Jiedu Tongluo Decoction Attenuates Myocardial Fibrosis Through Inhibition of The TGF-β1/Smad2/3 Pathway

Background: Jiedu Tongluo (JDTL) decoction is a traditional Chinese medicine (TCM) formula containing three herbal ingredients that is widely used to treat myocardial brosis (MF). This study aimed to investigate the molecular mechanism of action of JDTL decoction for the treatment of MF. Methods: The chemical constituents of Traditional Chinese medicine were analyzed by HPLC, Forty Wistar rats were randomly divided into normal control group, model group, Jiedu Tongluo decoction low-dose and high-dose treatment groups, with 10 rats in each group. Rat myocardial brosis model was induced by subcutaneous injection of 5 mg•kg-1 isoproterenol hydrochloride. 5 and 50 g•(kg.d) -1 were given intragastric administration for 7 days. Hematoxylin-eosin (HE) and Masson staining were performed for histological evaluation of myocardial tissue, MTT method detects the activity of cardiac broblasts, The alkaline water method is used to determine the content of hydroxyproline in myocardial tissue and myocardial broblasts, Immunohistochemical method was used to detect the expression of myocardial tissue transforming growth factor (TGF) β1, p smad2/3, and type III collagen (Col III) expression, Immunouorescence to detect the expression of α-SMA/Vimentin in myocardial tissue and myocardial broblasts; Western blot was used to detect the protein expression levels of TGFβ1 and psmad2/3 in cardiac broblasts. Results: In this study, six compounds of JDTL decoction were identied by high-performance liquid chromatography (HPLC). Hematoxylin and eosin (HE) and Masson staining showed that in the isoproterenol hydrochloride (ISO)-induced MF rat model, JDTL treatment protected the myocardial structure and inhibited type III collagen (COL3) expression(p<0.05). The immunohistochemistry (IHC) results also showed that JDTL treatment signicantly reduced the expression of vimentin, alpha-smooth muscle actin (α-SMA), and transforming growth factor-beta 1 (TGF-β1) as well as the phosphorylation of Smad2/3 in the rat MF model(p<0.05). Rat cardiac broblasts (RCFs) were used for the following assays performed in vitro: hydroxyproline detection, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, wound-healing test, western blot, and double immunouorescence staining; the results of these assays conrmed the inhibitory effects of JDTL decoction on the proliferation, migration, and transdifferentiation abilities of RCFs as well as the molecular mechanisms underlying these effects, including the inhibition of the TGF-β1/Smad2/3 pathway through downregulation of TGF-β1 expression and phosphorylation of Smad2/3 as well as inhibition of vimentin and α-SMA expression(p<0.05). Conclusion: JDTL decoction can prolong the process of MF through inhibition of the TGF-β1/Smad2/3 signaling pathway.


