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Abstract

Objective: Immune inflammatory cells and cytokines play an important role in the occurrence and development of diabetic nephropathy (DN). Acteoside has been reported to regulate the inflammation and immune response. The study aims to investigate the effects of acteoside on the expressions of MCP-1 and TGF-β₁ on nephropathy in diabetic mice.

Methods: C57BL/6J mice in the model group were given a single intraperitoneal injection of STZ (150 mg/kg). Model mice were divided randomly into two groups: 5 without treatment, 5 treated with acteoside. After continuous administration for 8 weeks, serum, urine, and kidney tissue were collected, then, ralated biochemical parameters, pathological characteristics and MCP-1 and TGF-β1 mRNA or protein were detected. The NRK-52E cells were divided into three groups as follows: the normal control group (NC group), the high glucose group (HS group), the high glucose+acteoside group (HS+ACT group). The expressions of MCP-1 and TGF-β₁ in the mRNA and protein levels were assessed with RT-PCR, western blot and ELISA.

Results: The expressions of MCP-1 and TGF-β₁ were significantly enhanced in DN group and dramatically reduced after acteoside treatment. Compared with those in NC group, the expressions of MCP-1 and TGF-β₁ in NRK-52E cell of HS group were significantly enhanced, while both were significantly decreased in HS+ACT group compared with HS group.

Conclusion: Our findings indicate that Acteoside has protective effects on DN via inhibiting the expressions of MCP-1 and TGF-β₁.

Keywords

acteoside diabetic nephropathy MCP-1

Introduction

Diabetic nephropathy (DN) is one of the most common and severe chronic complications of diabetes,¹ and is the leading cause of end-stage renal disease (ESRD) worldwide. DN is also an independent risk factor for cardiovascular diseases. Therefore, it is of great significance to delay the progression of DN in time and to find effective therapies. The pathogenesis of DN is complex, and the exact mechanism is still not understood, which is currently believed to be the result of multiple factors. With the further understanding of the pathogenesis, more and more evidences show that inflammation is also a recognized pathogenesis factor of DN,^2–4 inflammatory responses such as immune inflammatory cells and cytokines play an important role in the occurrence and development of DN.^5,6 The inflammatory response of diabetic nephropathy is initiated by the infiltration and activation of monocytesand macrophages in renal tissue, and the increased expression of monocyte chemoattractant protein-1 (MCP-1) and transforming growth factor-β (TGF-β) are two important factors leading to the infiltration of monocytes and macrophages into renal tissue.

As a member of the inflammatory chemokine superfamily, MCP-1 has chemotaxis and activation effects on a variety of inflammatory cells, including mononuclear macrophages, and mediates the infiltration of glomerular macrophages in diabetic nephropathy, and the number of infiltrated macrophages is proportional to the degree of renal interstitial fibrosis.⁷ MCP-1 is one of the most important inflammatory factors involved in kidney damage and plays a key role in aggravating the progression of diabetic nephropathy.

TGF-β₁ is the most important cytokine involved in the pathogenesis of diabetic nephropathy. The diabetic environment enhances the expression of TGF-β₁ induced by oxidative stress, while TGF-β₁ promotes cell hypertrophy and accumulation of extracellular matrix (ECM) and epithelial-to-mesenchymal transition (EMT). Among the many regulatory factors of EMT, TGF-β₁ is considered to be an important factor that can deteriorate renal tubular interstitial fibrosis.⁸

At present, the formation mechanism of renal interstitial fibrosis has been thoroughly studied, but the therapeutic methods and efficacy are still limited. Traditional Chinese medicine (TCM) has certain advantages in the prevention and treatment of renal interstitial fibrosis due to its characteristics of multi-pathway and multi-target. TCM plays an important role in the treatment of DN, showing comprehensive treatment advantages in anti-inflammatory, anti-oxidation, reducing urinary protein, protecting renal function and delaying renal fibrosis.⁹ Among them, acteoside has the functions of protecting renal function, immune regulation, anti-oxidation, anti-hypertension through reducing urinary protein and improving the degree of fibrosis and so on.¹⁰ However, the detailed mechanism and whether it is related to anti-inflammatory is not clear. On the basis of previous studies and known clinical efficacy, this original study established a diabetic nephropathy mouse model to observe the expressions of MCP-1 and TGF-β₁ in renal tissue and to explore the therapeutic effect and mechanism of acteoside on DN.

