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 Table of Contents  
ORIGINAL ARTICLE
Year : 2021  |  Volume : 8  |  Issue : 1  |  Page : 2

Tangshen formula attenuates renal fibrosis by downregulating transforming growth factor β1/Smad3 and LncRNA-MEG3 in rats with diabetic kidney disease


1 Department of Clinical Medicine, Beijing University of Chinese Medicine; Department of Pharmacology, Beijing Key Laboratory for Immune-mediated Inflammatory Diseases, Institute of Clinical Medical Sciences, China-Japan Friendship Hospital, Beijing, China
2 Department of Pharmacology, Beijing Key Laboratory for Immune-mediated Inflammatory Diseases, Institute of Clinical Medical Sciences, China-Japan Friendship Hospital, Beijing, China

Date of Submission16-Mar-2021
Date of Decision31-Mar-2021
Date of Acceptance11-Jun-2021
Date of Web Publication08-Sep-2021

Correspondence Address:
Prof. Ping Li
Department of Pharmacology,Beijing Key Laboratory for Immune-mediated Inflammatory Diseases, Institute of Clinical Medical Sciences, China-Japan Friendship Hospital, Beijing
China
Prof. Ting-Ting Zhao
Department of Pharmacology,Beijing Key Laboratory for Immune-mediated Inflammatory Diseases, Institute of Clinical Medical Sciences, China-Japan Friendship Hospital, Beijing
China
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/imna.imna_22_21

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  Abstract 


Background and Objective: The traditional Chinese Tangshen formula (TSF) has been reported to ameliorate diabetic kidney disease (DKD) in humans and animals. However, the effect of TSF on renal fibrosis remains unclear. Transforming growth factor-β1 (TGF-β1)/Smad3 signaling and lncRNA MEG3 are important in renal fibrosis. In this study, we examined the therapeutic effect of TSF on renal fibrosis and explored whether it was related to the modulation of TGFβ1/Smad3 signaling and lncRNA MEG3 expression. Materials and Methods: Experiments were performed in rats in vivo and in the HK2 cells in vitro. DKD was induced in rats by uninephrectomy combined with a single streptozotocin injection. The HK2 cells were stimulated by high glucose (HG) to explore the mechanism of TSF effects in vitro. Results: TSF significantly attenuated renal injury by lowering proteinuria and renal histological damage in DKD rats. TSF reduced collagen deposition by decreasing the expression of the fibrotic indicators collagen I, collagen IV, and fibronectin at the protein and mRNA levels, which suggested that TSF ameliorated DKD by decreasing renal fibrosis. Furthermore, TSF decreased TGF-β1 expression and suppressed the levels of phosphorylated Smad3 and Smad2/3 in vivo. Moreover, TSF downregulated the lncRNA MEG3 level in DKD rats. TSF reversed the upregulation of collagen I and fibronectin expression and downregulated Smad2/3 phosphorylation in the HK2 cells stimulated with HG. Conclusions: TSF ameliorates renal fibrosis in rats with DKD by suppressing TGF-β1/Smad3 signaling and lncRNA MEG3 expression.

Keywords: Diabetic kidney disease, LncRNA MEG3, renal fibrosis, the Tangshen formula, transforming growth factor β1/Smad3 signaling pathway


How to cite this article:
Zhou XF, Wang Y, Luo MJ, Zhao TT, Li P. Tangshen formula attenuates renal fibrosis by downregulating transforming growth factor β1/Smad3 and LncRNA-MEG3 in rats with diabetic kidney disease. Integr Med Nephrol Androl 2021;8:2

How to cite this URL:
Zhou XF, Wang Y, Luo MJ, Zhao TT, Li P. Tangshen formula attenuates renal fibrosis by downregulating transforming growth factor β1/Smad3 and LncRNA-MEG3 in rats with diabetic kidney disease. Integr Med Nephrol Androl [serial online] 2021 [cited 2021 Dec 6];8:2. Available from: http://www.journal-imna.com/text.asp?2021/8/1/2/325662




  Introduction Top


Diabetic kidney disease (DKD) is a key factor that leads to end-stage renal disease,[1] accompanied by renal fibrosis,[2] which, in turn, is characterized by excessive extracellular matrix deposition, fibrotic tissue replacement of functional parenchyma, and interstitial fibrillary collagen.[3]

Renal fibrosis is a complex process that depends on the interaction of different cell types and activation of several profibrotic signaling pathways, particularly the transforming growth factor-β1 (TGF-β1) pathway.[4],[5],[6] TGF-β1 regulates renal fibrosis by activating downstream signaling molecules, such as Smad2/3 and Smad2/3-dependent noncoding RNA. Therefore, specific targeting of the latter may be a promising approach for treating renal fibrosis.[7] Recently, experimental evidence has shown that overexpression of long noncoding RNA (lncRNA) MEG3 (GENE ID: 55384) inhibited cell proliferation, stimulated by TGF-β1, and induced apoptosis.[8],[9],[10]

The Chinese herbal medicine Tangshen formula (TSF) treats DKD,[11] effectively alleviating proteinuria and improving the estimated glomerular filtration rate in patients.[12] However, the underlying mechanism by which TSF improves renal fibrosis in DKD is still unclear. Here, we examined the therapeutic effect of TSF in rats with DKD and renal fibrosis. In particular, we explored whether TSF modulates the TGF-β1/Smad3 signaling pathway and lncRNA MEG3.