Introduction
Myocardial brosis (MF) is a common pathological phenomenon associated with the development of hypertensive heart disease, coronary heart disease, myocarditis, arrhythmia, cardiomyopathy, chronic pulmonary heart disease, and other cardiovascular diseases (Kong P, 2014). MF can cause microcirculation disorders and in ammatory reactions as well as cardiac systolic and diastolic function disorder, which can cause heart failure (NG, 2015), malignant arrhythmia, and sudden cardiac death in the worst cases. The pathogenesis of MF is complex, and there are many side effects of current western medicine-based anti-MF therapies.
Studies have revealed that extracellular matrix (ECM) deposition is considered as a cause of MF occurrence and that cardiac interstitial cells are the main source of the ECM, accounting for about 75% of the total number of cardiac cells, mainly cardiac broblasts (Jin X, 2019). Cardiac broblasts play important roles in the regulation of ECM homeostasis and in the maintenance of structural, mechanical, biochemical, and electrical properties of the heart. In addition, cardiac broblasts also in uence the function of cardiomyocytes through a variety of ways, such as direct cell-cell interactions or the secretion of various growth factors. In the case of heart injury, alterations of the cardiac environment, such as changes in the ECM, the increased amounts of growth factors and cytokines, and higher levels of mechanical stress, can induce the transdifferentiation of myocardial broblasts to myo broblasts, leading to destruction of the normal physiological structure of the myocardial interstitium and promoting MF (Pardali E, 2017). Therefore, the suppression of proliferation and transdifferentiation of myocardial broblasts caused by injury or stress may be a potential and effective therapeutic approach for preventing MF. Recently, broblast-based therapies have become a research hot-spot in cardiac disease.
Progress has been made in this eld, including cell therapies and microRNA-, peptide-and drug-based interventions. Furthermore, traditional Chinese medicine (TCM) also has been found to be effective in broblast-targeted therapy.
Dr. Huang Yongsheng, a well-known doctor of traditional Chinese medicine (TCM) throughout China, believes that cardiac function disorder caused by injury or stress may be due to the internal injuries of qiqing because the stagnation of qi in the body will induce conversion of the stagnated qi into re. At the same time, the stagnation of qi will also cause blood circulation to be blocked, causing blood stasis, which can induce heat. The heat can be turned into re, so the re and the blood stasis work together to form a toxin that is obstructed in the veins, thus blocking the blood circulation. If the re poison and the blood stasis cannot be terminated in the body rapidly and completely, the blood will be damaged, followed by putrefaction, resulting in a coronary blood supply disorder, heart injury, and eventually MF.
Based on the overall concept mentioned above, Dr. Huang has developed a novel TCM-based therapy for MF, which includes different herbs that can promote qi, improve blood circulation, disperse re, and detoxify, leading to smooth venation, harmony of qi and blood, and removal of toxins and pathogens. The mixture of these herbs (including Prunella vulgaris, Achyranthes bidentata, and Fructus paeoniae) is called the Jiedu Tongluo (JDTL) decoction, which has been widely used in the treatment of MF and in animal models .
To better understand the mechanisms underlying the effectiveness of the JDTL decoction in broblasttargeted therapy, this study aimed to investigate its effects on MF in a rat MF model that was established by subcutaneous injection of isoproterenol hydrochloride (ISO). The mechanism of action of JDTL, as it relates to regulation of the myocardial transforming growth factor-beta 1 (TGF-β1)/Smad2/3 signaling pathway, was also analyzed in order to provide new ideas and methods for treating patients with MF.

Animal model establishment and drug administration
After seven days of adaptive breeding, 40 Wistar rats were randomly divided into the following four groups: normal control group (Ctrl), model group (ISO), low-dose JDTL group (Jie L), and high-dose JDTL group (Jie H), with 10 rats in each group. Except for the Ctrl group, the rats in the other three groups were injected subcutaneously with ISO at 5 mg/kg body weight, while the Ctrl control group was injected subcutaneously an equal volume of saline. From the second day of model establishment, the experimental rats in the Jie L and Jie H groups were given JDTL decoction at 5 g/kg and 50 g/kg, respectively, by gavage daily for 7 consecutive days; meanwhile, the Ctrl and model groups were given (by gavage) an equal volume of normal saline daily.
To evaluate the effect of JDTL (alone) on normal rats, another batch of 30 Wistar rats was randomly divided into the following three groups: normal control group (Ctrl), low-dose JDTL (alone) group (Jie aL), and high-dose JDTL (alone) group (Jie aH), with 10 rats in each group. Rats in the Jie aL and Jie aH groups were given JDTL at 5 g/kg and 50 g/kg daily by intragastric administration, respectively; while the rats in the Ctrl group were intragastrically administered with an equal volume of normal saline daily for 7 consecutive days.
The rats were anesthetized and euthanized at 1-2 h after the last intragastric administration. Blood samples were collected, and the serum was inactivated at 56 ℃ for 30 min for future use.

