Study on the Mechanism of Bu-Shen-He-Mai Granules in Improving Renal Damage of Ageing Spontaneously Hypertensive Rats by Regulating Th17 Cell/Tregs Balance

Methods Blood pressure and urine biochemical indices were recorded. Renal blood flow was evaluated by renal ultrasonography. Transmission electron microscopy (TEM) and HE staining were used to assess kidney and spleen morphology. Renal fibrosis was assessed using Masson staining. Serum levels of IL-6, IL-10, and IL-17A were measured using ELISAs. The density of RORγ and Foxp3 in the spleen was observed by immunofluorescence staining. The levels of Th17 cells and Tregs in blood were detected via flow cytometry. Transcriptome sequencing was performed to screen the targets of BSHM granules in hypertensive kidneys. Results BSHM granules decreased SBP by 21.2 mm·Hg and DBP by 8.8 mm·Hg in ageing SHRs (P < 0.05), decreased the levels of urine mALB, β2-Mg, and NAG (P < 0.01), and improved renal blood flow and arteriosclerosis. BSHM granules increased IL-10 expression (P < 0.05) while decreasing IL-6 (P < 0.01) and IL-17A (P < 0.05) levels. BSHM granules improved Foxp3 density and the number of Tregs (P < 0.01) and reduced RORγt density and the number of Th17 cells (P < 0.01). Transcriptome sequencing identified 747 differentially expressed (DE) mRNAs in kidneys after BSHM treatment. GO analysis suggested that BSHM granules act through immunoregulation. Conclusions BSHM granules attenuated hypertensive renal damage in ageing SHRs, by significantly increasing Tregs and decreasing Th17 cells.