  Materials and Methods Top


Herbal formulation and components

The preparation and standardization of TSF (lot number 180408) were based on the established guidelines in the Pharmacopeia of the People's Republic of China (Edition 2015), consigned by the Beijing Institute of Clinical Pharmacy. TSF was prepared from the extracts of the following seven natural herbs: Astragalus membranaceus (Fisch.) Bunge, burning bush twigs (Euonymus alatus [Thunb.] Siebold.), Rehmannia root (Rehmannia glutinosa [Gaertn.] Libosch.), bitter orange fruit (Citrus aurantium L.), cornus fruit (Cornus officinalis Sieb and Zucc.), rhubarb root and rhizome (Rheum palmatum L.), and notoginseng root and rhizome (Panax notoginseng (Burk.) F. H. Chen) at a ratio of 10:5:4:3.4:3:2:1 (W/W). A previous study identified six representative chemical components of TSF for quality control, including neohesperidin, aloe-emodin, calycosin-7-O-β-D-glucoside, naringenine-7-rhamnosidoglucoside, loganin, and naringenin.[13]

Reagents

The antibodies against the following proteins were used: Collagen type I (#1310-01; SouthernBiotech, Birmingham, AL, USA), collagen type IV (#1340-01, SouthernBiotech), fibronectin (#sc-69681, Santa Cruz Biotechnology, Dallas, TX, USA), total Smad3 (#sc-101154, Santa Cruz), phosphorylated Smad3 (p-Smad3, #sc-517575, Santa Cruz), phosphorylated Smad2/3 (p-Smad2/3, #AP0326P, Bioworld Technology, Inc.), and TGF-β1 (#BS1361, Bioworld). ELISA Quantitation Set kit (#E101) was obtained from Bethyl Laboratories (Montgomery, TX, USA). Carboxymethylcellulose sodium (#C8621) and penicillin-streptomycin (#P1400) were purchased from Solarbio (Beijing, China). RPMI-1640 medium (#R8758) was supplied by Sigma-Aldrich (St. Louis, MO, USA). Trypsin-EDTA (0.25%) (#25200056) was purchased from Gibco (Life Technologies Corporation, USA).

Animals

Eighteen 8-week-old male Wistar rats weighing 200 ± 20 g were supplied by HFK Bio-Technology Co. Ltd. (Beijing, China). They were randomly assigned to two groups based on their body weight: sham group (n = 6) and diabetic group (n = 12). The rat model was established according to an established protocol.[14] Briefly, rats in the DKD group underwent right uninephrectomy. The sham group underwent sham operation: laparotomy and manipulation of the renal pedicles but without any damage to the kidney.[15] One week after uninephrectomy, DKD rats received a single intraperitoneal injection of streptozocin (STZ, 40 mg/kg, Sigma-Aldrich, St. Louis, MO). Three days after the STZ injection, all 12 rats developed hyperglycemia (blood glucose levels >16.7 mmol/L) and were randomly assigned to the DKD group and DKD + TSF group. There were three groups in this study and the treatments they received were as follows. In the DKD + TSF group (n = 6), TSF was administered daily by oral gavage at a dose of 1.2 g/kg bodyweight for 20 weeks. The DKD group (n = 6) received vehicle. In addition, the sham control rats that received sham operation without STZ injection (n = 6) received gavage with the same volume of vehicle (distilled water) as rats in the DKD + TSF group. All rats were housed at a humidity level of 65%–75%, temperature of 20°C–25°C, 12 h:12 h light/dark cycle and were given chow and water ad libitum. Bodyweight was measured weekly. Every 4 weeks, rats were transferred to individual metabolic cages (Fengshi Inc., Suzhou, JS, China) for 24 h to collect urine. The level of urinary albumin was expressed as the ratio of urinary albumin to creatinine (UACR) measured by a competitive ELISA method according to the manufacturer's instructions (Bethyl Laboratories, Montgomery, TX, USA). Blood glucose was measured every 4 weeks by tail vein blood sampling using a OneTouch Ultra Blood Glucose Monitoring System (LifeScan, Milpitas, CA, USA). Rats were sacrificed after overnight fasting at the end of the 20th week of treatment. Serum and tissue samples were collected for further analyses.

Animal experiments were performed in accordance with the Guidelines of the National Institutes of Health Guide for the Care and Use of Laboratory Animals (Edition 2011). The protocol was approved by the Ethics Committee of the China-Japan Friendship Institute of Clinical Medical Sciences (Approval No. 2010-A10).