Materials and method

Animals

A total of fifteen 8-week-old male C57BL/6J mice with body weight ranging from 18 to 22g were purchased from the Experimental Animal Centre of Guizhou Medical University. And some of the mice (n = 10) were used as diabetic model. All mice were housed with a 12-h artificial light/dark cycle at a controlled temperature (23°C–25°C) and humidity (55%–65%), and provided with food at libitum for 2 weeks adaptation before initiation of the study. The care and use of experimental animals were performed in conformity of all applicable international, national, and institutional guidelines.

The DN model and groups

After adaptive feeding for 2 weeks with metabolic cage, the mice were randomly divided into 2 groups, including 5 mice in the control group (N group) and 10 mice in the model group, all of which were given free drinking water and ordinary feed. The model group was given a single peritoneal injection of streptozocin (STZ, Sigma, Germany), the dosing concentration is 150 mg/kg body weight, diluted in 0.1 m pre-cooling citrate buffer pH4.5.¹¹ The same amount of citrate buffer was administered to control animals. Random blood glucose level was determined in a drop of tailvein blood using the glucose oxidase method on a blood glucosemeter (Sinocare Inc, Hunan, China) after 48 h. The diabetic model was successfully prepared if they had plasma glucose concentrations exceeding 16.7 mmol/L for 3 times. Urine was collected and the ratio of urinary albumin to urinary creatinine (≥30 mg/g) in urine was detected for three consecutive times, when the result ≥30 mg/g, it means the DN model is successful.¹² Model mice were randomly divided into two groups as follows (n = 5 per group): in the acteoside treatment group (ACT-DN group), the mice were given a gavage of acteoside (Jintaihe pharmaceutical CoLtd, Chengdu, China) 20 mg/kg/day, diluted in 0.9% (w/v) normal saline, acteoside was administered for 8 weeks after the onset of diabetes, and in the diabetic nephropathy group (DN group), the mice were left untreated. DN group was given the same amount of normal saline for 8 consecutive weeks.

Cell culture and groups

The adherent culture of NRK 52E cells (ATCC®CRL-1571 TM, Courtesy of Houfanfan Academician team of Southern Medical University) was performed in DMEM (Gibco, USA) medium containing 1 g/L glucose with 20%FBS (Hyclone, USA) at 37°C and 5% CO2. The NRK 52E cells were first digested by 0.25% trypsin, and then inoculated into a 6-well plate placed on cover glass when fusing to 70%–80%, which were next divided into 3 groups as follows: control group (1 g/L glucose), high glucose group (4.5 g/L glucose), and high glucose+acteoside group (4.5 g/L glucose+20 mg acteoside). Cells were harvested at each time point of 48 h and 72 h.

Sample collection

After 8-weeks admonition of the drug, animals were transferred to individual mouse metabolic cages to collect random and 24-h urine samples. And the non-fasting blood glucose levels of the mice in each group were examined. Next, mice were anesthetized with chloral hydrate. Aortic blood was obtained for the detection of urea nitrogen and creatinine, and their kidneys were rapidly excised. The right kidney tissue was rinsed with phosphate buffered saline (PBS) and then frozen in liquid nitrogen. The left kidney was fixed overnight in 10% (w/v) formalin (pH 7.4) and then stained with HE, PAS and MASSON.

The detection of biochemical indexes

The 24-h urine protein was determined by ELISA according to instructions of the kit (BioAssay Systems, USA). bUrea nitrogen and serum creatinine were detected by colorimetric method, and the operation steps were carried out according to instructions of the kit (BioAssay Systems, USA).

Histological examination

Renal tissue was fixed overnight in 10% (w/v) formalin (pH 7.4), dehydrated with gradient alcohol, transparent with xylene, soaked and embedded in paraffin, then cut into 2–4 μm sections using a microtome, which were stained with HE (Solarbio Life Sciences), PAS (Sigma, Germany) and MASSON (Solarbio Life Sciences) for histopathologic evaluation.

