Intervention Mechanism of Niao Du Kang Mixture on the EMT Process of Peritoneal Fibrosis Based on the Wnt/β-Catenin Signaling Pathway

Methods Quantification of 24-hour urine protein (24 h-Upro) and serum creatinine (Scr) levels was performed. The protein and mRNA expression levels of E-cadherin, α-SMA, collagen I, β-catenin, Wnt-1, and LEF-1 in peritoneal tissue were measured. In addition, the pathological morphology and ultrastructure of peritoneum were observed. Results After 5/6 nephrectomy + high glucose peritoneal dialysate + lipopolysaccharide (LPS) treatment, the Scr and 24 h-Upro of rats increased compared with normal rats, and the peritoneal tissue was damaged and thickened, showing fibrotic changes. Compared with the model group, the Scr and 24 h-Upro levels and the levels of E-cadherin, α-SMA, Wnt-1, collagen I, and LEF-1 protein expression in each Niao Du Kang mixture dosage group decreased. The protein expression of β-catenin and the mRNA expression of E-cadherin, α-SMA, Wnt-1, collagen I, β-catenin, and LEF-1 decreased in the high and medium Niao Du Kang mixture dose groups. Hematoxylin and eosin staining showed that the peritoneum of the rats was not only thicker in the model group than in the sham operation group (P < 0.01) but also accompanied by apparent inflammatory cell infiltration, tissue edema, and fibrosis. Compared with the model group, all the Niao Du Kang mixture groups demonstrated various degrees of mitigation in peritoneal thickness and fibrosis (P < 0.01). The strongest effect was observed in the medium-dose group. Transmission electron microscopy showed that the degree of injury of the peritoneal mesothelial cells was ranked as follows: model group > positive drug group > Niao Du Kang mixture high-dose group. Conclusions The Niao Du Kang mixture may effectively decrease the peritoneal thickness and fibrosis degree through its effect on the Wnt/β-catenin signaling pathway involved in EMT. The present study provides data that assist in elucidating the potential function of the Niao Du Kang mixture in treating or preventing PF.


Introduction
Peritoneal dialysis (PD) is one of the replacement therapies for end-stage renal disease. However, peritoneal tissue is exposed to dialysis fuid with poor biocompatibility for a long time, and repeated peritonitis can lead to peritoneal damage, resulting in peritoneal fbrosis (PF) [1], lowering dialysis efciency, and eventually leading to ultrafltration failure. Peritoneal fbrosis is closely related to infammation, angiogenesis, and EMT, and these processes interact. EMT plays an important role not only in organ development and wound healing but also in diseases including cancer and tissue fbrosis in the peritoneum. Mesothelial to mesenchymal transition (MMT) in the peritoneal membrane is indicative of its failure and is a trigger for peritoneal fbrosis. During this transition, mesenchymal cells migrate from the superfcial mesenchymal layer to the inferior mesenchymal layer, where they produce the extracellular matrix (ECM) that promotes fbrosis [2]. Activation of the Wnt/β-catenin signaling pathway is thought to play a key role in the EMT process [3].
Previous studies have shown that the Niao Du Kang mixture can signifcantly improve renal function in rats with unilateral ureteral ligation, improve the EMT of renal tubular cells on the ligation side, and signifcantly reduce the degree of renal interstitial fbrosis in unilateral ureteral obstruction (UUO) rats. Te efect is achieved by inhibiting the P38/extracellular regulated protein kinases (ERK) mitogen-activated protein kinases (MAPK) pathway [4]. Te Niao Du Kang mixture can inhibit the expression of phosphoinositol-dependent protein kinase 1 (PDPK1) by upregulating the expression of mir-129-5p and then inhibiting the PI3K/AKT pathway to improve renal fbrosis [5]. Additionally, the Niao Du Kang mixture can improve the dialysis efciency of patients with PD, delay the deterioration of renal function, and inhibit peritoneal fbrosis and renal microinfammation. After three months of clinical observation, it was found that compared with the patients in the peritoneal dialysis group, the levels of fbronectin and transforming growth factor-β1 (TGF-β1) in peritoneal dialysis fuid and the levels of high mobility group-1 (HMGB1), high-sensitivity C reactive protein (hsCRP), and interleukin-6 (IL-6) in serum were lower in patients in the peritoneal dialysis combined with Niao Du Kang mixture group [6]. However, its mechanism of action remains unclear. In the current study, we used 5/6 nephrectomy + high glucose peritoneal dialysis fuid-+ lipopolysaccharide (LPS) method to replicate peritoneal fbrosis in a rat model. Niao Du Kang mixture was used as an interventional drug, and we observed its infuence on the structure of peritoneal tissue and explored whether its intervention mechanism is related to the Wnt/β-catenin signaling pathway. Hitachi 7080 automatic biochemical analyzer (Sekisui Medical Technology Co., Ltd., Japan); Ci-L microscope (Nikon, Japan); ECL gel documentation system (Life Technologies, USA); GET96-PLUS PCR device (Hangzhou Boheng Technology Co., Ltd.); Real-time PCR device (Roche, Switzerland); HT7700 transmission electron microscope (HITACHI, Japan).

