Shen Qi Wan-Containing Serum Alleviates Renal Interstitial Fibrosis via Restraining Notch1-Mediated Epithelial-Mesenchymal Transition

Objective Shen Qi Wan (SQW) is the most classic prescription for the clinical therapy of chronic kidney disease in China. Nevertheless, the function of SQW in renal interstitial fibrosis (RIF) has not been clearly clarified. Our purpose was to explore the protective function of SQW on RIF. Methods After intervention with SQW-containing serum alone at increasing concentrations (2.5, 5, and 10%) or in combination with siNotch1, the transforming growth factor-beta (TGF-β)-induced HK-2 cell viability, extracellular matrix (ECM)-, epithelial-mesenchymal transition (EMT), and Notch1 pathway-associated protein expressions were assessed by cell counting kit-8, qRT-PCR, western blot, and immunofluorescence assays. Results SQW-containing serum intensified the viability of TGF-β-mediated HK-2 cells. Besides, it augmented the collagen II and E-cadherin levels, and weakened the fibronectin, α-SMA, vimentin, N-cadherin, and collagen I levels in HK-2 cells triggered by TGF-β. Moreover, it is found that TGF-β led to the upregulation of Notch1, Jag1, HEY1, HES1, and TGF-β in HK-2 cells, which was partially offset by SQW-containing serum. Furthermore, cotreatment of SQW-containing serum and Notch1 knockdown further apparently alleviated the Notch1, vimentin, N-cadherin, collagen I, and fibronectin levels in HK-2 cells induced by TGF-β. Conclusion Collectively, these findings elucidated that SQW-containing serum attenuated RIF via restraining EMT through the repression of the Notch1 pathway.


Introduction
In recent years, with the increasing incidence of diseases such as diabetes, hypertension, and obesity, the incidence of chronic kidney disease (CKD) is also growing year-by-year [1]. Te global estimated prevalence of CKD is 13.4%, which brings a huge health and economic burden to patients [2]. Te mounting medical and economic burden brought by CKD has been highly concerning to the World Health Organization [3]. In the next 20 years, the impact of CKD on health will rise from 16th to 5th in the world [4]. Regardless of the cause of CKD, renal fbrosis is the ultimate coconsequence and can be hardly reversed [5]. Currently, the treatment options for CKD or renal fbrosis are limited, such as control of protein, salt, and lipid intake, control of blood pressure and blood sugar, and renin-angiotensin system (RAS) blockade, but the efcacy is not satisfactory [6]. Terefore, actively exploring the pathogenesis of CKD and developing efective antifbrotic drugs has become an urgent problem in the feld of nephropathy.
Renal interstitial fbrosis (RIF) is a clinicopathological change characterized by the excessive accumulation of extracellular matrix (ECM) in the renal interstitium, destruction of the renal structure, and loss of function and is a common pathway for all CKD progression to the end-stage renal disease (ESRD) [7,8]. Te severity of RIF is a signifcant basis for judging the prognosis of kidney diseases [9]. Previous studies on the mechanism of RIF mainly focus on infammation, apoptosis, proliferation, and activation of fbroblasts, as well as oxidative stress, cytokines, and the signaling cascade [10,11]. At present, studies have clarifed that epithelial-mesenchymal transition (EMT) is also involved in the pathological repair after kidney injury and is related to the progression of RIF to CKD, which is another important mechanism for the formation of RIF [12][13][14]. In RIF, many cytokines have been discovered to be participated in the EMT process, including transforming growth factor β (TGF-β), platelet-derived growth factor (PDGF), and connective tissue growth factor (CTGF) [15][16][17]. Among many EMT-inducing factors, TGF-β1 is considered to be one of the strongest EMT-inducing factors [18]. TGF-β1 can induce the transdiferentiation of myofbroblasts from diferent sources such as macrophages, epithelial cells, and fbroblasts by activating the classic Smaddependent and non-Smad pathways Notch/Jagged, Wnt/ β-catenin, and Hedgehog pathways, and promote the formation of RIF [19][20][21][22]. Exploring how to delay or even reverse RIF via the EMT process so as to better protect kidney function has always been a research hotspot.
With the development of traditional Chinese medicine (TCM), TCM compounds exhibit certain advantages in the treatment of CKD due to their multiple components, multiple targets, and low side efects [23,24]. From the perspective of TCM, "kidney-yang defciency" is the internal condition for the development of renal fbrosis. Early treatment of the warming kidney and benefting qi drugs can delay the process of renal fbrosis. Shen Qi Wan (SQW) is a representative prescription for tonifying the kidney in Chinese medicine and is derived from Jingui Yaolüe, written by Zhang Zhongjing. SQW is composed of Radix Rehmanniae Recens 24 g, Dioscoreae Rhizoma 12 g, Corni Fructus 12 g, Alismatis Rhizoma 9 g, Poria 9 g, Moutan Cortex 9 g, Cinnamomi Ramulus 3 g, and Aconiti Lateralis Praeparata Radix 3 g [25]. A study confrmed that the Alismatis Rhizoma, the component of the SQW, inhibited the TGF-β1 and ANG-induced expressions of collagen I, fbronectin, α-SMA, vimentin, and E-cadherin in cells, attenuating the process of EMT [26]. In addition, Dioscoreae Rhizoma, which is related to the SQW, was found to inhibit the invasion and migration of HepG2 cells by reversing TGF-β1-induced EMT [27]. Our previous data confrmed that SQW can evidently reduce adeninemediated RIF, which is closely related to the EMT, and its mechanism is related to the repression of TGF-β1/Smad signaling [25]. Nevertheless, the mechanism by which SQW alleviates RIF is unclear.
TGF-β1 induces the secretion of fbrotic factors in HK-2 cells, a commonly used cell model of renal fbrosis [28]. Terefore, in this study, HK-2 cells induced by TGF-β1 were used as the experimental model in vitro to explore the efect of SQW-containing serum on the EMT, ECM, and Notch1 pathways of HK-2 cells. Tis study aims to clarify that SQWcontaining serum may weaken EMT by interfering with the conduction of the Notch1 pathway to have a synergistic anti-RIF efect and provide a new theoretical basis for the application and research of SQW in RIF.