Introduction
e latest study (PURE) showed that 22.3% of cardiovascular disease (CAD) events and deaths can be attributed to hypertension [1]. At the same time, hypertension is one of the main risk factors for the development and progression of chronic kidney disease (CKD) and end-stage renal disease (ESRD) [2] e intravascular pressure caused by long-range hypertension exceeds the self-adjustment of renal vessels leading to remodeling and wall thickening of small renal vessels, lumen stenosis, and a reduction in renal blood flow and the glomerular filtration rate, which in turn produces renal parenchyma injury [3]. Notably, recent studies have found that renal function in patients with hypertension decreased unexpectedly, requiring intensive antihypertensive therapy [4,5]. e role of inflammation and immunity in the pathogenesis of hypertension has been widely recognized [6]. e infiltration of innate and adaptive immune cells in the kidney, vessel wall, and perivascular regions and other inflammatory processes, such as elevated cytokine release, reactive oxygen species (ROS) generation, and the expression of adhesion molecules are consistent features of hypertension [7,8]. Several reports have provided evidence of a direct role of Ang II in the modification of T cell balance toward a more proinflammatory 1 phenotype, as indicated by increased production of the 1 cytokine IFN-c [9,10] and a decrease in 2-mediated responses, including IL-4 production, in Ang II-infused rats [9]. Madhur et al. [11] verified that Ang II-induced hypertension was associated with increased 17 cells and IL-17 production. Both T regulatory lymphocytes (Tregs) and their production of IL-10 show vascular protective effects and limit Ang II-mediated oxidative stress and vascular damage [12]. Ang IIinduced endothelial dysfunction was exacerbated in IL-10 KO mice (IL-10 −/− ) [13]. Increased circulating levels of IL-6 and IL-21 and TGF-β receptor activation in CD4+ T cells were found to induce the development of 17 cells and prevent the polarization of Tregs, which caused endothelial dysfunction and hypertension via an imbalance in 17 cells/Tregs [14,15]. Tacrolimus treatment reduced the number of Tregs and increased the level of IL-17, which was closely related to the hypertension caused by tacrolimus [16][17][18]. Valorie et al. [19] proposed that the Tregs and 17 cell imbalance caused by tacrolimus contributes to the development of endothelial dysfunction and hypertension.
us, regulating the 17 cell/Tregs balance is a significant therapeutic strategy for the prevention and treatment of hypertensive renal damage.
Many scholars have tried to apply a variety of immunomodulatory drugs, such as PDTC, infliximab, and Ni-0101, to reduce blood pressure and improve vascular function to treat hypertension and ameliorate the damage to corresponding target organs. However, these immunomodulatory drugs may lead to cytokine release syndrome, allergic reactions, and an increased risk of cardiovascular events. erefore, safety is still a serious obstacle to clinical application. e human immune system develops during the congenital embryonic stage and gradually continues to develop and improve with age. Ageing directly affects the immune response. e immune homeostasis mechanism of the body gradually collapses after 65 years of age, and T cells are the main age-related immune cell population [20]. Ageing T cells have clear pathogenic potential in cardiovascular diseases, such as hypertension, atherosclerosis, and myocardial infarction [21], in which the imbalance of 17 cells/ Tregs plays an important role.
In traditional Chinese medicine (TCM), the kidney is considered the origin of the congenital constitution. e essence hidden in the kidney is the innate essence, also known as the "kidney yuan," which transforms the innate qi of the human body, that is, the original power of life activities. As people age, the kidney qi becomes deficient, and the functions of the human body decrease, which causes dizziness. From the perspective of TCM, the immune system is also formed by the innate essence in the kidney. With the passage of time, immune function also declines. erefore, we can regard kidney deficiency as the common basis for elderly individuals suffering from vertigo and abnormal immune function. Tonifying the kidney, that is, tonifying "kidney yuan," is a common method for treatment of ageingrelated hypertension and immune imbalance.
In the past few years, due to recent progress in TCM, the topic of treatment of hypertensive kidney damage with TCM has rushed to the forefront of medical treatment approaches and public health [2]. Recently, clinical research [5] demonstrated that prescriptions could inhibit the glomerular and tubular hyperplasia caused by high BP in SHRs, reduce urinary albumin and β2-microglobulin, and improve renal function. Our previous study confirmed that Bu-shen-hemai (BSHM) decoction inhibits atherosclerosis by improving antioxidant and anti-inflammatory activities in ApoE-deficient mice [22]. Clinical studies [23,24] have found that the antihypertensive regimen of basic antihypertensive drugs combined with BSHM Decoction can more effectively reduce the urinary protein/creatinine ratio in patients with hypertension than the basic regimen of chemical antihypertensive drugs alone, which suggests that BSHM decoction can improve renal damage. In the present study, we focused on the immunoregulation activity of BSHM granules to improve renal damage in ageing spontaneously hypertensive rats (SHRs).
e dispensing granules of BSHM granules were purchased from TCM Pharmacy of Affiliated Hospital of Shandong University of Traditional Chinese Medicine (Jinan, China, Certificate: Luyao Zhizi z20120014).
BSHM treatment group: Rats were perfused with 2 g/kg/ d BSHM granules, equivalent to 40 g/kg/d crude herbs and equivalent to 2 times the common clinical dosage. e chief botanical composition is shown in Table 1.

Animals.
Animal welfare and experimental procedures were conducted in strict accordance with the principles of "Laboratory animal-Guide-lines for ethical review of animal welfare (GB/T 35892-2018)" (General Administration of Quality Supervision, Inspection and Quarantine of the People's Republic of China, 2018), and approved by the Faculty of Animal Ethics Committee of Affiliated Hospital of Shandong University of Traditional Chinese Medicine (Jinan, China). Eighty 18-month-old SHRs (SPF level, weighing 320-350 g) and twenty 18-month-old Wistar-Kyoto (WKY) rats were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Certificate: SCXC (Jing) 2016-0006). All rats were housed in an airconditioned room with a 12 h/12 h light/dark cycle at a temperature of 21 ± 1°C and humidity of 50 ± 5% and had access to food and water ad libitum. All rats were fed a standard chow diet for 8 weeks. Body weight and blood pressure were recorded every 2 weeks. SBP and DBP were detected with an ALC-NIBP noninvasive blood pressure analysis system. e measurements were repeated five times in parallel, and then, the average SBP and DBP values were recorded.
After 1 week of adaptation, 20 SHRs were randomly divided into two groups to confirm the efficacy of BSHM: the BSHM group (2 g/kg/d BSHM granules, equivalent to two times the common clinical dosage) and the SHR group. Ten WKY rats were used as the control group (n � 10). e WKY group and SHR group were intragastrically administered the same volume of saline.
Sixty SHRs were randomly divided into six groups to study the pharmacology of BSHM (n � 10): the BSHM group (2 g/kg/d BSHM), SHR group, BSHM + SR0987 group (2 g/ kg/d BSHM, 7.14 mg/kg, administered in a single subcutaneous injection), BSHM + S6281 group (2 g/kg/d BSHM, 4.4 mg/kg, administered via an intraperitoneal injection every second day). Only SR0987 group (7.14 mg/kg, administered in a single intraperitoneal injection), and only S6281 group (4.4 mg/kg, administered via an intraperitoneal injection every second day). e SHRs in the model group and the WKY rats were intragastrically administered the same volume of saline (n � 10).

