Kangxianyixin Granule Improves Myocardial Fibrosis by Suppressing RhoA/ROCK1 Pathway

Background: Kangxianyixin granule (KXYX), a traditional Chinese medicine prescription, has been clinically used to treat dilated cardiomyopathy (DCM) for many years. Myocardial brosis (MF) is a major pathological feature of DCM and independent predictor of adverse cardiac outcomes. The present study investigated the effect and the possible mechanism of KXYX on myocardial brosis. Methods: Male wistar rats of DCM was induced by furazolidone (Fz) (0.3 mg/g/day, gavage) for 8 weeks and treated with KXYX (3.6, 1.8, 0.9 g/kg/day) or captopril for another 4 weeks. The cardiac function indices were evaluated using ECHO. Myocardial morphology was visualized using H&E and masson staining. Then, the effect of differentiation of Ang II-induced cardiac broblasts (CFs) to myobroblasts was detected using a-SMA and Vimentin immunohistochemical staining. The expression of RhoA, ROCK1, p-MLC were observed using western blot. The mRNA level of RhoA, ROCK1, MLC were assayed by quantitative RT-PCR. Result: Fz-induced rats had changes in the structure and function of the left ventricle as well as myocardial bers broken and loosely arranged, cardiomyocytes necrosis in the myocardial tissue. High level expressions of brosis were observed in the model rats. The expressions of RhoA, ROCK1, p-MLC were elevated in dilated heart and Ang (cid:0) -induced CFs, while KXYX can rescued cardiac dysfunction and remodeling, inhibited the process of MF and down-regulated RhoA/ROCK1 signaling which suppressed the differentiation of broblasts into myobroblasts. Conclusion: Our study demonstrated that KXYX attenuated the development of myocardial brosis by down-regulating the expression of RhoA/ROCK1 signaling pathway in vivo and vitro. Thus, we provided an underlying mechanism of KXYX function in DCM therapy.


Background
Dilated cardiomyopathy (DCM) is a cardiomyopathy characterized by ventricular enlargement and systolic dysfunction, which is a common cause of heart failure [1]. Myocardial brosis (MF) plays a major role in ventricular remodeling which causes cardiomyopathy progression [2]. Large areas of brotic tissue can be detected in the biopsy of patients with DCM [3]. The process of MF mainly includes broblasts proliferate and transform to myo broblasts in the myocardial tissue and collagen bers excessive deposit in the extracelluar matrix (ECM). These changes lead to ventricular stiffness, and diastolic dysfunction, which exacerbate the heart failure [4]. Therefore, blocking the development of MF may be the key to DCM treatment. Drugs or interventions which can speci cally target brosis may break through the current clinical treatment limitations.
Rho kinase (ROCK), a downstream effector molecule of RhoA, is a transfer station for a variety of intracellular signaling pathways [5]. Rho kinases, including ROCK1 and ROCK2, promote cytoskeletal migration and recombination in normal physiological conditions. But excessive activation of ROCK1 promotes the transformation and differentiation of myocardial broblasts, which leads to the imbalance of collagen metabolism, and then leads to MF [6]. The RhoA/ROCK1 signaling pathway can induce broblasts to release brogenic cytokines such as TGF-β1, CTGF and α-SMA [7]. All the cytokines stimulate myo broblasts to secrete collagen bers and promote the development of MF [8][9][10]. The downregulation of RhoA/ROCK signaling pathway may be a new method for the treatment of MF.
Traditional Chinese medicine has a long history in the treatment of heart failure. KXYX is an effective prescription for DCM heart failure. The symptoms in DCM patients include fatigue, palpitations, chest tightness, shortness of breath and edema. Chinese medicine believes that these symptoms are closely related to qi de ciency and blood stasis. Therefore, KXYX combines a variety of herbs responsible for their multiple e cacies such as tonifying qi and invigorating the circulation of blood, Clinical observation showed that KXYX alleviated symptoms, rescued cardiac function and postponed ventricular remodeling in DCM patients [11,12]. In our previous studies, KXYX had multi-target effects on DCM by means of properties of inhibiting cardiomyocyte apoptosis [13], improving myocardial energy metabolism [14], antithrombosis, reducing DCM rat myocardial tissue pathological damage and reducing the expression level of myocardial interstitial collagen [15,16]. However, the speci c therapeutic mechanism of KXYX is still unclear. Therefore, this study used furazolidone-fed rats and angiotensin treated broblasts, and observe the anti brogenic effect of KXYX on MF in vitro and in vivo.