High-performance liquid chromatography (HPLC) ngerprint analysis
The composition of the JDTL decoction (water extract) was analyzed by HPLC, according to a standard procedure. First, the water extract was separated via a ZORBAX SB-C18 column (4.6 mm × 250 mm, 5-µm pore size, Agilent, Santa Clara, CA, USA), with the following conditions: mobile phase, acetonitrile (A) and 0.4% phosphoric acid/water (B); column temperature, 30 °C; ow rate, 1 mL/min; UV detection wavelength, 203 nm; and injection volume, 5 µL. The authorized software Agilent 1200 was used for chromatographic data collection and analysis of speci c components. Several chemicals, such as caffeic acid, oleanolic acid, ursolic acid, rosmarinic acid, rutin, hyperoside, β-ecdysterone, chlorogenic acid, and ginsenoside RO were used as standards to determine if they were components of the JDTL decoction.

Hematoxylin and eosin (HE) staining and Masson staining
The ventricular muscle tissues were collected from the experimental rats in each group and were used to make 5-µm-thick para n-embedded sections. After dewaxing with xylene, HE staining and Masson trichrome staining were performed on the ventricular muscle tissue sections. The images of the stained sections were recorded, and the degree of MF was analyzed using ImageJ software (U. S. National Institutes of Health, Bethesda, MD, USA).

IHC staining
The collected myocardial tissue of the experimental rats was xed with 4% paraformaldehyde for IHC staining. The expression of TGF-β1, p-Smad2/3, and COL3 in the different groups was analyzed using a standard IHC staining procedure (primary antibody dilution, 1:300). The secondary antibody was then added, according to the instructions of the IHC kit (PV-6000; Zhongshan Jinqiao Biotech Co., Beijing, China). The staining results were visualized by the DAB chromogenic method.

Cell culture and experimental groups
RCFs were cultured with high-glucose DMEM containing 10% FBS. Cells in the logarithmic growth phase were divided into ve groups: normal control group, TGF-β1 stimulation group, low-dose JDTL group, high-dose JDTL group, and SB431542 group. The cells in the normal control group were cultured in medium containing 2% normal rat serum. In the TGF-β1 stimulation group, TGF-β1 was added to the medium containing 2% normal rat serum at a nal concentration of 10 -8 µg/mL. In the low-dose and high-dose JDTL groups, the cells were cultured in medium containing 2% normal rat serum, low-dose or highdose JDTL, and TGF-β1 (10 -8 µg/mL). In the SB431542 group, both TGF-β1 (10 -8 µg/mL) and SB431542 (10 -5 µg/mL) were added into the medium containing 2% normal rat serum.

Cell viability assay
RCFs were plated into 96-well plates at a density of 2 × 10 3 cells/well. The corresponding medium of each group, as mentioned above, was added to the wells of the different groups, and the cells were incubated with 5% CO 2 at 37 ℃ for 24 h. The proliferation activity of the cells was determined using an MTT assay, which is a colorimetric assay that involves the reduction of the tetrazolium dye MTT into its insoluble formazan). The optical density (OD) of each well was measured with a microplate reader at a wavelength of 490 nm to determine the viability of the RCFs for evaluating their proliferation potentials. Meanwhile, the blank control wells (with culture medium only, but without cells) were used for zeroing. The cell proliferation activity was calculated as follows: cell proliferation activity (%) = 100 × (OD of experimental group [-blank control group] / (OD of control [-blank control group]).

Wound-healing assay
RCFs were placed into a six-well plate at a cell density of 4 × 10 5 cells/well and cultured to near con uence. In the wells of the different groups, the old culture medium was replaced with the corresponding medium of each group, as mentioned above. At 24 h after treatment, the cells were scratched and washed with phosphate-buffered saline three times to remove the suspended cells, followed by an additional 24-h culture in the corresponding medium of each group. The image of each well was recorded with an optical microscope. Comparison of the width of the scratch between the different groups was performed to evaluate the wound-healing ability of the treated cells by using ImageJ software.

Hydroxyproline content determination
To assess the degree of cell brogenesis, hydroxyproline released in the cell culture supernatant of the RCFs was measured by a spectrophotometric method (alkaline water method).