Model Preparation and Drug Administration.
Eighty male Wistar rats, adaptively fed for seven days, were randomly divided into the sham operation group, model group, positive drug group, and Niao Du Kang mixture high, medium, and low-dose groups. Te peritoneal fbrosis model was established by 5/6 nephrectomy + high glucose peritoneal dialysate + lipopolysaccharide (LPS). One week after modeling, 4.25% glucose peritoneal dialysate (30 ml/ kg) was intraperitoneally injected once daily for 28 consecutive days, and LPS (0.6 mg/kg) was intraperitoneally injected on days 8, 10, 12, 22, 24, and 26. Intragastric administration of drugs began simultaneously with the intraperitoneal injection of dialysate. Te positive drug group received intragastric administration of Niao Du Qing granules, 2.5 g/kg (converted according to the clinical dosage used in humans), which is a traditional Chinese medicine formula that is used for chronic renal failure. Te high, medium, and low dosage groups received intragastric administration of the Niao Du Kang mixture at 20, 10, and 5 ml/kg, respectively (relative to 12, 6, and 3 times the clinical dosage used in humans), with a gavage volume of 10 ml/kg. Purifed water was provided to the sham operation and model groups that was equal to their volume by gavage for 28 consecutive days.

Detection of Serum Creatinine.
To induce anesthesia, 45 mg/kg of sodium pentobarbital was injected intraperitoneally. Blood was collected from the abdominal aorta, left standing for 1-2 h, and centrifuged at 3000 revs. min −1 for 10 min to separate the serum. Te supernatant was collected and used to measure the level of Scr by the enzymatic method.

Detection of 24 h Urine Protein.
Metabolic cages were used to measure the amount of protein in rat urine. After dry fasting, urine was collected for 24 h. Te total volume of urine within 24 h was recorded, the supernatant was removed after centrifugation, and then, the 24 h-Upro was determined by the midpoint method.

Hematoxylin-Eosin (HE) Staining to Observe the
Pathological Morphology of Peritoneal Tissue. Te fxed peritoneal tissue was dehydrated, embedded in parafn, sliced, and stained with HE. Te peritoneal thickness was observed and measured under a light microscope. Tree peritoneal thicknesses were randomly measured in each visual feld and averaged.

Western Blot Detection of Expression of E-Cadherin, α-SMA, Collagen I, β-Catenin, Wnt-1, and LEF-1 Proteins.
An appropriate amount of peritoneal tissue was removed from storage at −80°C, and the tissue was then ground and centrifuged, and the supernatant was aspirated and used to measure the protein concentration. To prepare the samples, 5x protein loading bufer and normal saline were added, and the samples were transferred into sterile centrifuge tubes for protein denaturation. After the stacking gel and separating gel were prepared and constructed, the samples were loaded to carry out sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with constant circulating membrane and skim milk blocking bufer. Te ratio of the target band to the internal reference gray value was determined using Image J software to represent the expression level of the tested protein.