Cell culture

Human kidney tubular epithelial (HK2) cell line from ATCC (Rockville, MD, USA) was used in vitro experiments. HK2 cells were cultured in RPMI 1640 medium, containing 10% fetal bovine serum (Gibco, USA) and 1% penicillin-streptomycin in a humidified incubator containing 5% CO2 at 37°C. The cell groups were as follows: the normal glucose group (5.5 mmol/L glucose in the medium), the high glucose (HG) group (30 mmol/L glucose; #G8270, Sigma-Aldrich); HG + TSF 62.5 group (30 mmol/L HG stimulation and 62.5 μg/mL TSF intervention); HG + TSF 125 group (30 mmol/L HG stimulation and 125 μg/mL TSF intervention); HG + TSF 250 group (30 mmol/L HG stimulation and 250 μg/mL TSF intervention).

Cell viability detection

The effect of TSF on cell viability was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. After being incubated for 24 h in 96-well plates, HK2 cells were incubated with RPMI-1640 medium containing TSF at the concentrations of 62.5, 125, 250, 500, 1000, 2000, and 4000 μg/mL for 48 h. Then, 20 μL of 5 mg/mL MTT solution was added to each well, and the cells were further incubated for another 4 h. After supernatant removal, 100 μL of dimethyl sulfoxide was added to each well, and the formazan crystals were dissolved. The 96-well plates were shaken on a shaker for 15 min. A microplate reader (BioTek, Winooski, VT, USA) was used to measure the absorbance at 490 nm.

Measurement of biochemistry

The UACR was measured every 4 weeks in rats kept for 24 h in metabolic cages for urine collection. The serum was prepared for creatinine (Scr), urea and albumin (Alb). An automatic biochemistry analyzer machine (CD-1600CS, Abbott Labs, USA) was used to determine these biochemical indicators. Urine albumin level was measured by an ELISA Quantitation Set kit (Bethyl Laboratories Inc.), according to the manufacturer's instructions.

Renal tissue pathology and immunohistochemistry

The kidney tissue sections were immediately fixed in 10% phosphate buffered formalin solution and then embedded in paraffin. The embedded kidney tissue was then sliced into 3-μm thick sections, mounted on a slide, and stained with periodic acid–Schiff and Masson's trichrome. The degree of glomerulosclerosis, defined as the mesangial matrix percentage, was evaluated after observation of 20 cortical fields at ×400.[16] Image-Pro Plus software (version 6.0; Media Cybernetics, Warrendale, PA, USA) was used to measure the area of each glomerulus and mesangial matrix. The degree of fibrosis presented as a percentage of fibrotic area was evaluated in observations of 10 cortical fields at ×200. Image-Pro Plus software (version 6.0) was used to measure the area of each cortical field. Immunohistochemistry indicators included collagen Type I, collagen Type IV, fibronectin, TGFβ1, and p-Smad2/3. The immunohistochemistry protocol was based on previously established methods.[17] Sections were developed to produce a brown product with a positively stained area after staining. The sections were then counterstained with hematoxylin. After dehydration and mounting the renal slides, we used Image-Pro Plus software (version 6.0) to quantify the deposition of collagen I, collagen IV, and fibronectin in 20 random glomeruli, excluding the arterial lumen space, under ×400 magnification. The number of p-Smad2/3+ cells was counted in 20 random glomeruli. Cells positive for p-Smad2/3 in the tubulointerstitium was also counted in 20 random fields under ×400 magnification and expressed as cells per mm2.[18]

Extraction of RNA and real-time polymerase chain reaction analysis

RNA was extracted with TRIzol reagent (Invitrogen, USA) from rat kidney cortical tissues, which were frozen at −80°C. Template cDNA was prepared by a kit (Mei5 Biotechnology Co., Ltd). Real-time polymerase chain reaction (PCR) was used to analyze the expression levels of the genes of interest according to the manufacturer's instructions (ABI system, USA). The primer sequences used in this study are listed in [Table 1].
Table 1: Primers used for quantitative real-time polymerase chain reaction

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Western blot analysis

RIPA lysis buffer (EpiZyme, Shanghai, China) was used to extract protein from the kidney tissue and cells. Protein concentrations were measured by the BCA Protein Assay Kit assay (#PC0020, Solarbio, Beijing, China) according to the manufacturer's instructions. Equal amounts of total protein were denatured at 99°C for 5 min. Then, we followed the process previously described.[13] For western blotting, antibodies against collagen I, collagen IV, fibronectin, Smad3, and p-Smad3 were used at a concentration of 1:1000. The blots were imaged by enhanced chemiluminescence (Amersham Pharmacia Biotech, Amersham, UK) with a ChemiDoc XRS system (Bio-Rad, Hercules, CA, USA). Finally, the semi-quantitative analysis of the protein bands was performed using ImageJ software.

Immunofluorescence

For cell immunofluorescence, coverslips of HK2 cells were washed with 1×PBS and then fixed in 4% paraformaldehyde for 15 min. Triton X-100 (0.5%) was used to permeate the cells for 20 min at room temperature. After three washes with 1×PBS, the cells were blocked with 1% bovine serum albumin for 1 h at room temperature. They were then stained with antibodies at the following concentrations: anti-fibronectin antibody (1:100), anti-p-Smad2/3 antibody (1:50), and anti-Smad2/3 antibody (1:50). Then, they were stained with secondary antibodies with following concentrations: 1:200 Alexa Fluor 488 goat anti-mouse antibody and 1:200 Alexa Fluor 488 goat anti-rabbit antibody. The resulting images of cells were captured by confocal laser scanning microscopy.

