Effects of Ziyin Qianyang Formula on Renal Fibrosis through the TGF-β1/Smads Signaling Pathway in Spontaneously Hypertensive Rats

Objective The aim of the study is to explore the effects and mechanisms of action of Ziyin Qianyang Formula (ZYQYF) on renal fibrosis in spontaneously hypertensive rats (SHRs). Methods Forty SHRs were randomly divided into a model group, Ziyin Qianyang Formula regular-dose and high-dose groups (ZYQYF-R, 20 g/kg; ZYQYF-H, 40 g/kg), and a western medicine group (enalapril 10 mg/kg), and 10 Sprague-Dawley rats were selected as the normal group. The rats received continuous gavage administration for 6 weeks and systolic blood pressure (SBP) measurements were obtained every fortnight. The serum levels of urea, serum creatinine (sCr), and uric acid (UA) were measured; the pathological morphology and collagen content of the kidneys were observed by hematoxylin-eosin (HE) and Masson staining; and the serum Ang II level was measured by an enzyme-linked immunosorbent assay (ELISA). Transforming growth factor (TGF)-β1, Smad-2, Smad-3, and Smad-7 protein and mRNA expressions in kidney tissues was evaluated by western blotting and reverse transcription-polymerase chain reaction. Results The ZYQYF-H group showed significantly a lower renal weight and renal weight/body weight than the model group. The enalapril and ZYQYF-H groups showed significantly lower SBP than other groups after 6 weeks of administration. The ZYQYF-H group showed better improvement than the ZYQYF-R and enalapril groups in glomerular and tubular morphology and better reductions in inflammatory cell infiltration and collagen volumetric fraction. The ZYQYF-H group also showed better reductions in serum UA and Ang II levels; collagen-I, collagen-III, and p-Smad2/Smad-2 protein expression; and Smad-2 mRNA expression and a better increase in Smad-7 protein and mRNA expression than the enalapril group. Besides, the degree of renal function and fibrosis improvement was positively correlated with the dose of ZYQYF. Conclusion ZYQYF can significantly reduce SHR blood pressure, protect renal function and structure, and improve renal fibrosis by regulating Smad proteins through TGF-β1.


Introduction
Essential hypertension (EH) is a widespread disease of the cardiovascular system and is mainly characterized by an increase in arterial pressure [1]. Despite the availability of various therapeutic agents for hypertension, the incidence of this disease has continued to increase; the number of people with hypertension is expected to grow by 15%-20% each year and reach nearly 1.5 billion patients worldwide by 2025 [2]. Chronic hypertension can cause damage to the kidneys in a variety of conditions and is the second leading cause of end-stage renal disease (ESRD) [3]. Terefore, treatment of hypertension while delaying renal damage is important.
Renal fbrosis is an important pathological manifestation of hypertensive nephropathy [4,5]. Te pathogenesis of hypertensive kidney damage is mainly related to imbalance of extracellular matrix (ECM) production and degradation [6,7]. Increased activity of the transforming growth factor-β1 (TGF-β1)/Smads signaling pathway leads to abnormal proliferation and accumulation of fbroblasts in the kidney, and the resultant secretion of large quantities of ECM can cause fbrotic damage in the kidney [8][9][10]. Our group's previous study showed that Ziyin Qianyang Formula (ZYQYF) had a good antihypertensive efect and could reduce the damage to kidney tissues by regulating the Ang II/TLR4/NF-κB signaling pathway and reducing the expression of infammatory factors such as interleukin (IL)-6, IL-8, and tumor necrosis factor (TNF)-α [11,12]. However, the efect of this formula on renal fbrosis remains poorly understood. Te formula is particularly suitable for patients with "Yin defciency and Yang hyperactivity" syndrome, which is characterized by the highest serum levels of angiotensin II (Ang II), a powerful factor in inducing the diferentiation of periglomerular cells into fbroblasts, among various syndromes [13]. Terefore, the main objective of this study was to investigate the mechanism of action of ZYQYF in improving renal fbrosis in spontaneously hypertensive rats (SHRs) by regulating the Ang IImediated TGF-β1/Smads signaling pathway.