ANIMAL AND GROUP
All animal experiments were approved by the animal care and use committee of Henan hospital of Chinese Medicine. (PZ-HNSZYY-2019-021) one hundred male wistar rats(4-week old) were purchased from Beijing Charles River experimental animal technology Co., Ltd Beijing,China . The rats were randomly divided into the normal group (n = 10) and the DCM group (n = 90). All of them were allowed free access to food and water. DCM group received a gavage of furazolidone (Tianjin Lisheng, China) solution (50 mg/ml, 0.3 mg/g for 8 weeks) [17]. Left ventricular end-diastolic diameter (LVEDD) and ejection fraction (LVEF) was measured by echocardiographic examination to con rm the model successful or not [18,19]. The successful DCM rats (n = 62) were randomly divided into 5 group, including model, KXYX high, KXYX medium, KXYX low and captopril groups. The daily gavage of KXYX was 3.6 g / kg, 1.8 g / kg, 0.9 g / kg, respectively [20]. captopril group was 10.125 mg / kg (Shanghai Sine, China). normal group and model group were given the same amount of normal saline. The indicated treatment was administered orally daily for 4 weeks after furazolidone gavage.

Echocardiography (ECHO)
Rats were anesthetized with intraperitoneal injection of 1% pentobarbital sodium (30 mg/kg). Cardiac function and dimensions were measured by echocardiography with an Acuson Cypress system and a 7L3 probe (Siemens, Germany) after 4 weeks of KXYX treatment for rats, M-mode echocardiography was used to measure the left ventricular end-diastolic diameters (LVEDD)and left ventricular end-systolic diameters(LVESD) on the parasternal long axis views. Next, ejection fraction (EF)and fractional shortening (FS) were calculated. EF was calculated by the Teichholtz method. The mean values were obtained from at least three different cardiac cycles.

H&E and masson staining
After echocardiography examination, the hearts were removed by thoracotomy, the remaining blood was washed in pre-cooled PBS, and xed in 4% paraformaldehyde solution for 48 hours. After dehydration, heart tissue specimens were embedded in para n, and cut into5 µm sections. Hematoxylin eosin staining (HE) and Masson staining were performed according to the standard protocol. The myocardial collagen deposition in each group were observed under optical microscope. 5 elds were randomly selected to observe and record the images.

Cell culture
The ventricles of neonatal rats were cut into pieces under aseptic conditions, digested into single cells in 0.1% collagenase, and collected every 5 min. After centrifugation, all the cells were cultured in DMEM (DMEM, Gibco, USA) medium with 10% calf serum (Gibco, Australia), incubated in incubator of 5%CO 2 at 37℃ (Thermo Fisher Scienti c, USA) for 24 h, and myocardial broblasts (CFs) were obtained with differential attachment method. Passage as cells grow to a near-fused state. The adherent CFs were spindle-shaped, transparent cytoplasm, large oval shape nucleus under the microscope. the cultured cells identi ed by SP staining with vimentin. Fibroblasts of passage two were inoculated into a 6-well plate and cultured for 24 h, and the serum containing KXYX or fasudil were added to the culture for 2 h, washed with PBS, and then treated with Ang II for 24 h, and then samples were collected for further test.
Preparation of medicated serum SD Rats weighing 220 ± 10 g were chosen and randomly divided into KXYX group (KXYX serum) and normal group (normal serum). Rats in KXYX groups were treated gavage with a KXYX solution at a dosage of 1 ml/100 g/day (n = 10) while the normal group received the same volume of normal saline (n = 10) for 10 days. The blood from abdominal aortic were collected one hour after the nal treatment. After that, centrifuged at 3500 rpm for 15 min, and retained the supernatant. Serum was heated at 56 °C for 30 min, lter with a 0.22 µm lter membrane, stored at − 80 °C.