Statistical analysis

All quantitative data were expressed as the mean ± standard error of the mean. One-way analysis of variance followed by the X post hoc test was used to compare differences between the groups. GraphPad Prism software (GraphPad Prism, version 7.0, San Diego, CA, USA) was used for the analysis and plots. Differences were considered to be statistically significant if post hoc test P < 0.05.


  Results Top


Tangshen formula attenuated proteinuria and histological damage in rats with diabetic kidney disease

All DKD rats involved in this experiment developed high UACR [Figure 1]a, low body weight [Figure 1]b, and hyperglycemia (blood glucose >16.7 mmol/L) within the 20-week study period [Figure 1]c. TSF gavage decreased UACR in the DKD + TSF group at weeks 12 and 20 [Figure 1a]. Rats in the sham group gained weight during the experiment; however, rats in the DKD group displayed lower body weight gain, and the TSF treatment did not alleviate this phenotype [Figure 1]b. Likewise, TSF treatment failed to significantly reduce elevated blood glucose level in the DKD + TSF group [Figure 1]c. Serum urea and Scr levels were increased in DKD rats, but they were significantly lower in the DKD + TSF group [Figure 1]d and [Figure 1]e. In addition, serum Alb level was lower in DKD rats than in the sham group, whereas TSF treatment reversed this downward trend [Figure 1]f.
Figure 1: Tangshen formula attenuates proteinuria and renal functions in diabetic kidney disease rats. Effects of Tangshen formula on urinary albumin to creatinine (a), body weight (b), and blood glucose (c) in rats within 20-week study period. Effect of Tangshen formula on kidney functions: UREA (d), Scr (e), ALB (f) in the week 20. Data are presented as mean ± standard error #P < 0.05, ##P < 0.01, ###P < 0.001, diabetic kidney disease group versus sham group; *P < 0.05, **P < 0.01, diabetic kidney disease + Tangshen formula group versus diabetic kidney disease group

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Periodic acid–Schiff and Masson's trichrome staining of kidney tissues indicated that DKD rats developed expansion of the mesangial matrix in the glomeruli and the fibrotic matrix deposition in the tubulointerstitium [Figure 2]a. These histological features were significantly ameliorated by TSF treatment [Figure 2]a, [Figure 2]b, [Figure 2]c.
Figure 2: Tangshen formula attenuates renal injury in diabetic kidney disease rats. Histology (Masson's trichrome staining, original magnification [×200]; scale bars, 100 μm; Periodic acid–Schiff staining, original magnification [×400], scale bars, 50 μm.) (a) and semi-quantification of tubular injury (b) and mesangial matrix (c). Data are pr esented as mean ± standard error #P < 0.05, ##P < 0.01, ###P < 0.001, diabetic kidney disease group versus sham group; *P < 0.05, **P < 0.01, diabetic kidney disease + Tangshen formula group versus diabetic kidney disease group

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Tangshen formula inhibited renal fibrosis markers in diabetic kidney disease rats

We examined the therapeutic effect of TSF on renal fibrosis in rats with DKD. Immunohistochemistry, western blotting, and real-time PCR revealed that DKD rats developed renal fibrosis, demonstrating a significant accumulation of collagen I, collagen IV, and fibronectin in the renal cortex [Figure 3] and [Figure 4]. All these effects were significantly attenuated by the treatment with TSF [Figure 3] and [Figure 4].
Figure 3: Tangshen formula attenuates renal fibrosis in diabetic kidney disease rats. Immunohistochemistry of collagen I (Col-I, original magnification (×400), scale bars, 50 μm), collagen IV (Col-IV, original magnification (×400), scale bars, 50 μm), and fibronectin (Fn, original magnification (×400), scale bars, 50 μm) (a) Quantitative analysis of Col-I (b), Col-IV (c), and Fn (d). Data are expressed as means ± standard error for each group of 6 rats. *P < 0.05, **P < 0.01, diabetic kidney disease + Tangshen formula group versus diabetic kidney disease group; #P < 0.05, ##P < 0.01, ###P < 0.001, diabetic kidney disease group versus sham group

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Figure 4: Tangshen formula inhibits renal fibrosis in diabetic kidney disease rats. Western blot of Col-I, Col-IV, and Fn (a) and semi-quantitative analysis of Fn (b), Col-I (c), and Col-IV (d). Real-time polymerase chain reaction of Col-I (e), Col-IV (f), and Fn (g). Data are expressed as means ± standard error for each group of 6 rats. #P < 0.05, diabetic kidney disease group versus Sham group; *P < 0.05, diabetic kidney disease + Tangshen formula group versus diabetic kidney disease group

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Tangshen formula treatment downregulated the transforming growth factor β1/Smad3 signaling pathway and the Lnc RNA MEG3 to attenuate diabetic renal fibrosis