Elisa

TGF-β₁ protein in renal tissue and cultured cells was determined by ELISA. The operation was carried out according to the instructions of the kit (BioAssay Systems, USA).

Western blot

RIPA lysate lysed renal tissue cells to extract proteins, and Bradford method was used to determine the total protein concentration. The protein sample was mixed with 2×SDS loading buffer 1:1 and denatured at 100°C. SDS-PAGE electrophoresis was used to separate proteins and the protein bands were transferred by wet method. Rabbit anti-MCP-1 (1:1000, abcam, Britain) and β-tubulin (1:1000, abcam, Britain) were sealed at room temperature for 1h by 5% skimmed milk powder, incubated overnight at 4°C, and washed. Horseradish peroxidase-labeled secondary antibody was washed at room temperature for 2 h. The reaction time was 2min with electrochemical luminescent agent, and the exposure was developed. The relative expression of the target protein was calculated by the gray scale ratio of the target protein to β-tubulin.

RT-PCR

The expressions of MCP-1 (ThermoFisher, USA) and TGF-β₁ (ThermoFisher, USA) mRNA in renal tissues were detected. Total RNA was extracted according to instructions of the MCP-1 kit, and D260 and D280 were measured with the nucleic acid protein quantitative analyzer, and repeated for 3 times. The RNA purity was determined according to the D260/D280 value. Reverse transcription system (24 μL): 5×ReactionBuffer (5 μL), dNTP (1.25 μL), M-MLV RV (1 μL), Nacclease-free water (13.75 μL), RNA (3 μL). Reaction condition: 42°C, 60min; 70°C, 2min; 1 cycle. RT-PCR reaction system (25 μL): 2×SYBR Premix Ex TAQTM (12.5 μL), DH2O (8.5 μL), cDNA (2 μL), PCR Forward Prime (1 μL), PCR Rervise Prime (1 μL), reaction condition: 95°C, 3 min, 1 cycle; 95°C, 20s, 60°C, 40s, 40 cycles. Results analysis: 3 duplicates were repeated for each sample, and the mean value was taken. Results after internal reference GAPDH homogenization were analyzed by 2-ΔΔCt with the target gene expression in the normal control group as the reference factor. The reverse transcription reaction, RT-PCR reaction and result analysis of TGF-β1 mRNA expression detection in renal tissue were the same as that of MCP-1, reaction condition: 95°C, 3min, 1 cycle; 95°C, 20s, 68°C, 60s, 40 cycles.

Statistical analysis

Statistical analyses were performed using SPSS17.0 software. Measurement data are presented as mean±standard deviation, count data were tested by X2. Two groups were compared by a t test, and multiple comparisons between groups were performed by one-way analysis of variance (ANOVA) followed by Student-Newman-Keuls post-test. Only values of p < .05 were considered statistically significant.

Results

Pathological changes in diabetic kidneys

Light microscopic detection of the kidney tissue was performed to identify pathological changes. The glomerular hypertrophy and hypercellularity, mesangial matrix accumulation, tubular injury, interstitial infiltration of mononuclear cell were seen in mice of non-treated DN group. Pathological changes such as the glomeruli and tubular lesions in ACT-DN group were lighter than those in mice of DN group, and had alleviated to different degrees (Figure1).

Figure 1.

Pathological changes of renal tissues of mice in each group (×400). Abbreviations: NC, normal controls; DN,diabetic nephropathy; ACT-DN, Asteoside-treated DN.

The biochemical parameters of each group

STZ-induced diabetic mice model was developed and evaluated with metabolic parameters. Compared with the control group, blood glucose, 24-h urinary protein quantification, serum creatinine and urea nitrogen were significantly increased in mice of the model group. The biochemical indexes of the ACT-DN group were greatly reduced such as 24-h urinary protein quantification, serum creatinine and urea nitrogenin comparison with those in the DN group, nevertheless, treatments with asteoside had no significant effect on blood glucose level (Figure 2).

Figure 2.

Blood glucose,24-hr urinary protein quantification, serum creatinine and urea nitrogen levels in each group. Abbreviations: NC, normal controls; DN, diabetic nephropathy; ACT-DN, Asteoside-treated DN; *P < .05,***P < .001 vs. NC group; #P < .05,###P < .001 vs. DN group.