Immuno uorescence
The broblasts were xed in 4% paraformaldehyde for 30 min at room temperature, washed three times with PBS, and permeabilized with 0.3% Triton X-100 for 30 min. After blocking with BSA at room temperature for 30 minutes, slide the slides with anti-αSMA, Vimentin antibodies (Wuhan proteintech Biotechnology Co., Ltd.,) at After overnight incubation at 4℃, washing 3 times with PBS, the slides were incubated with FITC uorescent secondary antibody (Wuhan proteintech Biotechnology Co., Ltd.,) at 37 °C , protected from light for 2 h. After counter-staining the nuclei with DAPI, the images were observed and recorded using a uorescence microscope.

Cell viability
Fibroblasts were grown in 96-well plates, and the cell density in each well was controlled at 1 × 10 4 cells / well for 24 hours. After that, each well was given different concentrations of KXYX (0-1.0 mg / ml) and cultured for 24 hours. 10 was added to each well. ul of CCK-8, and then detect the absorbance at 450 nm in each well to calculate the growth and viability levels of KXYX cells at different concentrations. The optimal intervention concentrations of angiotensin II and fasudil were measured in the same way.
WB protein was extracted from heart tissue which was homogenized in RIPA lysis buffer, and quantitated by protein quanti cation BCA kit (Biyuntian Biological, China). The total protein was separated on SDS-PAGE gel, and then transferred to a PVDF membrane. the membrane was blocked with 5% skim protein powder at 37 ° C for 1 h. Membrane with primary antibody at 4 °C overnight. The primary antibodies against α-SMA,COL-1,RhoA,ROCK1,MLC, CTGF(proteintech),p-MLC Cell Signaling Technology, USA After washing, the membrane was coupled with a speci c HRP-conjugated secondary antibody (Wuhan Sewell Biotechnology, China) for 1.5 h. The membrane was subsequently washed. Relative luminescence intensity was analyzed by gel image system (Bio-Rad Laboratories, USA).

RT-PCR
Extract the total RNA solution according to the kit's protocol (Qiagen). The total RNA concentration and purity were measured and reverse transcribed into cDNA. Real-time PCR was performed using cDNA as the template, normal group as the reference group, β-actin as the internal reference, and the relative expression of mRNA was expressed by 2-ΔΔCt, and the relative quantitative analysis of ROCK1, MLC, and CTGF mRNA expression was performed. Data was analyzed with Bio-Rad CFX Manager software.

Statistical analysis
Page 6/19 SPSS for windows 22.0 was used for statistical analysis and GraphPad Prism 6.0 software was used for statistical graph. Measurement data are expressed as mean ± standard deviation (SD). analyzed by oneway analysis of variance (ANOVA) test is used for comparison of multi-sample means that follow a normal distribution, and LSD test is used for comparison between groups. A value of P < 0.05 was considered statistically signi cant.

Results
KXYX improve heart function in rats After 4-week treatment, the Echo of each model rat was measured. LVEDD and LVESD were increased in the model group than normal group, and EF and FS decreased as well. These indicate pathological changes in the left ventricular structure of model rats. Compared with the model group, the KXYX high and KXYX medium group can reduce the effects of LVEDD and LVESD, and improve the effects of EF and FS, but the improvement in the KXYX low group is not signi cant. The results show that KXYX can improve cardiac function in DCM rats.

