Oxidative Stress and TGF-β1/Smads Signaling Are Involved in Rosa roxburghii Fruit Extract Alleviating Renal Fibrosis

Fibrosis is involved in the pathogenesis of kidney diseases. We previously discovered that Rosa roxburghii fruit (Cili) possesses antifibrosis property in chronic renal disease, but the mechanisms are unknown. We hypothesized that Cili might prevent fibrosis development through mediating TGF-β/Smads signaling, which is known to be involved in renal fibrosis. This study aimed to confirm the effects of freeze-dried Cili powder in a rat model of unilateral ureteral obstruction (UUO) and examine TGF-β/Smads signaling. Rats were randomized to (n=12/group): sham operation, UUO, UUO with losartan, UUO with moderate Cili dose (3 g/kg/d), and UUO with high Cili dose (6 g/kg/d). The rats were sacrificed after 14 days of treatment. Collagen deposition was tested using Masson's staining. TGF-β/Smads signaling was examined by qRT-PCR, western blot, and immunohistochemistry. Rats in the UUO group showed excessive deposition of collagen in kidney interstitium, accompanied with high levels of renal 8-hydroxy-2′-deoxyguanosine, renal malondialdehyde, blood urea nitrogen (BUN), serum creatinine (Scr), and proteinuria (all P<0.05). Cili powder efficiently alleviated the pathological changes and oxidative stress in the kidneys of UUO rats, and decreased BUN, Scr and proteinuria (all P<0.05). Cili powder also inhibited the upregulation of TGFB1, TGFBR1, TGFBR2, SMAD2, and SMAD3 and reversed the downregulation of SMAD7 in obstructed kidneys (mRNA and protein) (all P<0.05). In summary, the results suggest that Cili freeze-dried powder effectively prevents renal fibrosis and impairment in UUO rats, which is associated with the inhibition of oxidative stress and TGF-β1/Smads signaling.


Introduction
Renal fibrosis is a consequence of progressive kidney diseases such as chronic infection, obstruction of the ureters, hypertension, and diabetic nephropathy [1,2]. Renal fibrosis is characterized by tubulointerstitial fibrosis and glomerulosclerosis, both caused by the excessive deposition of collagens and extracellular matrix (ECM) components. Although the kidney can regenerate after acute injury, renal fibrosis is usually irreversible [3].
The traditional Chinese medicine (TCM) is based on a personalized approach aiming to restore the balance between Yin and Yang [10,11]. There are hundreds of TCM formulation against chronic kidney diseases (CKD) [12,13]. Some TCM components were studied (including Astragalus, Angelica sinensis, Rheum plantarum, Radix bupleuri, Cordyceps sinensis, and Tripterygium wilfordii [11]) and their effects include anti-inflammation, antioxidation, antifibrosis, anticoagulation, and immune system modulation [13,14]. On the other hand, some TCM components can be toxic to the kidney [11,15], hence the importance of characterizing them carefully.
Rosa roxburghii is a member of the rose family and is a wild plant from the Guizhou Province and distributed in south China. In TCM, the fruit of Rosa roxburghii (named Cili or CL) improves immune response, enhances digestive ability, and possesses antiaging effects [16]. Chemical analysis revealed that Cili is rich in antioxidants including superoxide 2 Evidence-Based Complementary and Alternative Medicine dismutase (SOD), vitamin C, vitamin E, and flavonoids [17,18]. Cili has a long history in China and has been proven to possess antioxidant [19,20], antiatherosclerosis [21], antitumor [22], and radioprotective [23] effects. Nevertheless, the exact molecular mechanisms responsible for these beneficial effects are not well understood.
Our preliminary data showed that freeze-dried Cili powder improved renal fibrosis and indexes of oxidative stress in 90 patients with stages 3-4 renal failure [24], but the exact mechanisms responsible for these effects of Cili on kidney diseases are unknown. We hypothesized that Cili could prevent the development of renal fibrosis through mediating TGF-/Smads signaling, which is known to be involved in renal fibrosis [4][5][6]. Therefore, this study established a renal fibrosis rat model induced by unilateral ureteral obstruction (UUO) in order to confirm the effects of freeze-dried Cili powder on renal fibrosis and examine TGF-/Smads signaling.

Materials and Methods
. . Animals. Sixty specific pathogen-free-(SPF-) grade male Sprague-Dawley rats (140-160 g) were provided by Shanghai Sippr/BK Laboratory Animal Ltd., Co. (Shanghai, China; certificate number: DA942C48-72F76A1E-466ABC83-FC6974). The rats were kept at the Experimental Center for Science and Technology, Shanghai University of Traditional Chinese Medicine, under controlled temperature (25 ∘ C), humidity (45-55%), and light (12 h light/dark cycle). The animals were allowed free access to food (standard chow) and water. All rats were kept one week before experiments. All experimental procedures were performed according to the local Animal Care Committee's guidelines.