The TGFβ1/Smad3 signaling pathway is a crucial mechanism of diabetic renal fibrosis.[19] LncRNA maternally expressed gene 3 (LncRNA MEG3) was reported to regulate TGFβ signaling pathway.[8] Therefore, we investigated whether TSF treatment alleviated diabetic renal fibrosis by affecting the TGF-β1/Smad3 signaling pathway and lncRNA MEG3. LncRNA MEG3 levels were upregulated in the renal cortex of DKD rats [Figure 5]a. Immunohistochemistry results showed that the levels of TGF-β1 and p-Smad2/3 in the DKD group were also markedly enhanced [Figure 5]b. Treatment with TSF significantly reversed the upregulation of lncRNA MEG3 and TGF-β1. Furthermore, treatment with TSF also downregulated p-Smad3 level as well as decreased the extent of Smad3 nuclear translocation in the diabetic kidney [Figure 5]a, [Figure 5]b, [Figure 5]c. The western blot results were similar: the level of p-Smad3 was upregulated in DKD rats, whereas TSF treatment decreased it, implying an inhibitory effect on Smad3 activation [Figure 5]d and [Figure 5]e.
Figure 5: Tangshen formula blocks activation of the transforming growth factor β1/Lnc RNA MEG3/Smad3 signaling pathway in diabetic rats. Tangshen formula treatment inhibits expression of the Lnc RNA MEG3 (a), immunohistochemistry of transforming growth factor β1 and p-Smad2/3(original magnification (×400), scale bars, 50 μm) (b), quantitative analysis of transforming growth factor β1, quantitative analysis of nucleated p-Smad2/3 in glomeruli, and tubulointerstitium (c), western blot of Smad3 and p-Smad3 (d), and semi-quantitative analysis of p-Smad3 (e). Data are expressed as means ± standard error for each group of 6 rats. #P < 0.05, ###P < 0.001, diabetic kidney disease group versus Sham group; *P < 0.05, ***P < 0.001, diabetic kidney disease + Tangshen formula group versus diabetic kidney disease group

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Tangshen formula downregulated the expression of collagen I, fibronectin, P-Smad2/3, and LncRNA MEG3 in HG-Stimulated HK2 cells

Accumulation of fibrotic in renal proximal tubular cells is considered to be a crucial process of renal fibrosis, which is also an important pathological process in DKD.[20] To investigate the effects of TSF on HK2 cells, various concentrations (ranging from 62.5 to 8000 μg/mL) for 48 h were measured. Exposure to TSF at concentrations (ranging from 62.5 to 250 μg/mL) did not result in any significant changes in the survival rate of HK2 cells [Figure 6]a. However, whenever the dose was over 500 μg/mL, TSF exhibited the cytotoxic effect. Hence, TSF <500 μg/mL was used in this following experiment.
Figure 6: Col-I and Fn in HK2 cells stimulated by high glucose. Dose-dependent effect of Tangshen formula on cell viability as determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay for 48 h (a). **P < 0.01 versus medium group. Western blot analysis of Fn and Col-I expression in HK2 for 48 h with different concentrations of high glucose (b). *P < 0.05, versus the NC group. Western blot analysis of Fn and Col-I expression in HK2 cells stimulated with 30 mM concentration of high glucose for 0 h, 12 h, 24 h, 48 h, 72 h (c). Data are expressed as means ± standard error for at least three independent experiments. *P < 0.05, versus the 0 h group

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To establish a suitable model of HG-stimulated fibrosis, HK2 cells were incubated with 20–35 mmol/L glucose for 0, 12, 24, 48, and 72 h. We also investigated the expression levels of the fibrotic indicators, collagen I, and fibronectin. On the basis of the results obtained, a HG concentration of 30 mmol/L and an incubation period of 48 h were chosen as optimal modeling conditions [Figure 6]b and [Figure 6]c and used in the subsequent experiments.

We then investigated the effects of TSF treatment on the HK2 cells stimulated with HG for 48 h. Immunofluorescence results and western blot data showed that TSF dose-dependently attenuated the expression of collagen I and fibronectin [Figure 7]a, [Figure 7]b, [Figure 7]c, [Figure 7]d.
Figure 7: After treatment with Tangshen formula for 48 h, the expression of Col-I and Fn were downregulated in the HK2 cells stimulated with high glucose. Representative immunofluorescence stained with Fn (green) (original magnification (×400), scale bars, 50 μm) (a). Western blot of Col-I and Fn (b). Quantitative analysis of Col-I (c) and Fn (d) (n = 3 experiments). The data were expressed as the mean ± standard error of the mean of three independent experiments. #P < 0.05, versus the NC group; *P < 0.05, versus the HG group

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To explore the mechanisms of TSF effect, we investigated the levels of p-Smad2/3 and lncRNA MEG3 in HK2 cells. We found that whereas HG stimulation augmented the levels of these molecules in HK2 cells, TSF treatment dose-dependently reversed these increases [Figure 8]a and [Figure 8]b.
Figure 8: The activation of Smad2/3 was upregulated in the HK2 cells stimulated with high glucose for treatment with Tangshen formula for 48 h. Representative immunofluorescence stained with Smad2/3 and p-Smad2/3 (green) (original magnification (×400), scale bars, 50 μm) (a). Tangshen formula treatment downregulated the expression of LncRNA MEG3 (b). The data are expressed as the mean ± standard error of the mean of three independent experiments. #P < 0.05, versus the NC group; *P < 0.05, versus the HG group

