects. A systematic review involving 6 randomized controlled trials with 527 people demonstrated that, compared with antihypertensive drugs alone, Chinese herbal formula with kidney-tonifying effects as adjunctive therapy significantly lowered SBP by 8.69 mmHg in patients with senile hypertension 43 . Another systematic review including 6 randomized trials and a total of 555 hypertensive participants revealed a significant reduction of 9.31 mmHg of SBP and 6.27 mmHg of DBP by Liu Wei Dihuang pill as complementary therapy when compared to antihypertensive drugs alone 44 . Thus, this study provided evidence that BSJYD could be used to lower BP and is beneficial for hypertension treatment. Considerable evidence suggesting the important role of autonomic nervous system in BP regulation and development of hypertension have been established [45][46][47] . HR is regarded as an independent risk factor for hypertension 48 . A clinic HR (more than 79 bpm) is a significant predictor of all-cause, cardiovascular, and noncardiovascular mortality in untreated older patients with isolated systolic hypertension 49 . The elevated resting HR should be paid more attention to in hypertensive patients 50 . In this experiment, HR in the Bh group was significantly decreased, suggesting that BSJYD is beneficial in inhibiting sympathetic nervous activity, thus lowering HR in SHRs. Biochemically, some active ingredients from the components of BSJYD, including hawthorn extract and paeonol, have a certain effect in reducing HR. Previous studies identified that oral administration of paeonol result in a reduction of 20.25 mmHg in SBP and HR simultaneously 51 . Therefore, BSJYD not only exhibited certain antihypertensive effects, but also lowered HR significantly, which is similar to the pharmacological mechanism of β -blocker.
The results of our study showed a dose-dependently suppression of cardiac hypertrophy remodeling effect by BSJYD. It has been recognized that, in the chronic hypertension, elevated BP contributes to wall thickening and functional changes chronically in response to specific conditions, including LVH and cardiac fibrosis 52 . Increased LVM or LVH is a frequent complication of arterial hypertension, which is also an indicator of end-organ damage. The presence of LVH is associated with an increased rate of cardiovascular morbidity and mortality independent of other cardiovascular risk factors and, notably, independent of BP values 53 . Interestingly, both BP reduction and LVH suppression were accompanied by favorable outcome in the present research. It was identified that SHR continued to develop further high BP level, which could also be seen in other studies on SHR model, leading to a higher incidence of LVH when compared with WKY rats 54,55 . Obvious formation of cardiac hypertrophy and over expression of EKR signaling pathway were identified in the C group. In our experimental study, after administration of BSJYD with 8 weeks, we were able to demonstrate marked reduction of LVMI, improvement in histological examination, and down regulation of EKR signaling pathway to some degree in the SHRs treated with BSJYD compared with untreated animals. It was suggested that the suppression effect of BSJYD on LVH may relate to the down-regulation of EKR signaling pathway.
In summary, the SHR treated with a daily dose of BSJYD over a period of 8 weeks demonstrated not only reduction in BP and HR with a dose-dependently manner, but also protection of target organ damage in hypertension. In this respect, our findings were consistent with previous reports about Chinese herbal medicines on SHR model [56][57][58][59][60] . We can draw the following conclusions from the present study: (a) BSJYD treatment could decrease BP and HR, and suppress LVH remodeling to a certain extent in SHR; (b) BSJYD treatment can inhibit the expression of BDNF, Ras, ERK1/2, and c-fox mRNA in LVH; and (c) a dose-dependent cardiovascular protective effects of BSJYD was identified.

Conclusions
Our present study suggested that BSJYD and captopril inhibit cardiac hypertrophy via a mechanism that may involve the regulation of the EKR signaling pathway. BSJYD treatment decreased BP and HR efficiently and reversed ventricular remodeling of SHRs. The mechanisms were possibly associated with the suppressive effect of BSJYD on the EKR signaling pathway. Therefore, our findings provided a theoretical basis for using BSJYD in the treatment of hypertension and its associated myocardial hypertrophy and fibrosis. BSJYD may be a new candidate cardioprotective drug for patients with hypertensive vascular diseases, which should be given priority for future preclinical and clinical studies.

Materials and Methods
Composition of BSJYD. BSJYD consists of 8 commonly used Chinese herbs as shown in Table 3. The main chemical components of BSJYD were described in Fig. 5. All of these herbs with granule forms were provided and identified by Neo-Green Pharmaceutical Co., Ltd (Sichuan, China). The powdered BSJYD compounds, stored at 4 °C, were dissolved in distilled water prior to use.     Administration of drugs. Sixty SHRs were trained for two weeks and then randomly allocated into five groups of twelve rats each. From the start of the third week, the rats were given drugs at 8:00 am every morning.
The doses and routes are as follows: BSJYD high dose group (6.75 g/ml, Bh), BSJYD middle dose group (3.375 g/ ml, Bm), BSJYD low dose group (1.6875 g/ml, Bl); 1 group which received captopril (0.125 g/ml, Ca); and 1 control group of untreated SHRs (C). Additionally, a control group of 12 WKY rats was established. Both control groups (C and WKY) were treated with distilled water. The drugs were administered for 8 weeks. All rats used in this study received humane care.

Measurement of SBP and heart rate (HR)
. SBP of all rats was measured using the indirect tailcuff plethysmographic method with a rat tail BP monitor (BP-100A, a noninvasive computerized tail-cuff system for measuring blood pressure in mice, Chengdu Technology & Market Co., LTD, Sichuan, China). Rats were kept calm and conscious till pulsatory signals from the arteria caudilis were displayed steadily. At least 10 determinations were made on each rat and the mean of 6 readings within a 5-10 mmHg range was taken as the SBP 61 . HR was recorded at the same time. BP and HR were measured every 2 weeks (4 times totally). , which were then incubated with primary antibodies at 4 °C. The membranes were further incubated with horseradish peroxidase-conjugated secondary antibodies (1:10000) for 1 hour at room temperature. ECL visualisation was performed, and the resulting images were captured using the Gene Gnome Gel Imaging System (Syngene Co.). Image J (NIH image, Bethesda, MD, USA) was used to analyze the gel images. Quantitative values were obtained from the threshold cycle value (Ct), the point at which a significant increase in fluorescence was first detected. Experiments were performed in triplicate for each data point, and the data were analyzed with the 2 −△△Ct method 63 . The results of RT-PCR were confirmed by gel electrophoresis.

Statistical analysis.
All experimental data were expressed as the mean ± SD. The data was statistically evaluated using one-way analysis of variance (ANOVA), and a post hoc analysis was performed using Fisher's least significant difference (LSD) test. The SPSS computer program (version 18.0) was used for data analyses. p value less than 0.05 was considered to be statistically significant.