. . Unilateral Ureteral Obstruction (UUO) Modeling and
Treatment. According to the principles of sample size estimation in Chinese medicine pharmacology research [25], "When using small animals (mice or rats) to carry out the experiments and comparing categorical data among 3-5 groups, the number of animals in each group should be no smaller than 8-10". Some other studies with a similar methodology also used 8-10 rats/group [26,27]. Being prudent, a sample size of 12 rats/group was used in the present study. The rats were randomized to (n=12/group): sham operation group (Sham), UUO group (UUO), UUO rats treated with losartan (UUO+losartan), UUO rats treated with moderate dose of Cili freeze-dried powder (UUO+CL moderate), and UUO rats that received high dose of Cili freeze-dried powder (UUO+CL high). Rats that underwent UUO and were treated with resveratrol were used as a positive control group.
UUO was performed as previously described [28]. Briefly, animals were anesthetized with 2% pelltobarbitalum natricum (0.6-0.8 mL/rat). The left kidney was exposed and separated through a 2-3 cm flank incision. The left ureter was ligated at two locations with 4-0 silk and cut between the two ligatures. In the sham-operated rats, the left ureter was separated but not ligated or cut.
All rats were treated on the next day after successful modeling. The conversion formula for the drug dose in rats (in g/kg) is [clinical dose in humans, in g/kg]×6.2 [29]. Our previous study found that the equivalent dose for patients with chronic renal failure at stages 3-4 was about 40 g/day of freeze-dried powder of Rosa roxburghii fruit (Pharmacy Department, The First Affiliated Hospital, Guiyang College of Traditional Chinese Medicine) by oral administration [24]. The reference human body weight is about 60 kg [29]. Therefore, the equivalent dose for rats using this kind of Cili freeze-dried powder should be 40 g/60 kg × 6.2 =4 g/kg. The content of active ingredients (flavonoids) is twice higher in commercially available Cili freeze-dried powder (GuizhouJincili Technology Development Co., Ltd., Guizhou, China) compared with home-made preparation, due to optimized manufacturing processes [30]. Hence, the equivalent dose for rats using the commercially available Cili freeze-dried powder is 2 g/kg. In the present study, the dose of Cili freeze-dried powder (Guizhou Jincili Technology Development Co., Ltd., Guizhou, China) was 3 g/kg/d for the UUO+CL moderate group (1.5 times the equivalent dose) and 6 g/kg/d for the UUO+CL high group (three times the equivalent dose). The rats in the sham and UUO groups were given the same volume of sterile saline. Losartan (losartan potassium, lot: L037309; Merck Frost, Montreal, Canada) was given by oral gavage at 10 mg/kg/d (20% concentration). All experimental procedures and care were performed at the same time, once per day, for 14 days. According to previous studies [31,32], early renal fibrosis is observed in UUO rats from 3 days after surgery, most of the quantitative pathological changes occur about 7 days after surgery, and obvious renal fibrosis is observed on the 14th day after surgery. Therefore, 14 days of treatment was used for the prevention of renal fibrosis in the present study. According to similar studies [33,34], 14 days of traditional Chinese medicine intervention in UUO model rats showed treatment effectiveness.
The rats were sacrificed on day 14 after gavage under general anesthesia with 3.5% isoflurane. Both kidneys were collected, rinsed with ice-cold saline, dissected and frozen in liquid nitrogen or fixed in 5% PBS-buffered formalin, rinsed with phosphate buffer, dehydrated using a graded series of ethanol and xylene, and embedded in paraffin.
. . Histopathology. Paraffin sections (4 m thick) were stained with hematoxylin and eosin (HE) for histopathological examination. Kidney fibrosis was evaluated by Masson's trichrome staining, as described previously [35]. The amount of collagen-positive (blue) pixels in the stained sections was quantified by automatic analysis using the Image-Pro Plus 6 software (Media Cybernetics, Rockville, MD, USA). Eight random fields on each section were selected for analysis. The area of interest (AOI) was acquired based on the total amount of stained tissue per high-power field. Integrated optical density (IOD) of blue pixels was determined and the percentage of blue pixels in the AOI was automatically calculated.
. . Immunohistochemistry. Paraffin-embedded kidneys were sectioned at 4 m. The sections were air-dried, dewaxed, and rehydrated in graded ethanol and phosphate buffered Three to five sections of each specimen were used for immunohistochemistry. A qualitative analysis was first performed; according to the location of the specific protein in the control group (e.g., the cortical part), the same location was analyzed in the treatment groups. Semiquantitative analysis was then conducted using eight randomly selected nonoverlapping fields (×400) from each section. The quantitative analysis was conducted using Image Pro Plus 6.0.
.  . . Biochemistry. The serum levels of blood urea nitrogen (BUN) and creatinine (Scr) were detected with assay kits from the Nanjing Jiancheng Bioengineering Institute (C013-1 for BNU and C011-1 for creatinine; Nanjing, China), according to the manufacturer's instructions. Twenty-four-hour urine samples were collected using stainless steel metabolic cages at indicated time points. Urine was centrifuged at 12,000 ×g for 15 min at 4 ∘ C and the supernatant was used for detection. Urine protein (Upro) concentrations were determined using a urinary protein assay kit (C035-2; Nanjing Jiancheng Bioengineering Institute; Nanjing, China).
. . Statistical Analyses. All data were verified using the Kolmogorov-Smirnov test and were found to follow the normal distribution. The results were expressed as means ± standard deviation. The differences among the groups were analyzed using one-way analysis of variance (ANOVA) followed by the least significant difference (LSD) post hoc test. All analyses were performed using SPSS 18.0 (IBM, Armonk, NY, USA). The differences were considered statistically significant at P<0.05 (two-sided).