Expression levels of MCP-1 and TGF-β1 protein in kidney tissues

Western Blot and ELISA were respectively performed to determined the expressions of MCP-1 and TGF-β1 protein level, which were both significantly higher in mice of the DN group in comparison with those of the NC group. Compared with the DN group, the expression levels of MCP-1 and TGF-β1 protein in ACT-DN group were obviously lower. MCP-1, in particular, was only a quarter of the level in the DN group (p < .05). (Figure 3).

Figure 3.

Expression levels of MCP-1 and TGF-β1protein by Western Blot and ELISA respectively in each group. Abbreviations: NC, normal controls; DN, diabetic nephropathy; ACT-DN, Asteoside-treated DN; *P < .05,**P < .01,***P < .001 vs. NC group;#P < .05, ##P < .01,###P < .001 vs. DN group.

Expression levels of MCP-1 and TGF-β1 mRNA in kidney tissues

Following the detection of MCP-1 and TGF-β1 protein, RT PCR were performed to determined the expressions of MCP-1 and TGF-β1mRNA level, which were both distinctly higher in mice of the DN group in comparison with those of the NC group (p < .05). Compared with the DN group, the expression levels of MCP-1 and TGF-β1 mRNA in ACT-DN group were obviously lower (p < .05). (Figure 4).

Figure 4.

Expression levels of MCP-1 and TGF-β1mRNA by RT-PCR in each group. Abbreviations: NC, normal controls; DN, diabetic nephropathy; ACT-DN,Asteoside-treated DN; *P < .05,**P < .01,***P < .001 vs. NC group;#P < .05, ##P < .01,###P < .001 vs. DN group.

Expression levels of MCP-1 and TGF-β1 protein in NRK-52E cell

To further confirm the expressions of MCP-1 and TGF-β1 and the therapeutic effects of asteoside on diabetic nephropathy, NRK-52E cells were next cultured in vitro. Similarly, the results of cell culture were consistent with those of vivo experiment. Western Blot and ELISA demonstrated that the expression levels of MCP-1 and TGF-β1 protein in high glucose group (HS group) were both greatly enhanced than those in normal control group as well. Compared with the HS group, the expression levels of MCP-1 and TGF-β1 protein in high glucose+acteoside group (HS+ACT group) were significantly decreased (p < 0.05). (Figure 5).

Figure 5.

Expression levels of MCP-1 and TGF-β1protein by Western Blot and ELISA respectively inNRK-52E cell Abbreviations: NC, normal controls; HS, high glucose group; HS+ACT, high glucose+acteoside group; *P < .05,**P < .01,***P < .001 vs. NC group;#P < .05, ##P < 0.01,###P < .001 vs. HS group.

Expression levels of MCP-1 and TGF-β1 mRNA in NRK-52E cell

RT-PCR results showed that the mRNA expression levels of MCP-1 and TGF-β1 in HS group were significantly higher than those in NC group. Compared with the HS group, they were markedly decreased in the HS+ACT group. (Figure 6).

Figure 6.

Expression levels of MCP-1 and TGF-β1mRNA by RT-PCR in NRK-52E cell Abbreviations: NC, normal controls; HS, high glucose group; HS+ACT, high glucose+acteoside group; *P < .05,**P < .01,***P < .001 vs. NC group;#P < .05, ##P < .01,###P < .001 vs. HS group.

Discussions

Diabetic nephropathy is one of the most common microvascular complications of diabetes, the early clinical manifestations of which were slight, and the pathological features were as follows: glomerular hypertropgy, increase of masangial cells and the accumulation of mesangial matrix, tubular epithelial hypertrophy with vacuolar degeneration and interstitial inflammatory cell infiltration in the early-stage of DN, which gradually progressed to interstitial fibrosis and end-stage renal disease. The pathogenesis of DN has not been completely elucidated. Numerous studies suggest that inflammatory cells infiltration, increased cytokines and inflammatory medium level occur in diabetes mellitus (DM),^13,14 moreover, both in early-stage of type 1 and type 2 diabetes kidney disease, there were monocyte/macrophage infiltration in renal tissue.¹⁵ Infiltrated monocyte/macrophage destructed the renal structure through releasing cytokines, inflammatory mediators and oxygen free radicals, which accelerated the process of glomerular sclerosis.