Immuno uorescence staining
Both cultured cells and collected tissue samples on the glass slide were xed with 4% formaldehyde and then incubated with mouse anti-α-SMA or vimentin antibodies (1:50) at 4 ℃ overnight. The next day, the slides were incubated with secondary antibody coupled with FITC or Cy3 (1:1000) at room temperature in the dark for 1 h, followed by DAPI nuclei staining for 10 min. Finally, the stained slides were examined using a uorescence microscope. The images of the stained slides were recorded and analyzed using ImageJ software.

Western blot analysis
The cultured cells were lysed with radioimmunoprecipitation assay buffer and centrifuged at 12,000 rpm for 15 min before the supernatant was collected. The protein concentration was measured with a BCA protein assay kit. After denaturation at 95 ℃ for 5 min, the protein samples (100 µg each) were separated by electrophoresis on 10% or 12% sodium dodecyl sulfate-polyacrylamide gels and transferred to a polyvinylidene uoride membrane. The membrane was blocked with 5% skimmed milk and then incubated with primary antibodies (1:1500 dilution) against total Smad2/3 and p-Smad2/3, respectively, at 4 ℃ overnight. The next day, after incubation with secondary antibody (1:1500 dilution) at room temperature for 1 h, the bands on the membrane were visualized by ECL detection. The western blot results were analyzed using ImageJ software.

Statistical analysis
In this study, all data were expressed as the mean ± standard deviation (X ± s). SPSS19.0 software (IBM Corp., Armonk, N.Y., USA) was used for statistical analysis. Group comparison was performed by one-way analysis of variance. The least signi cant difference method was used for pairwise comparison between groups. A value of p < 0.05 was considered as a signi cant difference.

Identi cation of the composition of JDTL decoction
The composition of the water extract of the JDTL decoction was determined by HPLC analysis to include six compounds, which were identi ed by the comparison of the retention times of the standard products, as shown in Fig. 1.

Comparison of the cardiac histological changes in the different groups
The HE staining results ( Fig. 2A) showed that in the normal control group (Group 1), the myocardial tissue was arranged in order, the nucleus size was uniform, and no obvious in ammatory in ltration or necrosis was found. However, in the other groups treated with ISO (Groups 2-4), the rat myocardial tissue was irregularly arranged, and myocardial necrosis of different sizes and with clear boundaries was found at 7 days after ISO injection ( Fig. 2A). The presence of a necrotic area suggests that there were obvious changes in MF due to increases of the myocardial collagen composition and myocardial broblasts. Masson trichrome staining also demonstrated that the necrotic area in the rat myocardial tissue was lled by blue-stained collagen components and broblasts (Fig. 2B). The results of image analysis revealed that compared with the normal control group, the collagen components in the myocardial tissue collected from either the model group or the JDTL treatment groups were signi cantly increased (p < 0.001). Moreover, compared to the model group, the collagen composition in either the low-dose or highdose JDTL treatment group was signi cantly reduced (p < 0.001); however, as mentioned above, there was no statistically signi cant difference of the collagen composition of the myocardial tissue between the two groups.
As shown in Fig. 2C, E (IHC staining), the expression intensity of COL3 of the myocardial tissue in the model group was signi cantly greater than that in the normal control group (p < 0.001). However, compared with the model group, the expression intensity of COL3 in the Jie H group was signi cantly decreased (p < 0.01).

Comparison of vimentin and α-SMA expression in rat myocardial tissue in the different groups
Vimentin is considered as a marker of broblasts because of its speci c expression in broblasts.
Comparison of the vimentin expression in the rat myocardial tissue in the different groups (Fig. 3A, D) showed that the expression intensity of vimentin in the rat myocardial tissue from the model group and the Jie L group was signi cantly greater than that from the Ctrl group (p < 0.001). However, compared to that from the model group and Jie L group, the expression of vimentin in the rat myocardial tissue from the Jie H group was signi cantly reduced (Fig. 3A, D, p < 0.001).
Although α-SMA is a marker of smooth muscle cells, its expression has been found in myocardial broblasts during the period of MF (Xiao L, 2017). In this study, α-SMA was used as a marker of myo broblasts, and its expression in the rat myocardial tissue was detected by IHC. The results ( Fig. 3B-C) showed that the expression intensity of α-SMA in the rat myocardial tissue from the model, Jie L, and Jie H groups was signi cantly greater than that from the Ctrl group (p < 0.001). However, compared to that in the model group, α-SMA expression in the Jie H group was signi cantly increased (p < 0.005).