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  Discussion Top


TSF was previously demonstrated to ameliorate diabetic renal injuries in spontaneous diabetic db/db mice,[13] Otsuka Long-Evans Tokushima Fatty rats,[21] and rats with DKD induced by a high-fat diet.[18] Renal fibrosis is the most vital pathological feature of DKD.[22],[23],[24] At present, the TGF-β1/Smad3 signaling pathway and lncRNAs have become the focus of pharmacological studies aimed at the alleviation of DKD.[23],[25] In the present study, we explored the effect of TSF on DKD induced by uninephrectomy combined with a STZ injection and explored the underlying mechanism of this effect in the HK2 cells stimulated by HG in vitro. Our data suggest important roles of the TGFβ1/Smad3 signaling pathway and lncRNA MEG3 during this process.

The occurrence of the excessive deposits of extracellular matrix and interstitial fibrillary collagen in the kidneys of DKD rats, which was related to the activation of the TGF-β/Smad3 signaling pathway and upregulation of lncRNA MEG3, was a novel finding of this study. Furthermore, we showed that TSF treatment attenuated diabetic kidney injury and ameliorated renal fibrosis through the TGFβ1/Smad3 and lncRNA MEG-dependent mechanism.

TGF-β has long been considered the main cytokine in the pathogenesis of renal fibrosis.[26] Smad3 is a prominent downstream effector of TGF-β signaling in tissue fibrogenesis. The balance in the activity of the Smad signaling pathway is essential for TGF-β-mediated renal fibrosis, in which Smad3 is pathogenic and Smad7 is protective.[19] In the present study, DKD was associated with a significant elevation of TGF-β1 and Smad3 levels, suggesting that the upregulation of these proteins aggravated pathological injury, resulting in the excessive deposits of extracellular matrix and interstitial fibrillary collagen in the kidneys of DKD rats. In addition, an upregulated expression of collagen I and fibronectin stimulated by HG was observed in the HK2 cell fibrotic model. TSF treatment alleviated both the diabetic kidney injury and HK2 cell collagen deposition. These effects were consistent with the pivotal role of angiotensin II in DKD;[27],[28] angiotensin II blocked angiotensin through suppressing TGF-β1/Smad3 signaling. Therefore, blockade of the TGF-β1/Smad3 signaling could be an important mechanism by which TSF ameliorates renal fibrosis in DKD.

Recently, lncRNAs have been recognized as functional regulators of fibrosis. LncRNA MEG3, located on chromosome 14q32.3,[29] has an important role in fibrotic processes, including idiopathic pulmonary fibrosis,[30] cardiac fibrosis,[31] and liver fibrosis.[32],[33] In addition, lncRNA MEG3 plays a significant role in diabetes: [34] it is involved in the regulation of glucose homeostasis and insulin synthesis. Genetic variation of lncRNA MEG3 may increase the risk of type 2 diabetes in the populations.[35] Besides, LncRNA MEG3 enhances hepatic insulin resistance through regulating miR-214/ATF4 axis.[36] In a rat model of DKD induced by lncRNA MEG3 over-expression, expression levels of fibrosis-related proteins and inflammatory cytokines were increased.[25] Data from a lncRNA MEG3 over-expression model indicated that lncRNA MEG3 promotes fibrosis and inflammatory response by regulating the miR-181a/Egr-1/TLR4 axis. The level of lncRNA MEG3 was also increased in the kidneys of DKD rats in our study. In addition, the expression of fibrotic indicators, including collagen I, collagen IV, and fibronectin, was upregulated in DKD. Furthermore, increased levels of lncRNA MEG3, collagen I, and fibronectin were also observed in the HK2 cells stimulated by HG. TSF treatment downregulated the increased levels of collagen I, collagen IV, fibronectin, and lncRNA MEG3 both in vivo and in vitro.

The genes encoding the components of the TGF-β pathway are direct targets of lncRNA MEG3, which regulates them by forming an RNA-DNA triplex structure.[37] In the present study, the expression of TGF-β1 and the levels of p-Smad3 and p-Smad2/3 proteins were up-regulated in DKD. However, TSF treatment reversed these DKD-induced changes. These findings suggest that TSF ameliorated renal fibrosis by regulating the TGF-β1/Smad3 signaling pathway and lncRNA MEG3.


  Conclusions Top


In conclusion, we demonstrated that TSF may be a novel therapeutic agent for relieving renal fibrosis in DKD that acts by downregulating the activity of the TGF-β1/Smad3 pathway and lncRNA MEG3 level.

Financial support and sponsorship

This work was funded by the National Natural Science Foundation of China, grant number ”NO. NO. 81620108031, 81973627.”

Conflicts of interest

Ping Li is the Co-Editor-in-Chief of the journal. The article was subject to the journal's standard procedures, with peer review handled independently of this editor and his research groups.