Materials and Methods
2.1. Animals. Forty 12-week-old male SHRs and 10week-old matched clean-grade Sprague-Dawley (SD) rats weighing 230 ± 10 g were obtained from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd (animal license number SCXK (Beijing): 2016-0011). All rats underwent systolic blood pressure (SBP) measurements after purchase, and SHRs meeting the corresponding blood pressure (SBP > 180 mmHg) criteria were included in the study. Tese rats were routinely housed at the Animal Experiment Center of Anhui University of Chinese Medicine at a temperature of 20-23°C and humidity of 50%-65%.

Drugs and Reagents
2.2.1. Drugs. Te composition of ZYQYF is shown in Table 1. All medicinal herbs used to prepare ZYQYF were purchased from the Second Afliated Hospital of Anhui University of Chinese Medicine (Anhui, China) and qualifed by Professor Dong Chang-wu (Anhui University of Chinese Medicine). All herbs were decocted by an automatic decoction machine. Te raw herbs (130 g) were thoroughly washed and soaked in distilled water (1.5 L) for 1 h and then decocted. Te extraction solution (0.5 L) was obtained after secondary fltration, equivalent to 0.26 g herbs/mL. Finally, the solution was evaporated to a concentration of 2.0 g herbs/mL (used for ZYQYF-regular-dose) and 4.0 g herbs/ mL (used for ZYQYF-high dose) by a rotary evaporator. Enalapril maleate (5 mg/tablet, H32026568, Yisu; Jiangsu Pharmaceutical Co., Ltd., State Drug Administration) tablets were powdered and prepared in a suspension of 1 mg/mL with 0.5% sodium carboxymethyl cellulose (CMC-Na) and stored at 4°C.

Animal
Grouping and Dosing. Ten SD rats were selected as the normal group, while 40 SHRs were randomly divided into the model group, Ziyin Qianyang Formula regular-dose group (ZYQYF-R group), Ziyin Qianyang Formula high-dose group (ZYQYF-H group), and western medicine group (enalapril group; n � 10 in each group). On the basis of the adult animal dosages identifed in a previous experiment [11], the regular and high doses of ZYQYF were set as 20 and 40 g/kg, respectively. Animals in the western medicine group received enalapril maleate 10 mg/kg by gavage, while those in the normal and model groups received the same volume of CMC-Na by gavage. Te rats were gavaged twice a day, at 9: 00 am and 4: 00 pm, for 6 weeks.

Sample
Collection. After 6 weeks of gavage and 12 h of fasting, all rats were weighed and anesthetized with pentobarbital sodium 40 mg/kg. Subsequently, 4-5 mL of blood was collected from the abdominal aorta and centrifuged at 3000 r/min and 4°C for 10 min, and the supernatant was separated and stored in a refrigerator at −80°C. After completion of blood collection, the left kidney was quickly obtained, rinsed with saline, dried on flter paper, weighed Using a biosignal acquisition and processing system, a rat pulse transducer was connected to measure the systolic blood pressure in the tail artery. Measurements were taken before and two, four, and six weeks after administration of the drug. Each rat was kept quiet for 30 min; the blood pressure measurements were repeated three times, with a 5-minute interval between each measurement; and the mean value of the measurements was calculated for subsequent analyses.

Measurement of Renal Function Indicators.
Te serum urea, creatinine (sCr), and uric acid (UA) levels were measured automatically by biochemical analysis instruments.

HE Staining to Observe the Morphological Changes of Kidney Pathology in Each Group of Rats.
Te left kidney tissue of rats was used to prepare conventional parafn sections with a thickness of 4 μm. After dewaxing, the sections were stained with hematoxylin for 3 min, rinsed in ultrapure water, stained with eosin solution for 1 min, rinsed with ultrapure water, and then air-dried and sealed with a flm. Te histopathological morphology of the rat kidney was observed under a light microscope.