KXYX inhibit myocardial brosis in DCM rats
H&E and Masson staining showed that cardiomyocytes swell, disorder, necrosis in the myocardial tissue of the model group were increased than the normal. myoglobin arranged disorderly, collagen and α-SMA expression increased can also be found in the model group. Myocardial edema was reduced in myocardial tissues in the KXYX high and middle groups, as well as myoglobin alignment improved. KXYX low group did not show signi cant effect, and still had a large amount of collagen deposition ( Fig. 2A, B, and C).
We use WB and QPCR to determine the expressions of CTGF, COL-1 and α-SMA in myocardial tissues.
The expressions in the model group increased, while the myocardial tissues in the rats treated with KXYX were reduced to varying degrees, most obviously in the high-dose group, and the reduction in the low-dose group was not signi cant (Fig. 2D, E, F). These results indicate that the improvement of cardiac function in DCM rats by KXYX may be achieved by inhibiting the myocardial brosis.

KXYX inhibits AngII induced myo broblast transdifferentiation
Vimentin is a protein that is speci cally expressed by broblasts. Using immuno uorescence microscopy, we can observe a large number of broblasts were isolated from the heart tissue of suckling rats. When the pressure is overloaded, broblasts can adopt an "active" state known as "myo broblast". Induced by AngII, CFs were transformed into myo broblasts into round shape, and brosis activation marker α-SMA was abundantly expressed in cells (Fig. 3A). AngII did not signi cantly affect the viability of CFs between 10-10000 nmol / L (Fig. 3B), and the effect of CCK-8 of KXYX on cell viability was measured at different concentrations, the cell viability was optimal when the concentration at 25 mg / ml (Fig. 3C). When induced by different concentrations of AngII, the expression of α-SMA and collagen (COL-1) was the highest at 10 nmol / L (Fig. 3D, E), so 10 nmol / L was used as the optimal concentration for cell model induction. KXYX can inhibit the expression of myocardial brosis factors such as CTGF connective tissue growth factor , COL-1 and α-SMA in Ang -induced CFs (Fig. 4).
KXYX Down-regulate the Expression of RhoA, ROCK1, p-MLC By detecting the expression of proteins and mRNA in the RhoA / ROCK1 signaling pathway in myocardial tissue, the expression levels of RhoA, ROCK1, and p-MLC in the myocardial tissue of the model group rats were signi cantly higher than those in the normal group; The reduction effect was seen in the KXYX high and middle dose groups, but the improvement in the low dose group was insigni cance (Fig. 5).
Similar results were showed in vitro (Fig. 6A). Then we use fasudil, an inhibitor of the RhoA / ROCK signaling pathway to further evaluate the in uence of these signaling pathways. Further validation was performed in the AngII-induced CFs with KXYX or fasudil. It can be seen that both KXYX and Fasudil have the ability to inhibit the expression levels of RhoA, ROCK1, p-MLC, and the combination of KXYX and Fasudil can further inhibit the effect (Fig. 6B, C, D, E).