 
  References Top

1.
Holt RI. Lest we forget the microvascular complications of diabetes. Diabet Med 2018;35:1307.  Back to cited text no. 1
    
2.
Ni WJ, Tang LQ, Wei W. Research progress in signalling pathway in diabetic nephropathy. Diabetes Metab Res Rev 2015;31:221-33.  Back to cited text no. 2
    
3.
Distler JH, Györfi AH, Ramanujam M, Whitfield ML, Königshoff M, Lafyatis R. Shared and distinct mechanisms of fibrosis. Nat Rev Rheumatol 2019;15:705-30.  Back to cited text no. 3
    
4.
Lovisa S, LeBleu VS, Tampe B, Sugimoto H, Vadnagara K, Carstens JL, et al. Epithelial-to-mesenchymal transition induces cell cycle arrest and parenchymal damage in renal fibrosis. Nat Med 2015;21:998-1009.  Back to cited text no. 4
    
5.
Macconi D, Remuzzi G, Benigni A. Key fibrogenic mediators: Old players. Renin-angiotensin system. Kidney Int Suppl (2011) 2014;4:58-64.  Back to cited text no. 5
    
6.
Feng M, Tang PM, Huang XR, Sun SF, You YK, Xiao J, et al. TGF-β mediates renal fibrosis via the Smad3-Erbb4-IR long noncoding RNA Axis. Mol Ther 2018;26:148-61.  Back to cited text no. 6
    
7.
Meng XM, Tang PM, Li J, Lan HY. TGF-β/Smad signaling in renal fibrosis. Front Physiol 2015;6:82.  Back to cited text no. 7
    
8.
Mondal T, Subhash S, Vaid R, Enroth S, Uday S, Reinius B, et al. MEG3 long noncoding RNA regulates the TGF-β pathway genes through formation of RNA-DNA triplex structures. Nat Commun 2015;6:7743.  Back to cited text no. 8
    
9.
Yu L, Kuang LY, He F, Du LL, Li QL, Sun W, et al. The role and molecular mechanism of long nocoding RNA-MEG3 in the pathogenesis of preeclampsia. Reprod Sci 2018;25:1619-28.  Back to cited text no. 9
    
10.
Mahpour A, Mullen AC. Our emerging understanding of the roles of long non-coding RNAs in normal liver function, disease, and malignancy. JHEP Rep 2021;3:100177.  Back to cited text no. 10
    
11.
Zhao H, Li X, Zhao T, Zhang H, Yan M, Dong X, et al. Tangshen formula attenuates diabetic renal injuries by upregulating autophagy via inhibition of PLZF expression. PLoS One 2017;12:e0171475.  Back to cited text no. 11
    
12.
Yang X, Zhang B, Lu X, Yan M, Wen Y, Zhao T, et al. Effects of Tangshen Formula on urinary and plasma liver-type fatty acid binding protein levels in patients with type 2 diabetic kidney disease: Post-hoc findings from a multi-center, randomized, double-blind, placebo-controlled trial investigating the efficacy and safety of Tangshen Formula in patients with type 2 diabetic kidney disease. BMC Complement Altern Med 2016;16:246.  Back to cited text no. 12
    
13.
Wang Q, Tian X, Zhou W, Wang Y, Zhao H, Li J, et al. Protective role of tangshen formula on the progression of renal damage in db/db Mice by TRPC6/Talin1 pathway in podocytes. J Diabetes Res 2020;2020:3634974.  Back to cited text no. 13
    
14.
Zhao TT, Zhang HJ, Lu XG, Huang XR, Zhang WK, Wang H, et al. Chaihuang-Yishen granule inhibits diabetic kidney disease in rats through blocking TGF-β/Smad3 signaling. PLoS One 2014;9:e90807.  Back to cited text no. 14
    
15.
Zhao T, Zhang H, Zhao T, Zhang X, Lu J, Yin T, et al. Intrarenal metabolomics reveals the association of local organic toxins with the progression of diabetic kidney disease. J Pharm Biomed Anal 2012;60:32-43.  Back to cited text no. 15
    
16.
Yuan H, Lanting L, Xu ZG, Li SL, Swiderski P, Putta S, et al. Effects of cholesterol-tagged small interfering RNAs targeting 12/15-lipoxygenase on parameters of diabetic nephropathy in a mouse model of type 1 diabetes. Am J Physiol Renal Physiol 2008;295:F605-17.  Back to cited text no. 16
    
17.
Zhang H, Zhao T, Gong Y, Dong X, Zhang W, Sun S, et al. Attenuation of diabetic nephropathy by Chaihuang-Yishen granule through anti-inflammatory mechanism in streptozotocin-induced rat model of diabetics. J Ethnopharmacol 2014;151:556-64.  Back to cited text no. 17
    
18.
Zhao T, Sun S, Zhang H, Huang X, Yan M, Dong X, et al. Therapeutic effects of tangshen formula on diabetic nephropathy in rats. PLoS One 2016;11:e0147693.  Back to cited text no. 18
    