Preparation of SQW Suspension.
In brief, 400 concentrated pills (each 4 pills is equivalent to 1.5 g of the original medicinal materials) were placed in a mortar to be ground into a fne powder. After dissolving with distilled water, they were placed in an ultrasonic instrument for 30 min to fully dissolve. Ten, SQW was concentrated to a fnal concentration of 2 g/mL of the original medicinal materials, followed by storage at 4°C for later use.

Preparation of SQW-Containing Serum.
Twenty rats were randomly separated into the SQW group (n � 10, 3 g/ kg) and the control group (n � 10). Te SQW group was given 3 g/kg SQW suspension (0.3 g/mL) by intragastric administration, twice a day, for 5 consecutive days. Te control group was given 0.9% saline, twice a day, for 5 consecutive days. During the administration period, all rats ate normally and fasted 12 h before the last administration. One hour after the last administration, pentobarbital sodium (40 mg/kg intraperitoneally) was applied to anesthetize the rats. Subsequently, the blood samples were acquired through the abdominal aorta. Te blood sample was allowed to stand for 60 min at 4°C, followed by centrifugation (3000 r/min for 15 min). Ten, the serum was harvested and inactivated at 56°C for 0.5 h. After fltration, we got the SQW-containing serum and normal serum. All serum was put at −20°C for further in vitro experiments.

Cell Viability Experiment.
Cell counting kit-8 (CCK-8, HY-K0301), supplied from MCE (USA), was earmarked for evaluating the cell viability. Te logarithmic growth period of HK-2 cells (10000 cells/well) was appended to the 96-well plates and put in the incubator for 24 h. After treatment, 10 μL CCK-8 solution was applied to stimulate cells for another 3 h. In the end, a microplate reader (CMaxPlus, MD, USA) was earmarked for examining the absorbance (450 nm).

Real-Time Quantitative PCR (qRT-PCR).
Total RNA extraction kit (R1200), bought from Solarbio (China), was applied to isolate total RNA from cells. Ten, a one-stepSuperRT-PCR mix kit (T2240, Solarbio, China) was applied to conduct the qRT-PCR reaction. Afterward, the signals were examined in a PCR system (EDC-810, Eastwin Life Sciences, Inc.). GAPDH was employed as the normalization control. Te relative levels of gene were counted utilizing 2 −ΔΔCT . Te primers are displayed in Table 1.

Western Blot.
Total protein from cells was extracted with RIPA bufer (abs9229, Absin, China). Te extracted protein was centrifuged and then quantifed with the BCA kit (pc0020, Solarbio, China). After electrophoresis, they were electrotransferred onto the nitrocellulose membrane. Ten, 5% nonfat milk was taken to block the membrane. After rinsing, the blocked membrane was subjected to primary antibodies at 4°C all night. Te next day, an antirabbit secondary antibody (ab7090, Abcam, UK) was added.