Real-Time PCR Detection of E-Cadherin, α-SMA, Collagen I, β-Catenin, Wnt-1, and LEF-1 mRNA Expression.
Te total RNA of ileal tissue was extracted with TRIzol reagent, the RNA concentration of each sample was measured, and RNA reverse transcription was performed. Te DNA concentration was measured after reverse transcription. Once confrmed, 1 μL of DNA was added to the mixture, and then, the contents were mixed with the centrifuge tube lid closed. Te level of mRNA expression in each group was determined using the delta-delta Ct method.

Observation of the Ultrastructure of the Peritoneal
Tissue by Transmission Electron Microscope. Te peritoneal tissue, fxed in 2.5% glutaraldehyde solution, was removed, embedded, polymerized, sliced, double-stained with uranyl acetate and lead citrate, observed under a transmission electron microscope, and recorded for image analysis.
2.6. Statistical Analysis. SPSS 19.0 was used for data analysis. Te measurement data are presented as x ± s. One-way analysis of variance was used for comparison among groups. P < 0.05 indicated that the diference was statistically signifcant.

Te Efect of the Niao Du Kang Mixture on the Levels of Scr and 24 h-Upro in Peritoneal Fibrosis
Rats. Compared with the levels of Scr and 24 h-Upro in the sham operation group, those in the model group signifcantly increased (P < 0.01). Compared with the levels of Scr and 24 h-Upro in the model group, those in the Niao Du Qing granules and Niao Du Kang mixture groups signifcantly decreased (P < 0.01) (see Table 1).

Te Efect of Niao Du Kang Mixture on the Pathological Morphology and Tickness of the Peritoneum in Rats with
Peritoneal Fibrosis. HE staining showed that the peritoneum of the rats in the sham operation group was thinner, and there was no apparent infammatory cell infltration or tissue edema. Te peritoneum of the rats in the model group was signifcantly thicker than that of rats in the sham operation group (P < 0.01), accompanied by apparent infammatory cell infltration, tissue edema, and fbrosis. Compared with the model group, all the groups that received the Niao Du Kang mixture demonstrated various degrees of mitigation in peritoneal thickness and fbrosis (P < 0.01). Te mediumdose group demonstrated the most optimal efect among all the groups that received the Niao Du Kang mixture (see Figure 1 and Table 2).

Te Efect of the Niao Du Kang Mixture on the Expression of E-Cadherin, α-SMA, Collagen I, β-Catenin, Wnt-1, and LEF-1 Proteins in the Peritoneal Tissue of Peritoneal Fibrosis
Rats. Te levels of E-cadherin, α-SMA, collagen I, β-catenin, Wnt-1, and LEF-1 protein expression were signifcantly higher in the model group than in the sham operation group (P < 0.01). Te levels of E-cadherin, α-SMA, collagen I, Wnt-1, and LEF-1 protein expression were signifcantly lower in the Niao Du Qing granule and Niao Du Kang mixture groups than in the model group (P < 0.01, P < 0.05). Te levels of β-catenin protein expression in the Niao Du Qing granule group and the Niao Du Kang mixture high and medium-dose groups were signifcantly lower (P < 0.01, P < 0.05) (see Figures 2 and 3).

Efects on the Ultrastructure of Peritoneal Tissue.
Peritoneal mesothelial cells in the model group displayed the most severe damage. Te peritoneal mesothelial cells in the positive drug group displayed the second most severe damage. Te Niao Du Kang mixture high-dose group displayed the least damage of peritoneal mesothelial cells, in which the cell membrane was intact, the cells were slightly swollen, the basement membrane was intact, and the intercellular space was tight (see Figure 5).