Biochemical Analysis.
Rats were placed individually in plastic metabolism cages for 24 h for urine collection. e volume of the urine was recorded, and the urine samples were centrifuged at 3,000 r/min for 10 min at 4°C. e supernatant was used to determine the microalbumin (mALB), β-N-acetylglucosaminidase (NAG) and β2-microglobulin (β2-Mg) contents via biochemical assays.
17 cells were labeled with CD4-FITC and IL-17A-PE; Tregs were labeled with CD4-FITC, CD25-PerCP, and FoxP3-PE. A rat peripheral blood lymphocyte isolation kit (Solarbio, P8630, Beijing, China) was used to separate lymphocytes according to the manufacturer's protocol. en, 6 μL cocktail stimulation solution and 4 μL GolgiStop protein inhibitor were added to the lymphocytes, and the mixture was gently mixed and incubated in the dark at 37°C for 5 h. e cell surface was stained with IL-17A monoclonal antibody ( ermo Fisher Scientific, 45-7177-82, Waltham, MA, USA) or Foxp3 monoclonal antibody ( ermo Fisher Scientific, 56-5773-82, Waltham, MA, USA), and the cells were fixed and permeabilized. Intracellular staining was performed. 17 cells and Tregs were detected via flow cytometry.

Tissue Collection.
Rats were sacrificed after treatment for 8 weeks. After anaesthesia with sodium pentobarbital (40 mg/kg, i.p.), blood was drawn via venepuncture and collected in tubes with EDTA as an anticoagulant, and plasma was separated via centrifugation. e spleens and kidneys were removed and placed on ice as soon as possible. e kidneys were subjected to haematoxylin-eosin (HE) staining, Masson staining, transmission electron microscopy, transcriptome sequencing, RT-qPCR, and Western blotting. e spleens were subjected to HE staining and immunofluorescence staining. Evidence-Based Complementary and Alternative Medicine 3

Masson Staining and HE Staining.
Renal fibrosis was assessed via Masson staining. Dewaxed slices were soaked in Masson dye solution (Servicebio, G1006, Wuhan, China) according to the manufacturer's instructions and then observed under a microscope (Nikon Eclipse E100, Nikon, Japan), and images were acquired and analyzed (Nikon DS-U3, Nikon, Japan). Collagen fibers were stained blue, while muscle fibers, cellulose, and red blood cells were stained red. e fibrotic area (%) identified via Masson staining was analyzed using ImageJ. e morphology of the spleen and kidneys was observed via HE staining. Dewaxed slices were stained with an HE dye solution set obtained from Servicebio (Servicebio, G1003, Wuhan, China) following the standard steps. e stained spleens and kidneys were observed under a microscope (Nikon Eclipse E100, NIKON, Japan), and images were acquired and analyzed (Nikon DS-U3, NIKON, Japan). e nuclei were stained blue, and the cytoplasm was stained red.

