Polysaccharides extracted from balanophora polyandra Griff (BPP) ameliorate renal Fibrosis and EMT via inhibiting the Hedgehog pathway

Abstract Renal interstitial fibrosis (RIF) is a crucial pathological change leading to chronic kidney disease (CKD). Currently, no effective medicines have been available for treating it. In our research, we examined the effects of polysaccharides extracted from Balanophora polyandra Griff (BPPs) on kidney fibrosis and epithelial to mesenchymal transition (EMT) in vivo and in vitro, aiming to explore the underlying mechanisms. By using the mice with unilateral urethral obstruction (UUO) as experimental subjects, we examined the medicinal values of BPPs on alleviating RIF. The effects of BPPs were evaluated by examining the histological staining and relative mRNA and protein expression levels of the related genes. The possible underlying mechanisms were further explored with human normal renal proximal tubular epithelia (HK‐2 cells) as in vitro model. In UUO mice, BPP treatment could significantly alleviate interstitial fibrosis through reducing the components (Collagens I, III and IV) of extracellular matrix (ECM), and reducing the activation of fibroblasts producing these components, as revealed by inhibiting the hallmarks (fibronectin and α‐SMA) of fibroblast activation. Furthermore, BPP administration increased the expression levels of matrix metalloproteinases (MMPs) and declined those of tissue inhibitors of metalloproteinases (TIMPs). BPPs markedly ameliorated EMT in both the kidneys of UUO mice and TGF‐β1 treated HK‐2 cells. Moreover, BPP treatment decreased the expression levels of several transcriptional factors involved in regulating E‐cadherin expression, including snail, twist and ZEB1. Additionally, the Hedgehog pathway was found to be closely correlated with renal fibrosis and EMT. Altogether, our results clearly demonstrated that BPP treatment effectively inhibited the Hedgehog pathway both in renal tissues of UUO mice and TGF‐β1‐treated HK‐2 cells. Thus, BPPs ameliorated RIF and EMT in vivo and in vitro via suppressing Hedgehog signalling pathway.


| INTRODUC TI ON
Chronic kidney disease (CKD) has become one of the important problems in global public health, as it can progress to end-stage renal disease (ESRD), thus, affecting billions of people's health in the world. 1 One of the important histopathologic changes in CKD is renal tubulointerstitial fibrosis characterized by deposition of large amounts of extracellular matrix (ECM), infiltration of different immune cells, disappearance of tubular epithelial cells and production of myofibroblast, leading to progressive renal impairment. [2][3][4] Matrix metalloproteinases (MMPs) entail a set of proteinases, namely matrilysins, stromelysins, gelatinases, collagenases and membrane-type MMPs, 5 which degrade the components of glomerular basement membrane (GBM) and ECM. The activities of MMPs are regulated by a series of mechanisms, including inhibition by tissue inhibitors of metalloproteinases (TIMPs). 6,7 During the pathogenesis of fibrosis, epithelial to mesenchymal transition (EMT) occurring in epithelia of renal tubule is a part of renal fibrosis. EMT is mainly manifested by the change of cellular phenotype of epithelia, such as the decreased expression of E-cadherin, and the acquirement of interstitial cellular phenotype, such as the elevated expression of N-cadherin. Growing lines of evidence indicate that EMT plays a key role in the activation of renal interstitial myofibroblasts and that EMT may be the promising therapeutic targets for preventing progression of renal fibrosis. 8,9 Hedgehog signalling pathway is present in all bilaterians. In mammals, this pathway is involved in signal transduction in embryonic cells required for proper cell differentiation. Thus, this pathway is one of the key regulators for the animal development. The hedgehog protein has at least three important protein ligands, namely, Sonic hedgehog (Shh), Indian hedgehog (Ihh) and Desert hedgehog (Dhh), which are embellished by lipid. They perform functions by the way of paracrine and autocrine in their secreting areas. The Shh signalling pathway is an evolutionarily conservative pathway involved in numerous biological and pathological processes, including embryonic development, tissue repair and even tumorigenesis and development. 10,11 It has been shown that Shh signalling pathway is highly activated during the pathogenesis of fibrotic diseases, suggesting the possible existence of a potential relationship between organ fibrosis and abnormality of Shh signalling. [12][13][14] Recent studies have manifested that Shh signalling pathway was also activated after renal injury and became an important mediator for progressive renal fibrosis. 12,15 Gli transcriptional factors include Gli1, Gli2 and Gli3, generally, only Gli1 can play a role in activating Shh signalling.
Gli1 is a direct downstream target and receptor for Shh signalling, leading to the specific induction of renal interstitial fibroblasts (RIF). Additionally, Gli1 deficient mice could be avoided from the development of UUO-induced RIF. 12,14 It was demonstrated that of Gli1, the RIF severity was alleviated. 12,14 Therefore, blocking the activation of Shh signalling can be helpful to inhibit RIF and prevent CKD progression.
Balanophora polyandra Griff, belonging to the family Balanophoraceae, is a natural medicinal mushroom living in association with the root system of many different host plants, especially the evergreen shrubs in Hubei, Hunan and Yunnan provinces in China. 17 It has been demonstrated that the chemical components of Balanophora polyandra Griff include phenylpropanoids, flavonoids, tannins and polysaccharides etc, which have medicinal functions, such as anti-inflammation, liver protection, anti-oxidation and anti-cancer as well as other biological effects. 18 More recently, we found that Balanophora polyandra Griff polysaccharides (BPPs) significantly inhibited the proliferation of ovarian cancer cells via P53mediated pathway. 19 However, to date, there have been no reports about the influence of BPPs on renal fibrosis and the underlying mechanisms. These deficiencies prompted us to investigate their medicinal effects on renal fibrosis in both TGF-β1-reduced HK-2 cells in vitro and in kidneys of the unilateral ureteric obstruction (UUO) mice in vivo. Furthermore, we also investigated the effects of BBPs on EMT, degradation and accumulation of ECM components and the expression levels and activities of several important transcriptional factors involved in Shh signalling pathway. Our results indicated that BPPs could prevent the activation of Shh signalling pathway and thus inhibit the EMT, and finally ameliorate kidney fibrosis.