Discussion
DCM is one of the most common causes of heart failure today, which has become one of the highest mortality cardiovascular diseases and deteriorates the life quality for patients all over the world [21]. This study shows that furazolidone-induced DCM rat models can better replicate the relevant pathological characteristics of human DCM, such as decreased cardiac function, ventricular pathological remodeling and expansion, and the occurrence of myocardial brosis [22]. The result is similar to previous studies. When myocardial brosis occurs, cardiomyocytes in the myocardial tissue appear swollen, disordered, necrotic, disordered myoglobin arrangement, increased collagen deposition and brotic markers such as α-SMA were signi cantly increased. During the development of DCM, the activation of renin-angiotensin-aldosterone system (RAAS), oxidative stress, and cytokines (such as TGF-β) can promote the proliferation and transdifferentiation of CFs [23].
In ammatory cells can secrete cytokines and act on CFs, resulting in increased collagen secretion. At the same time, the collagen synthesis and degradation of myocardial tissue are imbalanced, and myocardial brosis occurs. The replacement of cardiac muscle by brotic tissue, increasing inability of the ventricle to pump blood su ciently to meet the demands [24,25]. The transdifferentiation of CFs is the core link of the development of cardiac brosis.
In recent years, inhibiting the development of myocardial brosis has been a hot issue in clinical treatment and scienti c research. A variety of natural and synthetic drugs have been proven to play a protective role for heart by inhibiting the development of myocardial brosis. Statins have become the most effective drugs for the prevention and treatment of coronary heart disease. Their main mode of action is to treat atherosclerosis by lowering cholesterol levels. Some statins show the effect in preclinical and clinical studies to inhibit brosis and adverse myocardial remodeling [26][27][28]. Simvastatin show an inhibition for CFs transdifferentiation through TGF-β pathway. Elevation of locally angiotensin II level serves as a potent stimulant for CFs both through direct actions and through TGF-β mediated effects. aldosterone antagonists, angiotensin receptor blockers (ARB) and ACE inhibitors (ACEI) show the good effect on heart failure is closely related to the improvement of cardiac brosis [23]. Researches on the pharmacological mechanism of Chinese medicines have gradually increased. Studies have shown that astragaloside IV and astragalus polysaccharides which are the main effective components of astragalus, can inhibit myocardial brosis and protect heart [29,30]. Ginsenoside Re can improve isoprenalineinduced myocardial brosis by down-regulating the TGF-β1 / Smad3 pathway [31]. Salvianolic acid which is extracted from salvia miltiorrhiza reduces Ang II-induced cardiac brosis in rats by inhibiting the NF-κB pathway [32].
Connective tissue growth factor (CTGF) is a key mediator produced in the extracellular matrix under conditions of pathological brosis [33]. The RhoA / ROCK1 signaling pathway can directly or indirectly promote the expression of CTGF [34]. The RhoA / ROCK1 signaling pathway plays an important role in broblast proliferation and transdifferentiation, which can directly promote the secretion of collagen by myo broblasts and also activate other brosis-related signaling pathways [35]. KXYX can inhibit the transformation of myocardial broblasts to myo broblasts, inhibit the expression of α-SMA, COL-1, and CTGF, and also down-regulate MF mediated by the RhoA / ROCK1 signaling pathway.
RhoA / ROCK inhibitor fasudil can inhibit the development of myocardial brosis and has bene ts for cardiac remodeling [36]. ROCK1 can over-activate the phosphorylation of myosin light chains(MLC), which can promote broblast migration and MF [37,38]. Our research shows that KXYX can decrease phosphorylation of MLC, thereby indirectly inhibiting myocardial brosis. However, the mechanism of how to inhibit MF by regulating the phosphorylation of myosin light chain needs further research.

Conclusion
To concluded,KXYX inhibit the expression of RhoA, ROCK1, p-MLC in vivo and vitro, it can also directly inhibit expression of myocardial brosis factors such as α-SMA, COL-1, CTGF and alleviate MF and improve heart function in DCM rats. Myocardial brosis is an important mechanism of DCM. Delaying the development of myocardial brosis is a possible way for KXYX to exert cardioprotection.
There are still a few questions in our research that deserve further exploration. We found that KXYX can reduce the apoptosis of cardiomyocytes in DCM myocardial tissue, and we found that KXYX can protect the mitochondrial morphology of cardiomyocytes under electron microscope. How does KXYX achieve the protective effect on cardiomyocytes? This is the direction of our further research. Availability of data and materials All data and materials are available from the corresponding author on reasonable request.

Ethics approval and consent to participate
The animal experiments were approved by the animal care and use committee of Henan hospital of Chinese Medicine. (PZ-HNSZYY-2019-021).

Consent for publication
This manuscript is approved by all authors for publication.