Discussion
PF occurs through two pathological mechanisms: retroperitoneal fbrosis and infection and infammation caused by non-biocompatible solutions. Te peritoneum is a single layer of continuous epithelial cells with regenerative properties, namely, peritoneal mesothelial cells (PMCs). In the early stage of PF, PMCs possess enhanced abilities for invasion and migration and transform into myofbroblasts, which induce the deposition of the ECM in the mesothelial area. During a long period of PD therapy, the integrity of the patient's peritoneum is compromised. Myofbroblasts have been observed in the PF experimental model and in biopsy specimens of PD patients, and EMT-transformed myofbroblasts have also been observed in the peritoneal mesothelial region [7,8]. Aroeira et al. used glucose dialysate to establish a PF model, in which partial exfoliation of PMCs was observed after two weeks. PF and angiogenesis occurred after fve weeks, accompanied by peritoneal ultrafltration failure. Fluorescent cytokeratin and α-SMA were used to highlight the characteristics of PMCs and myofbroblasts, demonstrating that PMCs undergo EMT [9]. TGF-β1 is considered the most crucial cytokine involved in organ fbrosis during EMT. TGF-β1 can activate various signaling pathways in the body to exert biological efects, with the TGF-β1/Smads pathway being the most important one. In 2002, it was frst discovered in a rat renal fbrosis model that Wnt/β-catenin signal transduction regulates organ fbrosis [10]. Studies have shown that the Wnt/ β-catenin and TGF-β1/Smads signaling pathways interact and inter-regulate. TGF-β1 signifcantly upregulates Wnt/ β-catenin and its downstream target genes and silences β-catenin. Dkkl inhibits the hypersecretion of TGF-β1 and afects the transcription of the Smad protein, meaning that the fbrotic process that is regulated by TGF-β1 requires  β-catenin signaling activation [11]. In an obstructive renal injury rat model, the expression of most Wnt proteins was upregulated, which produced a large volume of FZD receptors and Wnt antagonists. Furthermore, β-catenin transcription was enhanced, and nuclear translocation was observed in the renal tubular epithelial cells and renal interstitium. Obstructing the Wnt/β-catenin signaling pathway can signifcantly downregulate the expression of α-SMA, which results in the inhibition of EMT and renal fbrosis [12]. Te purpose of the current study is to investigate whether the intervention mechanism of the Niao Du Kang mixture that afects the EMT process of peritoneal fbrosis is related to the Wnt/β-catenin signaling pathway based on animal experiments. Experimental studies have shown that [13] astragaloside IV (AS-IV) derived from A. membranaceus attenuates high glucose-induced EMT by inhibiting the TGF-β1/Smads pathway in renal tubular epithelial cells (PTCs). In mice with UUO, emodin can downregulate the expression of TGF-β1 and P-SMad3, decrease the production of fber markers (including collagen I, collagen III, β-catenin, and α-SMA), and relieve renal interstitial fbrosis [14]. Salvia may inhibit the expression of collagen and fbronectin that are related to the TGF-β1/Smad pathway, thereby decreasing the hypertrophy and dilation of renal tubules and glomeruli [15]. One of salvia's active ingredients, tanshinone, was shown to attenuate TGF-β1-induced fbrosis in rat fbroblasts and attenuate induced pulmonary fbrosis [16]. Te active ingredient of safower (hydroxysafor yellow A) can decrease the expression level of the TGF-β1 protein in lung tissue and inhibit bleomycin-induced pulmonary fbrosis [17]. Treatment with safower yellow signifcantly downregulated cell proliferation, migration, and the expression of p-ERK1/2, AP-1, collagen I, and collagen III. Safower yellow exhibits  Evidence-Based Complementary and Alternative Medicine anti-proliferative, anti-migratory, and pro-apoptotic activities in rat aortic adventitial fbroblasts [18].

Conclusions
In this experiment, a rat peritoneal fbrosis model was used to simulate patients with peritoneal fbrosis caused by long-term peritoneal dialysis. Tis experiment has proven that the Niao Du Kang mixture can efectively protect the residual renal function of model rats and reduce the peritoneal thickness and the degree of fbrosis. Te Western blot and RT-PCR results suggested that the Niao Du Kang mixture might protect the morphological structure of peritoneal mesothelial cells, inhibit the EMTprocess of peritoneal epithelial cells, and protect the peritoneal function of peritoneal dialysis patients through infuencing the Wnt/β-catenin signaling pathway on EMT. In conclusion, the Niao Du Kang mixture may inhibit the EMT process of peritoneal epithelial cells through the Wnt/β-catenin signaling pathway.

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