Masson Staining of Collagen Fibers in the Kidney Tissue of Rats in Each Group.
Parafn sections were dewaxed with water, stained with iron hematoxylin for 5-10 min, divided using ethanol fractionation, washed once with water, stained in Masson blue solution, and then washed with water. Te sections were then stained with acidic Lichon red line red dip for 5-10 min; washed with a weak acid working solution for 1 min; washed in phosphomolybdic acid solution for 1-2 min; washed in a weak acid working solution for 1 min; stained with aniline blue staining solution for 1-2 min; washed with weak acid working solution for 1 min; dehydrated with anhydrous ethanol; and then treated with xylene to make them transparent. Te expression of blue-green collagen fbers in rat kidney tissues was observed under a light microscope, and the collagen volumetric fraction (CVF) was analyzed using Image-Pro Plus 6.0 software.

Enzyme-Linked Immunosorbent Assay for the Determination of Ang II in Rat Serum.
Frozen serum samples were obtained, and the levels of Ang II in rat serum were determined on the basis of the kit instructions.

Western Blot to Detect the Expression of Collagen-I,
Collagen-III, TGF-β1, p-Smad2, Smad-2, p-Smad3, Smad-3, and Smad-7 Proteins in Rat Kidney Tissues. After washing 50 mg of rat kidney tissues from each group 2-3 times with PBS, lysis solution equal to 10 times the volume of the tissue was added (protease inhibitor was added a few minutes before use); the sample was then placed on ice and lysed for 30 min, centrifuged for 10 min using a centrifuge at 4°C and 12000 r/min, and the supernatant was obtained to extract the protein. Te concentration of the extracted protein was determined using the BCA method. Te protein solution was denatured by boiling, electrophoresed, transferred to a membrane, blocked with 5% skimmed milk powder, and phosphorylated proteins were closed with 1% BSA, treated with the primary antibody (1 : 1000 for all objective proteins, 1 : 5000 for internal reference protein) and secondary antibody (1 : 5000) in parallel, incubated and then developed in ECL, decolorized in PhotoShop, and analyzed by Alpha software for optical density values.

Determination of TGF-β1, Smad-2, 3, and 7 mRNA Expressions in Rat Kidney Tissues by Reverse Transcription-Quantitative Polymerase Chain Reaction.
Total RNA was extracted from rat kidney tissues by the Trizol method; cDNA was synthesized by reverse transcription; and TGF-β1 and Smad-2, 3, and 7 mRNA were amplifed by adding the corresponding primers. Using 0.2 mL PCR tubes, three tubes were prepared for each reverse transcription product. Te specifc reaction system included 7.5 μL of 2 × qPCR Mix, 1.5 μL of upstream and downstream primers, 2.0 μL of the cDNA template, and 4.0 μL of ddH 2 O. Te reaction conditions were as follows: 40 cycles of pre-denaturation at 95°C (10 min), denaturation at 95°C for 15 s, and annealing/extension at 60°C for 30 s. Te fuorescence signal was collected once for every 0.5°C temperature rise from 65°C to 95°C. Data analysis was performed using the 2 −ΔΔCt method. Te primer sequences are listed in Table 2.
2.6. Blinding Design. Te allocation of animals, execution of the experiments, and data analyses were performed by diferent personnel. Te statistical staf were unaware of the specifc groupings and were unblinded after the data statistics were analyzed and evaluated.

Statistical Methods.
All data were analyzed using SPSS 19.0 software, and the measurement data were expressed as mean ± standard deviation. Data showing a normal distribution were compared using one-way ANOVA, and the LSD method was used when the variance was the same in the twoway comparison, while Dunnett's T3 method was used when the variance was not the same. Diferences were considered statistically signifcant at P < 0.05.

Efects on Renal Weight and Body Weight.
In comparison with the normal group, the model and ZYQYF-R groups showed signifcantly higher renal weight (P < 0.01, P < 0.05), signifcantly lower body weight (P < 0.05), and signifcantly higher renal weight/body weight (RW/BW) (P < 0.01). Renal Evidence-Based Complementary and Alternative Medicine weight and RW/BW in the ZYQYF-H and enalapril groups were signifcantly lower than those in the model group (P < 0.05, P < 0.01). RW/BW in the enalapril group was signifcantly lower than that in the ZYQYF-R group () and showed no signifcant diference from that in the ZYQYF-H group (P > 0.05; Figure 1).