| Plant materials, extraction and the content of polysaccharides
The whole plant of Balanophora polyandra Griff was collected from Wufeng county, Hubei province, China in 2019. The sample (2W19080802) was stored in National Level-3 Research Laboratory of Chinese Traditional Medicine Pharmacology. Preparation and determination of polysaccharide extracts were performed as previously described by Ju et al. 19 Briefly, the dried whole plant (300 g) was chopped into small pieces and extracted with 2.0 L of distilled water by heating at 95℃ for 2.5 hours. This extraction was repeated for three times. After filtering with multiple layers of gauze, the filtrate was centrifuged at 1500 g for 10 minutes, and the supernatant K E Y W O R D S Balanophora polyandra Griff, EMT, hedgehog signalling pathway, matrix metalloproteinases, renal fibrosis fluid was collected, concentrated to 1.5 L, and precipitated with 2.5 volume of 95% ethanol (EtOH) at 4℃, overnight. Subsequently, the precipitate was dissolved in distilled water. The proteins were removed with the Sevag reagent (chloroform/butanol, 4:1). After freeze-drying of the deproteinized solution, approximately 26.5 g of polysaccharide extract was obtained, which was stored at −20°C for subsequent experiments. The total carbohydrate content of the polysaccharide extract was determined with phenol-sulphuric acid methods to be 54.0% (g/g) and expressed as anhydrous glucose equivalent. The detailed extraction procedures of BBPs and preliminary analysis of chemical compositions had been described in our lab's previously published papers. 19,20

| Cell culture
Human renal proximal tubular epithelial cells (HK-2 cells) were acquired from the Cell Bank of Chinese Academy of Sciences. The Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% foetal bovine serum (FBS) (Invitrogen) was used for cell culture. These cells were pre-treated with serum-free DMEM containing BPPs at gradient concentrations (25,50 and 100 μg/mL) for 24 hours, respectively. The doses were chosen according to our previous studies. 19,20 Thereafter, the cells were then induced with TGF-β1 at 10 ng/mL for 24 hours and collected after being detached by digestion with 0.5% trypsin for 2 minutes. The un-stimulated cells were used as the negative control group. GDC-0449 (Vismodegib), a specific Hedgehog inhibitor, was used at 100 nM, which served as the positive control group.

| Histopathologic evaluation
After being fixed with 4% paraformaldehyde in 0.1M PBS for 24 hours, the isolated renal tissues were conventionally embedded in paraffin. Paraffin sections of renal tissue (4 μm) were dewaxed by xylene, dehydrated by gradient ethanol (100%, 95% and 70% ethanol), and then stained with H&E. The changes in renal pathological parameters were examined under the light microscope (Olympus, BX61).

| Masson's trichrome staining
The Masson's staining was applied in the tissue slices according to the directions of Masson's trichrome staining kit (Beyotime Institute of Biotechnology, Haimen). The bluely stained areas were quantitatively analysed by Image Pro Plus6.0 software. Twenty different visual fields were randomly selected from each section, and blue area was taken as the positive staining. The ratio of the positive area to the total field of vision was taken as the renal fibrosis index.