Researchers have shown that inflammation is a key factor in the pathogenesis of DN,¹⁶ many cytokines are involved in the inflammatory response such as interleukin 6 (IL-6), TGF-β1, MCP-1 and so on, which play different roles^17,18 and constitute a complex cytokines network. Among them, MCP-1 and TGF-β1 are closely related to the progression of DN. At present, MCP-1 and TGF-β1 have became an important research direction in diabetes kidney disease (DKD).

As a member of the inflammatory chemokine superfamily, MCP-1 could be released by a variety of cells and is low-expressed under physiological conditions. However, in pathological conditions such as hyperglycaemia and oxidized lipoprotein, MCP-1 gene expression could be stimulated by mesangal cells and endothelial cells respectively. On one hand, the high expression of MCP-1 can stimulate the expression of interleukin1 (IL-1) and TGF-β1 produced by renal tissue cells through autocrine or paracrine forms and aggravate the injury of endothelial cells. On the other hand, mesangial cells can be directly activated to produce muscle fibrin, which lead to glomerular fibrosis. In addition, monocytes/macrophages in blood circulation can be aggregated and activated in the inflammatory area of renal tissue. Activated macrophages can release reactive oxygen species, inflammatory mediators and growth factors, which promote the increase and deposition of ECM and eventually lead to the occurrence of glomerulosclerosis.¹⁹ There were evidences that suggested that MCP-1 was a key factor in the inflammatory process of the early-stage of atherosclerosis and also exerted a central role in renal lesion, which accelerated the development of renal fibrosis and promote the progression of DN.

Transforming growth factor-β mainly distributed in the kidney was a core factor in the complex cytokine network associated with the pathogenesis of DN, which can regulate tissue damage and repair in normal conditions. Excessive production of TGF-β1 in diabetes was associated with chronic fibrosis. Furthermore, the expression of TGF-β1 has been implicated in oxidative stress enhanced by continuous hyperglycemia.²⁰ TGF-β1 can mediate TGF-β1/Smad signaling pathway to promote the aggregation of ECM, and to reduce the activity of the enzymes that degrade ECM, as well as to promote EMT, and eventually lead to glomerular hypertrophy and interstitial fibrosis. In addition, TGF-β1 can also induce the self-phagocytosis of renal tubular epithelial cells and promote the apoptosis of renal tubular epithelial cells, resulting in renal tubular damage.²¹ Meanwhile, TGF-β1 is correlated with the formation of urinary protein, leading to glomerulosclerosis.²² These effects above ultimately lead to the decrease of glomerular filtration rate and chronic renal failure.²³

The high expression of TGF-β1 upregulated and enhanced the transcription of MCP-1 through the nuclear transcription factor-κB (NF-κB) signaling pathway. Meanwhile, the high expression of MCP-1 can activate the p38MAPK signaling pathway through the inflammatory response factors released by activated monocytes/macrophages, which produced TGF-β1, thereby promoting glomerular fibrosis and leading to renal lesions. Studies have shown that when recombinant TGF-β1 was present in humans, the level of MCP-1 in renal interstitium was increased, and macrophages recruited by MCP-1 can in turn stimulate the production of TGF-β1 by interstitial myofibroblasts, which promoted the deposition of ECM and eventually caused renal interstitial fibrosis. Therefore, TGF-β1 and MCP-1 interacted in direct or indirect ways, creating a vicious cycle, aggravating the inflammatory response of DN, and promoting the progress of DN. In consequence, MCP-1 and TGF-β1 played an vital role in the development of diabetic nephropathy, which indicated that MCP-1 and TGF-β1could be used as biomarkers to evaluate the development of DN.^24,25 Inhibition of TGF-β1 and MCP-1 can effectively reduce the local aggregation of monocyte/macrophages and decrease ECM deposition as well as postpone the progression of DN.