19.
Tang PM, Zhang YY, Mak TS, Tang PC, Huang XR, Lan HY. Transforming growth factor-β signalling in renal fibrosis: From Smads to non-coding RNAs. J Physiol 2018;596:3493-503.  Back to cited text no. 19
    
20.
Li R, Guo Y , Zhang Y , Zhang X, Zhu L, Yan T . Salidroside ameliorates renal interstitial fibrosis by inhibiting the TLR4/NF-κB and MAPK signaling pathways. Int J Mol Sci 2019;20:1103.  Back to cited text no. 20
    
21.
Zhang H, Li P, Burczynski FJ, Gong Y, Choy P, Sha H, et al. Attenuation of diabetic nephropathy in otsuka long-evans tokushima fatty (OLETF) rats with a combination of chinese herbs (Tangshen Formula). Evid Based Complement Alternat Med 2011;2011:613737.  Back to cited text no. 21
    
22.
Meng XM, Nikolic-Paterson DJ, Lan HY. TGF-β: The master regulator of fibrosis. Nat Rev Nephrol 2016;12:325-38.  Back to cited text no. 22
    
23.
Gu YY, Liu XS, Huang XR, Yu XQ, Lan HY. TGF-β in renal fibrosis: Triumphs and challenges. Future Med Chem 2020;12:853-66.  Back to cited text no. 23
    
24.
Kato M, Natarajan R. Epigenetics and epigenomics in diabetic kidney disease and metabolic memory. Nat Rev Nephrol 2019;15:327-45.  Back to cited text no. 24
    
25.
Zha F, Qu X, Tang B, Li J, Wang Y, Zheng P, et al. Long non-coding RNA MEG3 promotes fibrosis and inflammatory response in diabetic nephropathy via miR-181a/Egr-1/TLR4 axis. Aging (Albany NY) 2019;11:3716-30.  Back to cited text no. 25
    
26.
Gu YY, Liu XS, Huang XR, Yu XQ, Lan HY. Diverse role of TGF-β in kidney disease. Front Cell Dev Biol 2020;8:123.  Back to cited text no. 26
    
27.
Wang W, Huang XR, Canlas E, Oka K, Truong LD, Deng C, et al. Essential role of Smad3 in angiotensin II-induced vascular fibrosis. Circ Res 2006;98:1032-9.  Back to cited text no. 27
    
28.
Jin D, Han F. FOXF1 ameliorates angiotensin II-induced cardiac fibrosis in cardiac fibroblasts through inhibiting the TGF-β1/Smad3 signaling pathway. J Recept Signal Transduct Res 2020;40:493-500.  Back to cited text no. 28
    
29.
Al-Rugeebah A, Alanazi M, Parine NR. MEG3: An oncogenic long non-coding RNA in different cancers. Pathol Oncol Res 2019;25:859-74.  Back to cited text no. 29
    
30.
Gokey JJ, Snowball J, Sridharan A, Speth JP, Black KE, Hariri LP, et al. MEG3 is increased in idiopathic pulmonary fibrosis and regulates epithelial cell differentiation. JCI Insight 2018;3:e122490.  Back to cited text no. 30
    
31.
Piccoli MT, Gupta SK, Viereck J, Foinquinos A, Samolovac S, Kramer FL, et al. Inhibition of the cardiac fibroblast-enriched lncRNA Meg3 prevents cardiac fibrosis and diastolic dysfunction. Circ Res 2017;121:575-83.  Back to cited text no. 31
    
32.
He Z, Yang D, Fan X, Zhang M, Li Y, Gu X, et al. The roles and mechanisms of lncRNAs in liver fibrosis. Int J Mol Sci 2020;21:1482.  Back to cited text no. 32
    
33.
Yu F, Geng W, Dong P, Huang Z, Zheng J. LncRNA-MEG3 inhibits activation of hepatic stellate cells through SMO protein and miR-212. Cell Death Dis 2018;9:1014.  Back to cited text no. 33
    
34.
Sathishkumar C, Prabu P, Mohan V, Balasubramanyam M. Linking a role of lncRNAs (long non-coding RNAs) with insulin resistance, accelerated senescence, and inflammation in patients with type 2 diabetes. Hum Genomics 2018;12:41.  Back to cited text no. 34
    
35.
Ghaedi H, Zare A, Omrani MD, Doustimotlagh AH, Meshkani R, Alipoor S, et al. Genetic variants in long noncoding RNA H19 and MEG3 confer risk of type 2 diabetes in an Iranian population. Gene 2018;675:265-71.  Back to cited text no. 35
    
36.
Zhu X, Li H, Wu Y, Zhou J, Yang G, Wang W. lncRNA MEG3 promotes hepatic insulin resistance by serving as a competing endogenous RNA of miR-214 to regulate ATF4 expression. Int J Mol Med 2019;43:345-57.  Back to cited text no. 36
    
37.
Mondal T, Subhash S, Vaid R, Enroth S, Uday S, Reinius B, et al. Author Correction: MEG3 long noncoding RNA regulates the TGF-β pathway genes through formation of RNA-DNA triplex structures. Nat Commun 2019;10:5290.  Back to cited text no. 37
    


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