SQW-Containing Serum Intensifed the Viability of TGFβ-Mediated HK-2 Cells.
In our research, we frst adopted HK-2 cells treated with 10 ng/mL TGF-β to test the function of SQW-containing serum. CCK-8 experiment clarifed that TGF-β led to the reduction of HK-2 cell viability (Figure 1(a)), P < 0.01). Importantly, the repression of TGFβ on HK-2 cell viability was reversed by SQW-containing serum in a concentration-dependent way (Figure 1(a)), P < 0.05). And diferent dosages of the SQW-containing serum have no impact on the HK-2 cell viability (Figure 1(b)).

SQW-Containing Serum Repressed the Notch1 Pathway, TGF-β, and N-Cadherin Level and Enhanced the E-Cadherin Level in HK-2 Cells Mediated by TGF-β.
In this part, we tested the Notch1 pathway, which is tightly related to RIF. Te western blot elucidated that the intervention of TGF-β led to the upregulation of Notch1, Jag1, HEY1, HES1, and TGF-β and the downregulation of E-cadherin in HK-2 cells, which was partially ofset by SQW-containing serum (Figures 4(a)-4(g), P < 0.05). Besides, we used immunofuorescence assay to assess the fuorescence intensity of the Ncadherin and the E-cadherin, the results reconfrmed that SQW-containing serum elevated the expression of Ecadherin while reduced the N-cadherin in HK-2 cells mediated by TGF-β ( Figure 5(a)-5(b), P < 0.05 or P < 0.01). Simultaneously, we also assessed the fuorescence intensity of Notch1 and Jag1. We discovered that SQW-containing serum weakened the fuorescence intensities of Notch1 and Jag1 in HK-2 cells mediated by TGF-β ( Figure 6, P < 0.05).
Since the high-dose group of SQW-containing serum has the most obvious protective efect on RIF, we chose 10% SQWcontaining serum for follow-up experiments.

Te Impacts of SQW-Containing Serum and siNotch1 on EMT-Related Factors in HK-2 Cells Mediated by TGF-β.
To further confrm that the protective efect of SQWcontaining serum on RIF was correlated with the repression of the Notch1 pathway, HK-2 cells transfected with Notch1 silencing were intervened with 10% SQWcontaining serum and 10 ng/mL TGF-β for 48 h. Notch1 silencing alone or combined with SQW-containing serum strongly alleviated the Notch1, vimentin, N-cadherin, collagen I, and fbronectin levels in HK-2 cells induced by TGF-