Comparison of TGF-β1 expression and the p-Smad2/3 level in rat myocardial tissues from the different groups
The IHC results (Fig. 4A, C) showed that compared to that from the normal control group, TGF-β1 expression in the rat myocardial tissue from the model, Jie H, and Jie L groups was increased signi cantly (p < 0.001, p < 0.01, p < 0.05, respectively). However, TGF-β1 expression in both the Jie L and Jie H groups was signi cantly less than that in the model group (p < 0.01, p < 0.001).
The IHC results (Fig. 4B, D) also revealed that the p-Smad2/3 level of rat myocardial tissue from the model group and the Jie L group was signi cantly greater than that from the normal control group (p < 0.001, p < 0.05). However, compared with the model group, the p-Smad2/3 level from the Jie H group was signi cantly reduced (p < 0.05). There was no statistically signi cant difference of TGF-β1 expression or the p-Smad2/3 level between the Jie L and Jie H groups.

Effects of JDTL decoction on the proliferation, migration, and transformation of RCFs
RCFs were used to investigate the effects of JDTL decoction on the proliferation, migration, and transformation of RCFs in vitro. Our study revealed that the cell proliferation activity in the TGF-β1 stimulation group was signi cantly increased, compared with the normal control group (p < 0.001).
The hydroxyproline content in the cell culture supernatants is shown in Fig. 5B. Similar to the results of the cell viability assay, the hydroxyproline content in the TGF-β1 stimulation group was signi cantly greater than that in the normal control group (p < 0.001); however, high-dose JDTL treatment (Jie H group) could signi cantly reduce TGF-β1-enhanced hydroxyproline accumulation in the cell culture supernatant (p < 0.05), the inhibition degree of which was close to that of SB431542 (p < 0.001).
Although there was no statistically signi cant difference of the cell viability and ECM secretion capacity between the Jie L and Jie H groups, the high-dose JDTL treatment was reasonably selected for the subsequent experiments due to its excellent inhibitory effects. The additional experiments included the wound-healing test. As shown in Fig. 5C-D, the cell migration rate of RCFs enhanced by TGF-β1 was remarkably reduced in the Jie H and SB431542 groups (p < 0.001, p < 0.01), although cell migration in these two groups was still faster than that in the normal control group (p < 0.001, p < 0.01).
Furthermore, the expression levels of vimentin and α-SMA in the cells treated with TGF-β1 were detected by double immuno uorescence staining. As shown in Fig. 5E-F, compared with the normal control group, the rate of co-expression of vimentin and α-SMA in the cells treated with TGF-β1 (TGF-β1 group) was dramatically increased (p < 0.001). However, treatment with either high-dose JDTL or SB431542 reduced TGF-β1-enhanced vimentin and α-SMA expression signi cantly (p < 0.05, p < 0.01).

JDTL decoction treatment decreased the ratio of Smad/p-Smad in RCFs
Since the Smad signaling pathway is mainly regulated by TGF-β1, our study focused on the effect of JDTL on TGF-β1-mediated activation of the Smad signaling pathway. Western blot analysis showed that compared with that in the normal control group, the ratio of p-Smad/Smad in the TGF-β1 group was signi cantly increased (p < 0.001). But compared to the TGF-β1 group, the ratio of p-Smad/Smad in both the JDTL and SB431542 groups was signi cantly decreased (p < 0.01, p < 0.05, Fig. 6), suggesting the inhibitory effect of JDTL on TGF-β1-promoted activation of the Smad signaling pathway. Our study also found that there was no statistically signi cant difference between the inhibitory effects of JDTL and SB431542.