Results
. . Analysis of the Freeze-Dried Powder of Rosa roxburghii. In order to ensure the consistency among batches of freeze-dried powder of Rosa roxburghii, samples were sent to and tested by the China National Analytical Center in Guangzhou (China). The results showed that the flavonoids content was the same (0.4%) for all three batches, while the polysaccharides contents were 5.5%, 6.2%, and 5.7%, respectively (mean of 5.8%). Table 2 presents the exact content of some nutrients. SOD activity was 2.88 × 10 4 U/g.

. . Cili Powder Protects the Obstructed Kidneys from Severe Oxidative Stress.
To determine the oxidative stress in the kidneys, the levels of 8-OHdG, MDA, and SOD were measured. The levels of 8-OHdG, MDA, and SOD were all increased by UUO modeling (all P<0.01) (Figure 4). Cili (both moderate and high doses) and losartan decreased the 8-OHdG, MDA, and SOD levels compared with the UUO group (all P<0.01) (Figure 4). The decrease of 8-OHdG of the UUO+CL high dose group was a little smaller than that of the UUO+losartan group (P<0.001) (Figure 4). Those results suggest that Cili prevented oxidative stress development after UUO, in a similar way as in the losartan group.

. . Cili Powder Alleviates Renal Fibrosis Which Is Associated with the Inhibition of TGF-/Smads
Signaling. Because the TGF-/Smads pathway plays a pivotal role in renal fibrosis, the effects of Cili powder on the mRNA and protein expressions of TGF-, TGF-receptors, and Smads were investigated in the kidney. In the UUO group, the mRNA levels of TGFB , TGFBR , TGFBR , SMAD , and SMAD were significantly upregulated (P<0.01), while the protective SMAD was decreased (P<0.01) (Figure 5(a)). Treatment with losartan, resveratrol, or Cili powder efficiently prevented the changes in TGFB , TGFBR , TGFBR , SMAD , SMAD , and SMAD ( Figure 5(a)) (P<0.01 vs. UUO without treatment).
At the protein level, Cili moderate and high dose, losartan, and resveratrol inhibited the expression of TGF-1, TGF-R1, TGF-R2, and Smad3, but high dose of Cili powder did not show a better antagonistic effect on the expression of any  Relative protein level (ratio to GAPDH) Relative protein level (ratio to GAPDH) Relative protein level (ratio to GAPDH) Relative protein level (ratio to GAPDH) TGF-/Smad proteins ( Figure 5(b)). According to immunohistochemistry, Cili, losartan, and resveratrol inhibited the expression of TGF-1, TGF-R1, and Smad3, while maintaining Smad7 expression in obstructed kidneys (Figures 6  and 7), which is consistent with the RT-PCR results. Taken together, the results suggest that Cili powder improved renal fibrosis, possibly through the inhibition of the TGF-/Smads pathway.