| Immunohistochemistry detection
The immunohistochemical staining was employed in the paraffin-embedded renal tissue sections. The sections from kidney (4 μm) were dewaxed by xylene, dehydrated by gradient ethanol, and co-incubated with 0.01 M citrate buffer (pH 6.0) for antigen retrieval. The endogenous peroxidase was inactivated with 3% H 2 O 2 and the sections were blocked using 5% bovine serum albumin (BSA) at 25℃ for 1 hour.
Afterwards, slices were incubated with primary antibodies specific to α-SMA (Santa Cruz Biotechnology, sc-53142, 1:500) and fibronectin (Santa Cruz Biotechnology, sc-29011, 1:500) at 4℃ overnight. The slices were washed with a PBS buffer, and incubated with corresponding secondary antibodies at 25°C for 1h. The slices were stained with freshly prepared diaminobenzidine (DAB) solution. After being counterstained with haematoxylin, the sections were differentiated with hydrochloric acid alcohol, dehydrated with gradient ethanol, and transparentized with xylene. Finally, the positive expressions of α-SMA and fibronectin were observed under the microscope. Ten different fields were randomly picked from each slice, and the relative positively stained area was acquired using Image Pro Plus 6.0 software.

| Quantitative real-time PCR
The total RNA was extracted from mouse renal tissue and HK-2 cells with Trizol reagent (Invitrogen) by following the instructions. The concentration of total RNA was assayed with Nanodrop at 260 nm and its purity was evaluated by the ratio of OD 260nm /OD 280 nm.
The RNA integrity was detected by running electrophoresis of RNA samples on 1.2% agarose gel after treatment with DNase. 2 μg total RNA was used for the reverse transcription to synthesize template cDNA. Real-time PCR assay was performed as follows: initial reaction at 95℃ for 10 minutes, then followed by 40 cycles of 95℃ for 15 seconds and 60℃ for 1 minutes. The 2 -ΔΔCt formula was used for the analysis of expression levels of related genes, and GAPDH was taken as the internal control. The sequences of specific primers for RT-PCR were listed in the supplementary Table S1.

| Western blot analysis
The renal tissues or HK-2 cells were homogenized with radioimmunoprecipitation assay (RIPA) buffer containing phenylmethylsulfonyl fluoride (PMSF, 100 mmol/L) and inhibitor of protein phosphatases.
The homogenate was centrifugated at 12 000 g and 4℃ for 15 minutes, and then the supernatant was saved and stored at −80℃.
Protein content was assayed with Bicinchoninic Acid (BCA) protein kit. A total of 50 g protein sample was diluted with 5 x loading buffer and denatured by heating at 100℃ for 5 minutes. The denatured protein samples were loaded and electrophoresed via sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), then transferred to polyvinylidene difluoride (PVDF) membrane, which were soaked in 5% skim milk for 1h, then incubated with corresponding primary antibody at 4℃ overnight. The membranes were washed with Tris-buffered saline, 0.1% Tween® 20 detergent (TBST) buffer for 3 times (10 minutes for each), and then incubated with the corresponding horseradish peroxidase-labelled secondary antibody at room temperature for 1h. Lastly, after being washed, the membrane was developed with enhanced chemiluminescence (ECL) reagent and then protein bands were visualized. The expression level of β-actin was employed for the internal normalization of each proteins. The relative expression level was expressed as the folds of induction or folds of suppression in relative to that of the corresponding control group.

| Statistical analysis
All the experimental data were expressed with mean ± standard deviation (SD). The experimental data were processed and analysed using softwares, including Image-proplus 6.0, GraphPad Prism 7 and SPSS13.0 software,respectively. The significance of the difference in the average values between groups was analysed by one-way ANOVA and t-test, and the difference between groups with P < .05 was considered statistically significant.

| Effects of BPPs on kidney morphology in UUO mice
In comparison with the sham group, the H&E staining of renal tissues of mice in the UUO group revealed obvious tubular dilation, atrophy and inflammatory infiltration in the renal interstitial area.
Meanwhile, the Masson staining results also showed more obvious collagen deposition in the obstructed kidneys ( Figure 1A and B).
With BPP administration, the renal tubular injury was improved as reflected by the gradual reduction of interstitial damage scores in both UUO + BPP-L and UUO + BPP-H group. The collagen deposition was also remarkably declined dose-dependently. The improved renal tubular injury and the declined collagen deposition in UUO-BPP-H group were closer than UUO-BPP-L group to that of the positive control (UUO + CPN) group ( Figure 1A and B).