Discussion
RIF and the degree of the renal tubular atrophy are signifcant factors determining the severity of diferent forms of renal diseases. Renal tubular epithelial cells (RTECs) are the major targets of renal interstitial damage [30]. Under the stimulation of continuous ischemia and hypoxia, proteinuria, and a variety of profbrotic factors, RTECs secrete multiple chemokines, infammatory factors, and vasoactive factors into the renal interstitium in order to adapt to the changes in the microenvironment so as to promote the infammatory response and fbrosis process of the renal interstitium [14]. TGF-β1 is considered to be a major agonist in the formation of renal fbrosis [31]. Meanwhile, HK-2 itself is also a target of TGF-β1. HK-2 can transform into cells with obvious myofbroblast morphology under the induction of TGF-β1, such as changes in cell polarity, actin flaments, and dense bodies, resulting in excessive deposition of ECM in the renal interstitium and formation of RIF [32,33]. Based on the important role of HK-2 cells and its correlation with TGF-β1, this study stimulated HK-2 cells with TGF-β1 to mimic RIF in vitro, and investigated the anti-RIF efect of SQW-containing serum. And this study demonstrated that SQW can attenuate the TGF-β-mediated EMT process in HK-2 cells to alleviate RIF in vitro. It was reported that during renal fbrosis, RTECs can diferentiate into mesenchymal cells and transform into fbroblasts, becoming a considerable source of ECM [34]. Te transformation of epithelial cells to mesenchymal cells is characterized by the absence of epithelial marker E-cadherin and the increase of mesenchymal marker α-SMA, N- cadherin, vimentin, and fbronectin [35,36]. Fibronectin is one of the main components of the ECM, which can be formed in the early stage of fbrosis and is one of the intuitive indicators of renal fbrosis [37]. N-cadherin is a cadherin expressed in proximal tubules and generates a pivotal function in maintaining the integrity and polarity of RTECs [38]. Under normal circumstances, there is basically no expression of α-SMA in the glomerulus, renal tubules, and renal interstitium, and α-SMA is considered as one of the symbols of EMT in TECs [39]. According to Lu et al.'s research, it is proved that with the induction of the TGF-β1, it notably augmented the expression of α-SMA, but ameliorated the expression of E-cadherin, indicating the completion of transformation from epithelial cells to mesenchymal cells in Madin−Darby canine kidney cells [40]. Consistently, we uncovered that TGF-β led to the downregulation of collagen II and E-cadherin and the upregulation of fbronectin, α-SMA, vimentin, N-cadherin, and collagen I in HK-2 cells. Terefore, inhibition of EMT and ECM can be an efective way to improve RIF.
At present, TCM has been widely utilized as an alternative therapy for kidney diseases. For instance, some scholars clarifed that the Huangkui capsule mitigated renal tubular EMT in diabetic nephropathy in rats through the repression of the NLRP3 infammasome and TLR4/NF-κB pathway [41]. In TGF-β1-inducedHK-2 cells, ganoderic acid weakened the ECM and EMT through the inhibition of the TGF-β/Smad and MAPK pathways [42]. Shan et al. illuminated that Astragalus membranaceus improved RIF via impeding tubular EMT in vivo and in vitro [43]. In our study, we proved the role of SQW in TGF-β-mediated HK-2 cells for the frst time. We discovered that SQWcontaining serum weakened the expressions of fbronectin and collagen I, and this function was accompanied by the suppression of EMT process by restraining α-SMA, vimentin, and N-cadherin levels and elevating the Ecadherin level in HK-2 cells triggered by TGF-β, unveiling that SQW may exhibit an anti-RIF efect by repressing ECM and EMT processes.
It was reported that Notch is also a considerable modulatory pathway for EMT and renal fbrosis [44]. Notch pathway is low or not expressed in the adult normal kidney tissue, but is reactivated in various kidney diseases [45]. Expression of the Notch receptor in RTECs cultured in vitro can drive epithelial cells to develop EMT [46]. In addition, the increase of Notch expression in the tubule epithelium is associated with the continuous increase of TGF-β1 [47]. Inhibition of the Notch1 pathway can alleviate EMT  Evidence-Based Complementary and Alternative Medicine induced by TGF-β1 and ameliorate RIF [48]. Some scholars found that the expressions of Notch1 and Jagged1 were signifcantly upregulated in the kidney tissues of IgA nephropathy, and Notch1, HEY1, and HES1 were signifcantly reduced after administration of the Notch1 pathway inhibitor DAPT, thereby alleviating RIF [49]. Notch pathway inhibitors were found to weaken glomerulosclerosis and RIF by decreasing the expression of Jagged1, Notch1, NICD1, HEY1, HES1α-SMA, and fbronectin in uremic rats [50]. Tus, inhibition of the Notch pathway can alleviate RIF to a certain extent. In this research, our results uncovered that TGF-β led to the upregulation of Notch1, Jag1, HEY1, HES1, and TGF-β in HK-2 cells, which was partially ofset by SQW-containing serum. To further explore whether the Notch1 pathway afects the efect of SQW on RIF, Notch1 silence was transfected into HK-2 cells. We confrmed that Notch1 silencing or cotreatment of SQWcontaining serum with Notch1 silencing mitigated ECM and EMT induced by TGF-β1 and improved RIF via weakening the Notch1 pathway.
In short, this study demonstrated that SQW attenuated the TGF-β-mediated EMT process in HK-2 cells, and its possible mechanism is associated with the inhibition of the Notch1 pathway. However, there are some shortcomings of the study, the overexpression of Notch 1 should be conducted to make the results more convincing, and there is    Figure 6: Te impact of SQW-containing serum on the fuorescence intensities of Notch1 and Jag1 in HK-2 cells mediated by TGF-β. Te impact of SQW-containing serum on the fuorescence intensities of Notch1 and Jag1 in HK-2 cells mediated by TGF-β was examined by immunofuorescence. ▲▲ P < 0.01 vs. control; ★ P < 0.05 and ★★ P < 0.01 vs. TGF-β.  Evidence-Based Complementary and Alternative Medicine a need to verify the mechanism in an animal model. Other potential mechanisms should be explored in subsequent studies to confrm the exact mechanism of SQW anti-RIF.

Data Availability
Te data used to support the fndings of this study are included within this article.

Consent
No written consent has been obtained.

Conflicts of Interest
Te authors declare that they have no conficts of interest. Evidence-Based Complementary and Alternative Medicine 9