Transcriptome Sequencing.
Total RNA was extracted from kidneys using a TRIzol reagent kit (Life Technologies, 15596-018, Carlsbad, USA) according to the standard protocol. Total RNA was further purified to avoid contamination. A total amount of 1 μg RNA per sample was used as input material for the RNA sample preparations. Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature in First Strand Synthesis Reaction Buffer (5X). First strand cDNA was synthesized using random hexamer primers and M-MuLV Reverse Transcriptase (RNase H-). Subsequently, second strand cDNA synthesis was performed using DNA Polymerase I and RNase H. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of the 3′ ends of DNA fragments, adaptors with hairpin loop structures were ligated to prepare for hybridization. To preferentially select cDNA fragments 370-420 bp in length, the library fragments were purified with AMPure XP beads (Beckman Coulter, Beverly, USA). en, PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers, and Index (X) Primer. Finally, PCR products were purified (AMPure XP beads), and library quality was assessed on an Agilent Bioanalyzer 2100 system. Gene Ontology (GO) enrichment analysis of differentially expressed (DE) genes was implemented using the cluster Profiler R package, in which gene length bias was corrected. GO terms with corrected P values less than 0.05 were considered significantly enriched in differentially expressed genes. e genes were ranked according to the degree of differential expression in the two samples, and then, the predefined gene set was tested to determine whether the genes were enriched at the top or bottom of the list. Gene set enrichment analysis can include subtle expression changes.

Reverse Transcription-Quantitative Polymerase Chain
Reaction (RT-qPCR). After extraction with TRIzol reagent ( ermo Fisher Scientific, 10296010, Waltham, MA, USA) and quantification with a NanoDrop 2000°c spectrophotometer ( ermo Fisher Scientific, Waltham, MA, USA), total RNA samples were reverse-transcribed using a Reverse Transcriptase Kit (Sparkjade, AG0303-B, Qingdao, China). e relevant expression of RNAs was determined using the 2 −△△CT method. e fold change was normalized to that in the WKY group, and β-actin was used as an internal reference.

Western Blotting Analysis.
For validation of the transcriptome sequencing data, key proinflammatory cytokine genes, such as IL-6, Smad3, Stat3, NF-κB, and TGF-β1, were chosen for Western blot analysis.
2.13. Statistical Analysis. Statistical analysis was performed using SPSS 26.0 software. e data are presented as the mean ± standard deviation (SD). Statistical analysis was performed with one-way ANOVA, followed by an LSD test. P < 0.05 was considered to indicate a significant difference.
e renal blood flow in the SHR group was much worse than that in the WKY group, with slow renal artery blood flow and renal arteriosclerosis: Vp, Vd, Vm, RI and PI (P < 0.01), as measured by renal ultrasonography. BSHM enhanced the blood flow in the renal artery (Vp increased by 24% (P < 0.01), Vd increased by 62% (P < 0.05), and Vm increased by 38% (P < 0.01)) and improved the degree of renal arteriosclerosis (PI was reduced by 28% (P < 0.05), and RI was reduced by 10% (P < 0.05)) ( Figure 2(b)).
Compensatory hypertrophy of renal tubules was observed in SHRs, but BSHM improved the morphology of kidneys (Figure 2(c)). TEM observation revealed that the mitochondria in SHR kidneys were obviously swollen, the density of cytoplasm was increased, and the nucleoplasm was condensed. BSHM effectively improved the morphology of SHR kidneys (Figure 2(d)). More collagen deposition was observed in SHR kidneys, which suggested an inclination toward renal fibrosis in SHRs (P < 0.01) (Figures 2(e) and 2(f )). BSHM decreased collagen deposition in the kidneys and improved renal fibrosis in ageing SHRs (P < 0.01) (Figures 2(e) and 2(f )).
Flow cytometry was used to determine the levels of both 17 cells and Tregs in peripheral blood. Compared with the WKY group, the 17 cell number was increased in the SHR group (P < 0.01), and that of Tregs decreased (P < 0.01). BSHM significantly increased the number of Tregs (P < 0.01) and decreased the number of 17 lymphocytes (P < 0.01) (Figures 3(c) and 3(d)).
us, we focused on the 17 cell/Tregs balance due to the potential activity of BSHM in immunoregulation in kidneys suggested by transcriptome sequencing.