Acteoside is the main component of total glycoside of Leucodiae, and its activated component is phenylethanolglycoside, which has critical biological function such as anti-inflammation, immunomodulation, anti-tumor, as well as anti-oxidation and other effects.²⁶ Experimental studies had demonstrated that acteoside used for a period of time can alleviate some pathological changes in the early-stage of DN such as glomerular hypertrophy, basement membrane thickening, and ECM accumulation.²⁷ In addition, in the simulation model of renal fibrosis with unilateral ureteral obstruction, acteoside can significantly reduce renal tubulointerstitial fibrosis.²⁸ Studies have found that severe renal interstitial inflammatory cell infiltration and fibrosis can occur in the DN rat model, and EMT was an crucial mechanism for the onset and progression of renal interstitial fibrosis.²⁹ Relevant studies have verified that acteoside can down-regulate TGF-β1 of diabetic renal tissue and inhibit EMT to delay the progression of renal fibrosis.^30,31 Our study implied that the treatment group with acteoside had obviously better effects on reducing 24-h urinary protein, urea nitrogen, creatinine as well as glomerular fibrosis than those of the model group, which was consistent with the previous research results. In terms of mechanism research, both in vivo and vitro experiments, the results consistently demonstrated that the expressions of MCP-1 and TGF-β1 in the model group were significantly up-regulated. Meanwhile, it was also found that acteoside could inhibit the expressions of MCP-1 and TGF-β1 in the renal tissue. Our findings suggested that acteoside played a role in the prevention and treatment of diabetic nephropathy by inhibiting inflammation and fibrosis key factors MCP-1 and TGF-β1. Furthermore, we also found acteoside reduced inflammatory cell infiltration in HE, PAS and MASSON staining sections. Recently, more and more emerging evidences suggest that acteoside has a wide range of therapeutic effects and comprehensive treatment value, along with less adverse reactions. In view of above-mentioned advantages, acteoside is a promising drug.

There may be some possible limitations in this study. Firstly, we did not calculate the sample size, and determination of sample size is only based on the literature, which may cause bias. Secondly, the study focused on inflammatory cytokines, fibrosis downstream signaling pathways were not shown. Thirdly, as reported in other studies, acteoside has many effects such as anti-oxidative stress, anti-inflammation, immunomodulation, present study only demonstrates a small part of anti-inflammatory cytokines and inflammation. Therefore, the mechanism of acteoside’s protective effect on diabetes nephropathy still deserves further studies.

Conclusion

To conclude, this study demonstrates that acteoside have a great protective effect on renal damage caused by DN. These beneficial effects are closely related to down-regulate the expressions of MCP-1 and TGF-β₁, which is of profound significance in the development of new strategies for the prevention and treatment of DN.

Footnotes

Acknowledgements

The authors would like to express their gratitude to the Central Laboratory of Guizhou Provincial People’s Hospital for providing experimental equipment.

Declaration of conflicting of interests

The author(s) declared no potential conflicts of interest with respect to the research,authorship,and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research,authorship,and/or publication of this article: This work was supported by grants from the National Natural Science Foundation of China (grant number 81760134 to Z. Y),Joint Foundation of Guizhou Provincial Science and Technology Department-Guizhou Provincial People’s Hospital (LH[2017]1102 to L. X,LH[2016]7170 to X. Y),Guizhou Science and Technology Support Plan Project(Guizhou Science and Technology Support Project [2019] No2801 to Z. Y),the Special Fund for Basic Scientific Research Operating of Central Public Welfare Research Institutes,the Chinese Academy of Medical Sciences (2019PT320003 to Z. Y),Guizhou high-level innovative talents program [ QKHPTRC(2018)5636 to Z. Y ],Guizhou Province Clinical Research Center for Kidney Disease (QKHPTRC[2020]2201 to Z. Y).

Ethics approval

The study was approved by the Ethics Committee of Guizhou Provincial People's Hospital (Guiyang,China). No. LH[2016]7170

Animal welfare

The study followed the guideline “Care and Use of Laboratory Animals formulated by the Ministry of Science and Technology of China”.

ORCID iD

Xin Lin

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Effects of acteoside on the expressions of MCP-1 and TGF-β 1 in the diabetic nephropathy mice