Efects on Systolic Blood
Pressure. Before administration, the blood pressure of SHRs in all four groups was signifcantly higher than that of the rats in the normal group (P < 0.01). In comparison with systolic blood pressure in the model group, systolic blood pressure was signifcantly lower in the ZYQYF-R group after 6 weeks of administration (P < 0.01), in the ZYQYF-H group after 4 and 6 weeks of administration (P < 0.05, P < 0.01), and in the enalapril group after 2, 4, and 6 weeks of administration (P < 0.01). After 2 and 4 weeks of administration, the systolic blood pressure in the enalapril group was signifcantly lower than those in the ZYQYF-R and ZYQYF-H groups (P < 0.05, P < 0.01). However, after 6 weeks of administration, the systolic blood pressure in the enalapril group was not signifcantly diferent from that in the ZYQYF-H group (P > 0.05; Figure 2).

Efects on the Serum Levels of Urea, sCr, and UA.
Te serum UA levels in the ZYQYF-H and enalapril groups were signifcantly higher than that in the normal group (P < 0.01), while the serum urea and sCr levels showed no signifcant diference (P > 0.05). Te serum urea, sCr, and UA levels in the ZYQYF-R, ZYQYF-H, and enalapril groups were signifcantly lower than those in the model group (P < 0.05, P < 0.01). In comparison with the ZYQYF-R group, serum urea, sCr, and UA levels were signifcantly lower in the ZYQYF-H and enalapril groups (P < 0.05, P < 0.01). Te decrease in serum UA levels was more signifcant in the ZYQYF-H group than that in the enalapril group (P < 0.01; Figure 3).

HE Staining.
Te normal group showed normal glomerular volume, no obvious atrophy, neatly arranged tubular structures, and no obvious infammatory cell infltration in the interstitium. In the model group, the glomerular cyst lumen was enlarged, internal atrophy occurred, the tubular lumen was dilated, the structure was unclear and disorganized, and a large number of infammatory cells were seen infltrating the interstitium. Te ZYQYF-R and enalapril groups showed diferent degrees of improvement in glomerular morphology, diferent degrees of reduction in tubular dilatation, neater alignment, and reduced infammatory cell infltration in the interstitium. In the ZYQYF-H group, the glomerular morphology was generally normal; the tubular structure was clear and neatly arranged; and only a few infammatory cells were seen infltrating the interstitium. Figure 4.

Masson Staining.
Te CVF was signifcantly higher in all SHR groups in comparison with the normal group (P < 0.01). However, the CVF in the ZYQYF-H and enalapril groups was signifcantly lower than that in the model group (P < 0.01, P < 0.05). Moreover, the CVF in the ZYQYF-H group was signifcantly lower than those in the ZYQYF-R and enalapril groups (P < 0.01, P < 0.05; Figure 5).

Efects on the Serum Level of Ang II.
In comparison with the normal group, the four SHR groups showed signifcantly higher serum Ang II levels (P < 0.01). In comparison with the model group, the ZYQYF-R, ZYQYF-H, and enalapril groups showed signifcantly lower serum Ang II levels (P < 0.01). In comparison with the ZYQYF-R group, the ZYQYF-H and enalapril groups showed signifcantly higher serum Ang II levels (P < 0.05, P < 0.01). Te serum Ang II level in the enalapril group was signifcantly lower than that in the ZYQYF-H group (P < 0.05; Figure 6).

Efects on Collagen-I, Collagen-III, TGF-β1, p-Smad2, Smad-2, p-Smad3, Smad-3, and Smad-7 Protein Expressions in Kidney Tissues.
Collagen-I and collagen-III protein expressions in all SHR groups were signifcantly higher than those in the normal group (P < 0.01). In comparison with the model group, the three treatment groups showed signifcantly lower expression levels of these two proteins (P < 0.01, P < 0.05), while the ZYQYF-H group showed signifcantly lower expression of these proteins in comparison with the ZYQYF-R and enalapril groups (P < 0.01).