BSHM Improves Urine Biochemical Indices and Renal Fibrosis in Ageing SHRs, and the Effect Was Expanded When Treatment Was Combined with S6281 and Offset When
Combined with SR0987. SHRs had higher urine levels of mALB, β2-Mg and NAG than WKY rats (P < 0.01), and BSHM administration decreased the levels of urine mALB, β2-Mg, and NAG (P < 0.01). Compared with the BSHM group, the urine levels of mALB, β2-Mg, and NAG were increased in the BSHM + SR0987 group (P < 0.01), but decreased in the BSHM + S6281 group (P < 0.01) ( Figure 5(d)). Shrinkage of glomerular loops, basement membrane thickening, and both atrophy and compensatory hypertrophy of renal tubules were observed in SHRs. Compared with the BSHM group, the BSHM + SR0987 group had a much worse renal morphology, while BSHM + S6281 improved the morphology of the kidneys ( Figure 5(e)). Based on Masson staining, compared with the BSHM group, renal fibrosis in the SHR group and BSHM + SR0987 group was more severe (P < 0.01), and BSHM + S6281 obviously alleviated renal fibrosis (P < 0.01) (Figures 5(f ) and 5(g)).

Immunoregulation of BSHM Was Expanded by Combined Treatment with S6281 and Offset by Combined
Treatment with SR0987. HE staining suggested that the spleen cell distribution in SHRs was disordered. BSHM effectively improved the splenic cell distribution, and the effect was expanded after cotreatment with S6281 and offset after cotreatment with SR0987 ( Figure 6(a)).

RT-qPCR.
qPCR results showed that IL-6, STAT3, TGF-β1, NF-κB, and SMAD3 expression levels were significantly higher in the SHR group than in the WKY group: IL-6 (43 times), STAT3 (25 times), TGF-β1 (21 times), NF-κB (7 times), and SMAD3 (10 times). BSHM + S6281 significantly reduced the expression levels of these indicators close to the levels observed in the WKY group. e expression levels of these indicators were much higher in the BSHM + SR0987 group than in the BSHM group. BSHM and BSHM + S6281 significantly reduced the expression of these indicators. More details can be found in Figure 7(a). Evidence-Based Complementary and Alternative Medicine

Western Blotting Analysis.
Western blot assays confirmed that BSHM and BSHM + S6281 decreased IL-6, SMAD3, Stat3, NF-κB, and TGF-β1 protein expression, and SR0987 increased the expression of these proteins (Figures 7(b) and 7(c)), which was consistent with the RT-qPCR data and in line with the GO analysis of transcriptome sequencing.