Discussion
TCM believes that the occurrence of MF is caused by emotional factors that bring out liver dysfunction so that the maintenance of the normal ow of qi fails and stagnation of qi occurs, eventually causing qi depression, which leads to blood stasis as well as the stasis of veins and collateral vessels. At the same time, qi stagnation can turn into re, blood stasis can turn into heat, and heat polarization can turn into re. After experiencing illness for a long period of time, the re stasis in the body becomes poisonous, which blocks the venation, damages the venation and nutrient-qi, causes damage to the heart blood, and then develops myocardial remodeling. Therefore, it is necessary to develop a TCM-based novel therapeutic approach for MF.
On the other hand, ISO belongs to the group of catecholamine drugs that have been used to establish an animal MF model. Injection of ISO into the abdominal cavity of an animal can increase the myocardial contractility and frequency by stimulating cardiac β-adrenergic receptors, destroying the balance of the myocardial oxygen supply, and increasing the concentration of angiotensin II in the circulatory system and myocardium, thereby promoting massive accumulation of collagen to reduce the compliance of the ventricular wall and the proliferation of collagen bers, which in turn leads to the occurrence of MF. The disordered arrangement of myocardial tissue and pyknosis of the nucleus are typical histological features of MF. Other histological features of MF include cardiomyocyte hypertrophy, cytoskeletal rearrangement, and damaged cardiomyocytes, which are replaced by collagen bers, leading to the accumulation of hydroxyproline in the myocardial tissue and elevated expression of COL1 and COL3 (Liu JC, 2018).
JDTL decoction is a water decoction containing three types of TCMs: P. vulgaris, A. bidentata, and motherwort fruit (at a ratio of 3:2:1, respectively). In this study, the main pharmacological components of JDTL decoction were identi ed by HPLC. Finally, we identi ed nine pharmacological components of JDTL decoction that were selected/judged based on the Chinese Pharmacopoeia and published literature (Li F, 2018, Commission, 2020. Among these nine standard compounds, six compounds were previously known to be a part of the JDTLT extract, and the remaining three compounds were newly discovered. In this study, JDTL decoction was used to treat ISO-induced MF rats. In this rat MF model, after 7 days of treatment, the degree of MF was found to be signi cantly reduced. In addition, the collagen composition in the myocardial tissue of the necrotic area was also signi cantly reduced, and the expression level of COL3 in the JDTL-treated group was signi cantly less than that in the model group (ISO only). Moreover, the in-vitro study showed that the treatment of RCFs with JDTL decoction signi cantly inhibited the cell proliferation activity and reduced the content of hydroxyproline in the cell culture supernatant, indicating that JDTL decoction has a potential inhibitory effect on MF.
Vimentin, an intermediate lament protein, is often considered as a protein marker of broblast activation and also as a representative factor of mesenchymal cells in the epithelial-mesenchymal transition process. It has been reported that vimentin can interact with actin during cell migration (Battaglia RA, 2018) as well as reduce epithelial cell activity via weakening cell adhesion and increasing cell exercise capacity, thereby destroying the dense structure between cells and the original cell cytoskeleton and leading to tissue brosis (Yiting Fu 2019). Furthermore, α-SMA exists in the striated muscle of humans, mice, and other mammals, including the bone, heart, and smooth muscle (Laura W, 2019). In addition, α-SMA is an effector protein downstream of the TGF-β1 signaling pathway and is involved in the activation of broblasts induced by TGF-β1. Smad, a protein that is downstream of the TGF-β1 signaling pathway, can regulate the gene expression of α-SMA to induce the transformation of broblasts into myo broblasts. Upregulation of α-SMA expression can promote the formation of the actin cytoskeleton and induce the differentiation of broblasts into myo broblasts. At the same time, the activation of broblasts, the synthesis and secretion of ECM components, and the cell proliferation ability are signi cantly enhanced, which are involved in tissue damage repair (Xouri G, 2010, Baum J, 2011) and brotic disease progression (TA., 2011). The current study revealed that in a rat MF model, after 7 days of JDTL decoction treatment, the expression level of vimentin in the necrotic area of myocardial tissue was signi cantly less than that in the model group (ISO only, without JDTL). The expression level of α-SMA in the JDTL treatment group was also signi cantly greater than that in the control group, suggesting that both myocardial broblasts and myo broblasts are the main cell types that accelerate MF. Previous studies have shown that the conversion of broblasts into myo broblasts and their migration seem to have a more important role in the process of MF (Wang. R. 2014, S. R. , Wang. 2002, Pan LL, 2017. In our study, RCFs were used as a tool to investigate the effect of JDTL decoction on cell migration and the phenotypic transformation process. The results of the wound-healing test showed that JDTL decoction treatment could signi cantly inhibit the migration of myocardial broblasts. Moreover, the results of double immuno uorescence staining showed that JDTL decoction treatment signi cantly reduced the proportion of the vimentin and α-SMA co-expressed cells, suggesting that this treatment could signi cantly inhibit the production of myocardial broblasts by inhibiting the degree of MF as well as cell migration and phenotypic transformation. According to previous reports, ISO can promote angiotensin II production to induce activation of the classical downstream TGF-β1/Smad signaling pathway (Zhao M, 2015). Once TGF-β1 is activated, it promotes the phosphorylation of Smad2 and Smad3, the downstream signaling molecules of TGF-β1, to form heterotrimeric complexes with their common mediator, Smad4, which enters the nucleus, resulting in the transcriptional regulation of target gene expression (Toma I, 2012). The role of TGF-β1 in the development of MF has been reported. TGF-β1 can induce the differentiation of cardiac broblasts into myo broblasts and the activation of myo broblasts; stimulate the synthesis of ECM components, such as collagen, bronectin, and proteoglycan; and promote the deposition of ECM (Zhang HX, 2012). The IHC results in this study showed that JDTL decoction treatment signi cantly inhibited the expression of TGF-β1 in the myocardial tissue of the rats with MF. In rat MF model, the phosphorylation of Smad2/3, a key target downstream of the TGF-β1 pathway, was also signi cantly reduced after JDTL decoction treatment. Furthermore, the in-vitro experimental studies demonstrated that JDTL decoction treatment signi cantly decreased the ratio of p-Smad2/3 in TGF-β1-treated RCFs, implying that this treatment could prevent MF by inhibiting the TGF-β1 pathway and thus downregulating Smad2/3 phosphorylation.