Discussion
Early renal impairment in patients with EH is often not clinically evident and the symptoms usually appear only when the renal damage is in the middle-to-late stages [14]. Elevated systolic blood pressure and mean arterial pressure are also the most important risk factors for renal impairment in the elderly [15]. Terefore, clinical management of hypertension is based not only on achieving blood pressure targets but also on the maintenance of target organ function. ZYQYF is composed of six herbs: Radix rehmanniae, Rehmannia glutinosa, Concha Haliotidis, Uncaria rhynchophylla, oyster, and Polygonum multiforum Tumb. From a modern medical point of view, the active ingredients of Radix rehmanniae and Rehmannia glutinosa can protect kidney tubule epithelioid cells and reduce serum urea nitrogen levels [16][17][18]. Concha Haliotidis can exert a hypotensive efect by infuencing the calcium channels and the expression of plasma membrane calcium ATPase (PMCA) mRNA [19]. Uncaria rhynchophylla (UR) is known to have signifcant hypotensive efects and can attenuate Ang IIinduced myocardial fbrosis by inhibiting the RhoA/ROCK1 signaling pathway [20]. Te oyster extract exhibits antihypertensive efects by inhibiting angiotensin-converting enzyme (ACE) and improves organ infammation, fbrosis, and apoptosis [21,22]. Tetrahydroxystilbene glucoside (TSG) is the main active component of Polygonum multiforum Tumb, and it can protect the kidney by inhibiting oxidative stress and reducing infammatory responses [23]. According to the theory of Chinese medicine, ZYQYF is utilized for "Zi Yin Bu Shen (nourishing Yin and strengthening the kidney)" and "Ping Gan Qian Yang (repressing hyperactive Liver Yang)." Te formula thus takes into account both the symptoms and root cause, allowing the smooth reduction of blood pressure and protecting renal function.
Tese results indicate that a decrease in body weight and an increase in renal weight in the SHR model group in comparison with the SD rats, an alteration that may be related to fbrosis of the kidneys, and that high doses of ZYQYF could alleviate these changes. SHRs typically develop hypertension at 4-6 weeks and gradually show features of hypertensive end-organ damage [24]. Te 12-week-old SHRs used in this experiment had signifcantly elevated blood pressure, elevated related renal function parameters, and pathological changes caused by renal damage, indicating that the animal model is consistent with the typical features of hypertensive renal damage. Te results of this study showed no signifcant decrease in systolic blood pressure in the ZYQYF-R and ZYQYF-H groups after 2 weeks of administration, although a signifcant decrease in systolic blood pressure was observed in both groups after 6 weeks of administration. Tus, ZYQYF has a defnite antihypertensive efect and the accumulation of this efect is needed over time. Te systolic blood pressure in the ZYQYF-H group was signifcantly lower than that in the ZYQYF-R group after 6 weeks of administration, indicating that the antihypertensive efect of ZYQYF was positively correlated with the concentration of the drug administered.
Urea is a sensitive indicator of glomerular fltration function and is often used to assess the degree of progression of renal disease [25]. sCr is also often used in clinical practice to assess the glomerular fltration rate [26]. Elevated serum levels of UA may lead to increased blood pressure through renal infammation, activation of the renin-angiotensinaldosterone system (RAAS), and downregulation of NO production, so serum UA levels are closely related to the regulatory system of the kidney [27]. Terefore, the serum levels of urea, creatinine, and UA are important indicators of the renal function. Te results of this study showed that ZYQYF signifcantly reduced the levels of urea, sCr, and UA in SHRs, and the degree of reduction was more obvious in the ZYQYF-H group than in the ZYQYF-R group, with the serum urea and sCr levels in the ZYQYF-H group close to those in normal rats. Tis fnding suggested that ZYQYF could efectively improve the renal function impairment of SHR and the degree of renal function improvement was positively correlated with dose. Te results also suggested that ZYQYF can reduce serum UA levels more efectively than enalapril. In addition, the results of HE and Masson staining in this test showed that ZYQYF signifcantly improved the pathological morphology of glomeruli and tubules in rats and reduced interstitial infammatory cell infltration and collagen fber deposition between tubular interstitial cells, suggesting that ZYQYF not only improved renal function but also had a protective efect on structural aspects of the kidney.