Discussion
Hypertension-related renal damage is the second leading cause of ESRD, and coexistent hypertension plays a predominant role in the progression of most chronic kidney diseases [26,27]. Currently, the mechanism of hypertensive renal damage is not fully understood. Immunoregulation, in   addition to genetics and the environment, is involved in the process of hypertensive renal damage. One characteristic of the relationship between activation of the innate and adaptive immune systems that causes inflammation of the kidneys and hypertension is an increase in the infiltration of immune cells, including macrophages and T lymphocytes, in renal interstitial tissue [28]. It has been reported that depleting both B and T cells, such as with mycophenolate mofetil, protects against hypertension and renal disease in SHRs [29,30]. Ageing directly affects the immune response. After 65 years of age, the immune homeostasis mechanism of the body gradually collapses. T cells are the main immune cell groups affected by ageing [20]. Ageing T cells have a clear role in pathogenicity in cardiovascular diseases, such as hypertension, atherosclerosis, and myocardial infarction [21].
BSHM is a TCM formula commonly used to treat ageing-related spontaneous hypertension in clinical practice, and the chief botanical components are HunagQi, HuangJing, SangJisheng, NvZhenzi, and NiuXi, which are used to tonify the kidney and replenish qi. e effect of BSHM on improving renal damage in ageing SHRs was investigated in the present study. Eight weeks of BSHM administration led to a successful decrease in SBP and DBP in ageing SHRs. Urinary mALB has been identified as an independent predictive factor of hypertension, cardiovascular complications, and other chronic renal diseases and is a reliable and sensitive indicator of early-stage renal injury [31]. β2-Mg, a protein with a relative molecular weight of 11,800, is mainly produced by human lymphocytes and serves as a sensitive indicator of the glomerular filtration rate and the reabsorption function of the proximal tubule that reflects renal injury [32][33][34]. Urinary NAG is a conventional marker used to assess renal injury [34]. BSHM significantly reduced the levels of urinary mALB, β-Mg, and NAG in SHRs, suggesting renoprotective properties of BSHM. BSHM improved renal artery blood flow and arterial stiffness, which are independent and robust predictors of chronic kidney and cardiovascular disease [35,36]. Alleviation of hyperplasia and hypertrophy of vascular smooth muscle layers contributed to an increase in arterial compliance and a decrease in blood pressure. Injury to renal tubules plays an important role in the development of various renal diseases [37], thus, we mainly focused on the structure of renal tubules in this study. e structural and fibrotic improvement in kidneys after BSHM treatment was supposed to be an important pharmacological mechanism of BSHM.
IL-6 induces 17 cell differentiation together with TGFβ, while inhibiting TGF-β-induced Treg differentiation, indicating that IL-6 is a very important factor in determining the 17 cell/Tregs balance [38]. Tregs demonstrate a prominent antihypertensive effect due to their ability to produce the anti-inflammatory cytokine IL-10 [28] and reduce oxidative stress in blood vessels [39]. IL-10 is an antiinflammatory factor secreted by Tregs and plays an important cardiovascular protective role in hypertension [12], while IL-17A is a proinflammatory factor secreted by 17 cells that contributes to the maintenance of elevated blood pressure [11]. e transcription factor forkhead box P3 (Foxp3) controls the phenotype and function of Tregs [28]. RORct is expressed in a subset of lamina propria Tcells and is required for the expression of IL-17 [40]. e ratio of Foxp3 and RORct contributes to the 17 cell/Treg balance. BSHM significantly increased the density of Foxp3 in the spleen, circulating Tregs and IL-10 but decreased the density of RORct in the spleen, circulating 17 lymphocytes, IL-6, and IL-17A, indicating that BSHM inhibited the inflammatory response and ameliorated the 17 cell/Treg balance. ese data suggest that the immunoregulatory effect of BSHM plays an important role in improving renal damage in ageing SHRs. erefore, SR0987 (a RORct agonist [41,42]) and sodium thiocyanate (S6281, an IL-10 secretion agonist) were used to verify the immunoregulatory effect of BSHM. In this study, separate SR0987 or S6281 treatment aggravated or alleviated renal damage in ageing SHRs, respectively.
e effects of BSHM were offset by BSHM combined with SR0987 but enhanced by BSHM combined with S6281.
BSHM significantly decreased systemic expression of IL-6, NF-κB and Stat3 in SHRs and expression in SHR kidneys, which suggests that the Toll-like receptor signalling pathway screened out by transcriptome sequencing might be a target of BSHM.
Recently studies [43][44][45] have confirmed that TGF-β1 is the central mediator of progressive renal fibrosis and acts as a negative regulator of the immune response [46]. Smad3 is the main mediator of the biological effects of TGF-β1 and plays a role in promoting fibrosis [44,47,48]. erapy targeting Smad3 may be a specific and effective treatment for renal fibrosis [48]. BSHM reduced both TGF-β1 and Smad3 expression, suggesting that BSHM brought greatly alleviated renal fibrosis and protected against the progression of hypertensive renal damage.
In conclusion, we demonstrated that BSHM granules attenuated hypertensive renal damage in ageing SHRs, improved renal structure and blood flow, and alleviated arteriosclerosis and fibrosis by significantly increasing Tregs and decreasing 17 cells and the levels of their related inflammatory factors. Because this was an in vitro study, the results only provide clues to guide further clinical studies, which are required to confirm our findings.

Conclusions
BSHM attenuated hypertensive renal damage in ageing SHRs, mainly by balancing 17/Treg lymphocytes and inhibiting the inflammatory response.

Data Availability
e data used to support the findings of this study are included within the article.