Discussion
Since fibrosis is involved in the pathogenesis of kidney diseases [1,2], since Cili possesses antifibrosis properties [16,19,20], and since our preliminary data showed that freeze-dried Cili powder improved renal fibrosis and indexes of oxidative stress in 90 patients with stages 3-4 renal failure [24], we hypothesized that Cili could prevent the development of renal fibrosis in rat models of UUO through TGF-/Smads signaling, which is known to be involved in renal fibrosis [4][5][6]. Therefore, this study aimed to confirm the effects of freeze-dried Cili powder on renal fibrosis and examine the TGF-/Smads signaling. The results showed that Cili freeze-dried powder could effectively prevent the phenotype of renal fibrosis in UUO rat models. The beneficial effects may result from the inhibition of oxidative stress and TGF-1/Smads signaling pathway. Therefore, we accepted the research hypothesis. The incidence of chronic kidney diseases is increasing, and current treatments are limited to angiotensin II receptor blockers and angiotensin converting enzyme inhibitors. Losartan is an angiotensin II receptor inhibitor and can inhibit interstitial fibrogenesis by modulating nitric oxide synthase (NOS) isoforms and cyclooxygenase-2 (COX-2) expression [36], decreasing oxidative stress in the kidneys [37,38] and inhibiting MCP1 and TGF-1 expression in rats with UUO [39]. Resveratrol is a wellknown kidney protective agent through potent reactive oxygen species scavenging abilities [40]. Resveratrol has been shown to protect against renal injury in models of diabetic nephropathy, drug-induced injury, aldosteroneinduced injury, ischemia/reperfusion injury, sepsis, and UUO [40]. Therefore, losartan and resveratrol were selected as positive controls for the prevention of renal fibrosis in the UUO model.
Cili, used as a dietary supplement, has been shown to enhance the antioxidant status [17][18][19][20]. Unlike losartan, the effects of Cili extract on chronic renal diseases are still poorly understood, which limits its wide application. We previously used freeze-dried Cili powder (oral administration) to treat 90 patients with stages 3-4 chronic renal failure [24]. Therapeutic effects were achieved based on the evaluation of syndrome improvement, renal fibrosis indexes, and oxidative stress indexes. The present findings in rats confirmed the effects of Rosa roxburghii fruit extracts in attenuating the development of renal fibrosis in mammals. In the present study, 8-OHdG and MDA in the UUO kidneys were increased as indicatives of the renal oxidative stress induced upon injury, which could be significantly reduced by the administration of Cili freeze-dried powder. In addition, there was a higher level of SOD in the UUO kidneys as compared to the normal ones, suggesting the endogenous regulation to counteract the renal oxidative stress. Cili freeze-dried powder restored the counterregulatory rise of renal SOD in UUO rats, possibly because the antioxidant effects have been achieved. The effects observed with Cili were comparable to those of resveratrol, a potent antioxidant [40].
Notably, high-dose Cili did not result in better efficacy in preventing renal fibrosis in the UUO rat model, as revealed by the markers of kidney function, histological examination, collagen III expression, and oxidative stress. We supposed that, apart from supplying more antioxidants, high-dose  Cili powder may also put a heavier excretion load on the obstructed kidney, as observed with some TCM preparations [11], limiting its efficacy at high doses, where benefits are limited by harms. A similar example is that high-dose vitamin C can induce hyperoxaluric nephropathy and progressive renal failure [41]. Therefore, since Cili is not a pure compound but a mixture of several compounds with different biological effects, a comprehensive analysis of each of the major or biologically significant Cili component will contribute to a better understanding of the effects of Cili on renal diseases.
Reactive oxygen species are important mediators of kidney damage and fibrosis, but they are not the only factors involved; many others are involved, including the various players (genes, proteins, and miRNAs) of the HIF, TGF-, Notch, PKC/ERK, PI3K/Akt, NF-B, and Ang II/ROS pathways [42]. The present results provided some clues for the mechanisms underlying the effects, with a focus on the TGF 1/Smads pathway. The positive control resveratrol was also shown to inhibit renal fibrosis through the TGF 1/Smads pathway [43], supporting the present study. As the dysregulation of the TGF 1/Smads pathway is central in the progression of renal fibrosis [44,45], any intervention that could modulate this pathway should play important roles in the management of the condition. Additional parameters (e.g., leukocyte infiltration [46], fibroblasts, and apoptosis [47]) need to be taken into account and to be investigated in the future. A better understanding of the mechanisms involved in renal fibrosis could lead to improved treatments in those patients.
A potential limitation of the present study was that Cili was given concomitantly with UUO. Hence, the effects observed in the present study were more preventive effects than treatment effects. Future studies should first establish renal fibrosis after UUO and then observe the effects of Cili on the kidneys. In addition, the involvement of TGF-1/Smads signaling pathway will need to be confirmed using knock-out models and/or siRNAs. Furthermore, a number of other pathways are involved in renal fibrosis (e.g., HIF, TGF-, Notch, PKC/ERK, PI3K/Akt, NF-B, and Ang II/ROS pathways [42]) and those pathways should be explored in order to gain a more comprehensive understanding of the effects of Cili on renal fibrosis.

Conclusions
In conclusion, the results demonstrate that Cili freeze-dried powder could effectively prevent renal fibrosis and injury in UUO rat models, suggesting the underlying mechanisms may involve the inhibition of oxidative stress and TGF-1/Smads signaling.

Data Availability
No data were used to support this study.

Conflicts of Interest
The authors declare that there are no conflicts of interest regarding the publication of this paper