Conclusion
Taken together, our study (including the in-vivo and in-vitro experiments) revealed that JDTL decoction has inhibitory effects on the transformation of broblasts into myo broblasts, the collagen deposition in myocardial tissue, and the MF process. The molecular mechanisms underlying the inhibitory effects of JDTL decoction include inhibition of the TGF-β1/Smad2/3 pathway through downregulation of TGF-β1 expression and phosphorylation of Smad2/3 as well as inhibition of the expression of vimentin and α-SMA. Our ndings provide a new theoretical basis and experimental support for the use of JDTL decoction to treat MF.  Figure 1 Identi cation of the components of JDTL decoction (water extract).    the high-dose JDTL group (Jie H). *** p < 0.001, ** p < 0.01 vs. the normal control group; ### p < 0.001, ## p < 0.01, # p < 0.05 vs. the TGF-β1 group, Δ p < 0.05 vs. the low-dose JDTL-treated group.

Figure 6
Inhibitory effect of JDTL on TGF-β1-promoted activation of the Smad signaling pathway in rat cardiac broblasts (RCFs). A. Western blot results of the p-Smad, Smad, and GAPDH levels in RCFs. Lane 1, the normal control group; Lane 2, the TGF-β1 group; Lane 3, the JDTL group; Lane 4, the SB431542 group. B.