| Effects of BPP on the expression Levels of MMP2, MMP9, TIMP1 and TIMP2 in the renal tissues of UUO mice
The ECM degradation is mainly catalysed by MMPs, including MMP2 and MMP9, which are related to renal fibrosis. On the other hand, the activation of MMPs is regulated by TIMPs, such as TIMP1 and TIMP2. Thus, the balance between accumulation and disposition of ECM is regulated by the relative activities of MMPs and TIMPs. Above results have already proven that BPP could inhibit ECM deposition. Therefore, we next determined the effects of BPP on the levels of

| Effects of BPP on the EMT in the renal tissues of UUO mice
It has been shown that the EMT has a potential effect on RIF development. 8

| Effects of BPPs on the expression levels of Shh, Gli1 and Smo involved in SHH signalling pathway in the renal tissues of UUO mice
Recent studies have demonstrated that the SHH signalling pathway is activated after renal injury and is a crucial mediator of the

| Effects of BPP on ECM accumulation and EMT in TGF-β1-treated HK-2 cells
It has been documented that TGF-β1 may be the trigger of the ECM accumulation and inducer of EMT in the fibrotic kidney. 21

| DISCUSS IONS
RIF is one of the main causes for the renal failure. However, effective medicines for treating RIF are currently unavailable and needed. Thus, in this study, to confirm the renal anti-fibrosis effect of BPP, we investigated the effects of BPPs on the renal tubulointerstitial injury score, fibroblast activation, collagen expression and ECM accumulation in UUO mice. Our results clearly showed that BPPs could ameliorate renal interstitial damage dose-dependently.
Especially, the interstitial injury score of the mice in BPP-H (450 mg/ kg.d) group was significantly decreased as compared with that of UUO group and was nearly close to that of the positive control group. To gain significant insights into the cellular and molecular mechanisms underlying the anti-fibrotic effects of BPPs, we investigated the effects of BPPs on the mRNA and protein expression levels of genes involved in ECM deposition and degradation, EMT and SHH signalling pathway in renal tissue of UUO mice. We also confirmed and extended the in vivo results of the effects of BPP on genes involved in ECM accumulation, EMT and SHH signalling pathway in TGF-β1-treated HK-2 cells. These experiments led us to make several interesting and important findings.
Firstly, we found that BPPs significantly reduced the accumulation and deposition of tubulointerstitial ECM components, including collagens I, III and IV, and the hallmarks of fibroblast activation, fibronectin (FN) and α-SMA. It is well known that the accumulation and deposition of ECM is the fundament of renal fibrogenesis. 22 The Masson's trichrome staining results showed that BPP administration remarkably reduced the Masson's staining positive region and accumulation of tubulointerstitial ECM (Figure 1 A and B). Fourthly, we found that BBP treatment could dose-dependently and significantly suppressed the levels of Shh, Gli1 and Smo, the important factors involved in the SHH signalling pathway, and enhanced the levels of Ptch1, the inhibitor of Shh signalling pathway. More and more studies have reported that Shh signalling pathway plays a crucial role in many diseases. 28,29 The functions of the Shh signalling pathway in the occurrence and development of CKD have also been studied extensively. Recent research has already shown that abnormal activation of the Shh signalling pathway could lead to renal fibrogenesis. 30 Additionally, several other groups have also confirmed that the Shh signalling pathway is abnormally activated during the EMT process. 31 Balanophora polyandra Griff is a natural medicinal mushroom and has been known to have numerous medicinal values. More recently, our group has demonstrated that BPPs significantly inhibited ovarian cancer cell proliferation via a P53-mediated pathway. 19 The present study clearly demonstrated that BPPs could also significantly ameliorate kidney fibrosis and EMT via inhibiting the Shh signalling pathway. These studies indicate the nutritional and medicinal values and potential applications of Balanophora polyandra Griff is as a functional food in clinical treatment for patients with chronic kidney disease and cancer. This prospective is worthy for further exploration.
The doses of Balanophora polyandra Griff crude drug reported in Chinese literature are in the range of 9-15 grams. We used the maximum human dose (15g/70kg) to calculate the conversion ratio of the body surface area between the human body (70kg) and the mouse (20g), which is 387.9, that is, the equivalent dose (mg/g) of the mouse is 38.67mg/20g. In addition, we calculated that the extraction rate of total extract of Balanophora polyandra Griff polysaccharides (BPP), which was 0.528. Therefore, the maximum dose of BPP in mice was calculated by the drug extraction rate, which was 1019.04 mg/kg. Therefore, we determined that the highest dose of BPP was 1000 mg/ kg while the lowest dose was 1/4 of the high dose, which was 250mg/ kg. Finally, according to the experimental results of animal tolerance, the dose of BPP was adjusted to: BPP-L, low dose of BPP (150mg/ kg.d); BPP-H, high dose of BPP(450mg/kg.d).

| CON CLUS IONS
In conclusion, these in vitro and in vivo experiments preliminarily demonstrated that BPP treatment could inhibit the hedgehog signalling pathway and EMT induction, resulting in the reduced ECM accumulation in the kidneys. These results also implicate that BPPs could be used as a potential therapeutic agent for preventing kidney fibrosis. Obviously, its potential applications in clinical treatment of chronic kidney disease still need further exploration.

ACK N OWLED G EM ENTS
This study was co-financed by the National Natural Science

CO N FLI C T O F I NTE R E S T
There is no declaration of any conflict of interest.