Ang II, an important efector regulated by RAAS, is one of the main mediators in the pathogenesis of EH. On the one hand, Ang II can act directly on the RAAS, stimulating the synthesis of cortisol, regulating the secretion of aldosterone and water and sodium reabsorption, and causing an increase in blood pressure [28]. On the other hand, it regulates the expression of TGF-β1 by inducing multiple pathways, which in turn regulates the TGF-β1/Smads signaling pathwaymediated renal fbrosis [29]. As seen in the experimental results, ZYQYF can signifcantly reduce the levels of collagen-I and collagen-III, indicating an improvement in the degree of Evidence-Based Complementary and Alternative Medicine renal fbrosis. TGF-β1 has a strong fbrogenic efect; it can lead to an imbalance om ECM production and degradation and induce proliferation and migration of renal fbroblasts, and its level signifcantly correlates with the progression of renal interstitial fbrosis [30]. Smad-2, Smad-3, and Smad-7 are the main substrates in the regulation of TGF-β1, of which Smad-2 and Smad 3 are receptor-regulated proteins and Smad-7 is an inhibitory protein. In the main process of TGF-β1/Smads regulation, the activation of TGF-β1 induces phosphorylation of Smad-2 and Smad-3, and the phosphorylated Smad-2 and Smad-3 bind to Smad-4 and signal into the nucleus, thus exerting the efects of cell proliferation, diferentiation, apoptosis, and fbrosis. Smad-7 mediates the negative regulatory mechanism of this pathway, regulating the stability of TGF-β1 by binding directly to Smads and inhibiting the activity of this pathway [31,32]. Te results of the present study showed that ZYQYF can signifcantly reduce the serum Ang II level, thereby reducing the activation of the TGF-β1/Smads signaling pathway induced by it. Additionally, the results of this study also showed that ZYQYF could efectively inhibit the expression of TGF-β1, Smad-2, and Smad-3 proteins and mRNA, and increase the expression of Smad-7 protein and mRNA in kidney tissues, thereby regulating the TGF-β1/ Smads signaling pathway-mediated renal fbrosis process, and   Figure 7: Efects on collagen-I (b), collagen-III (c), TGF-β1 (d), p-Smad2/Smad-2 (e), p-Smad3/Smad-3 (f ), and Smad-7 (g) protein expression in the kidney of rats in diferent groups. △ P < 0.05, △△ P < 0.01 vs. the normal group; ▲ P < 0.05, ▲▲ P < 0.01 vs. the model group; * P < 0.05, * * P < 0.01 vs. the ZYQYF-R group; # P < 0.05, ## P < 0.01 vs. the enalapril group. Data are expressed as mean ± SD (n � 10).
the fndings also indicated a dose-efect relationship. On the basis of these fndings, ZYQYF can be considered to reduce the degree of renal fbrosis by lowering the serum Ang II level and inhibiting the TGF-β1/Smads signaling pathway mediated by Ang II.

Conclusion
In summary, ZYQYF can signifcantly reduce systolic blood pressure in SHRs and show good protective efects on both the function and structure of the kidney. Its mechanism of action may involve inhibition of the activation of the Smads signaling pathway, reducing the phosphorylation of Smad-2 and Smad-3, and enhancing the expression of Smad-7 by decreasing the Ang II-mediated expression of TGF-β1, which in turn reduces the production of ECM and mitigates the degree of renal fbrosis, playing a role in the treatment of hypertensive renal damage. Te present study is limited to the efect of ZYQYF on renal fbrosis through the TGF-β1/ Smads signaling pathway, but the efect of other signaling pathways and the efect on renal vascular and renal hematological changes require further investigation.

Data Availability
Te data used to support the fndings of this study are available from the frst author Hui Xu upon reasonable request.

Ethical Approval
Te animal experiments in this study were approved by the Animal Ethics Committee of Anhui University of Chinese Medicine (No. 2022001).

Conflicts of Interest
Te authors declare that they have no conficts of interest.