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TIMPs system in streptozocin-induced diabetic nephropathy in rats
Author’s Accepted Manuscript Renoprotective effects of berberine through regulation of the MMPs/TIMPs system in streptozocin-induced diabetic nephropathy in rats Wei-Jian Ni, Hai-Hua Ding, Hong Zhou, Yuan-Ye Qiu, Li-Qin Tang www.elsevier.com/locate/ejphar
PII: DOI: Reference:
S0014-2999(15)30168-0 http://dx.doi.org/10.1016/j.ejphar.2015.07.040 EJP70139
To appear in: European Journal of Pharmacology Received date: 24 May 2015 Revised date: 13 July 2015 Accepted date: 16 July 2015 Cite this article as: Wei-Jian Ni, Hai-Hua Ding, Hong Zhou, Yuan-Ye Qiu and Li-Qin Tang, Renoprotective effects of berberine through regulation of the MMPs/TIMPs system in streptozocin-induced diabetic nephropathy in rats, European Journal of Pharmacology, http://dx.doi.org/10.1016/j.ejphar.2015.07.040 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Renoprotective effects of berberine through regulation of the MMPs/TIMPs system in streptozocin-induced diabetic nephropathy in rats1 Wei-Jian Ni1, Hai-Hua Ding1, Hong Zhou2, Yuan-Ye Qiu1, Li-Qin Tang1* Author names and affiliations: W.-J. Ni 1
Affiliated Anhui Provincial Hospital; Anhui Medical University, Anhui Province,
People’s Republic of China. E-mail: [email protected] H.-H. Ding 1
Affiliated Anhui Provincial Hospital; Anhui Medical University, Anhui Province,
People’s Republic of China. E-mail: [email protected] H. Zhou, 2
Anhui Provincial Cancer Hospital, Anhui Province, People’s Republic of China.
E-mail: [email protected] Y.-Y. Qiu 1
Affiliated Anhui Provincial Hospital; Anhui Medical University, Anhui Province,
People’s Republic of China. E-mail: [email protected] L.-Q. Tang 1
Affiliated Anhui Provincial Hospital; Anhui Medical University, Anhui Province,
People’s Republic of China. E-mail: [email protected] *Address for correspondence: L.-Q.Tang, Affiliated Anhui Provincial Hospital; Anhui Medical University, 17# Lujiang Road, Hefei 230001, Anhui Province, People’s Republic of China. E-mail: [email protected]
Tel/Fax: +86551-62283378-809
1
Acknowledgments
This work was supported by the National Natural Science Foundation of China (No.81102864), the Anhui Provincial Natural Science Foundation (China, No. 1508085MH179). 1
Abstract Berberine has proven protective effects on diabetic nephropathy, but the mechanism for its effects has not been comprehensively established. Hence, we aimed to explore the renoprotective mechanism of berberine on the accumulation of extracellular matrix, alterations of its major components and corresponding changes in the regulatory system, including the matrix metalloproteinases/tissue inhibitor of matrix metalloproteinases (MMPs/TIMPs) system, in diabetic nephropathy rats. In the experiments, diabetic nephropathy rats were treated with berberine (0, 50, 100, 200 mg/kg) respectively. The protein levels of transforming growth factor-β1 were then detected by Western blot, while fibronectin and type IV collagen levels were assessed using immunohistochemistry. Changes in the MMP2/9 and TIMP1/2 levels were detected using two forms simultaneously. In addition, we also measured the characteristics and biochemical indicators of the diabetic nephropathy rats. The results showed that berberine could ameliorate the fasting blood glucose, and the majority of biochemical and renal function parameters, but did not have an effect on body weight. Immunohistochemistry and Western blot examination revealed a significant increase in the MMP9 and TIMP1/2 levels, with an obvious decrease in MMP2 expression in the diabetic nephropathy rats. Berberine (100 and 200 mg/kg) could significantly improve the abnormal changes in the MMPs/TIMPs system. Meanwhile, reductions in the transforming growth factor-β1, fibronectin and type IV collagen expression levels were observed in the berberine treatment groups. Therefore, the renoprotective effects of berberine on diabetic nephropathy might be associated with changes in the extracellular matrix through the regulation of the MMPs/TIMPs system in the rat kidney.
Keywords: Diabetic nephropathy; berberine; MMPs/TIMPs system; ECM; renal fibrosis
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1. Introduction Diabetic nephropathy (DN), the major cause of end-stage renal disease (ESRD) worldwide, is one of the most common and severe chronic complications of diabetes mellitus (DM) (Sidaway, 2014). It is characterized by proteinuria, mesangial expansion, glomerular basement membrane (GBM) thickening, persistent fibrosis and a gradual decrease in renal function, which result from increasing extracellular matrix (ECM) deposition at the glomerular level (Kato and Natarajan, 2014). A number of factors are important for the development of DN, including hyperglycemia, hypertension, oxidative stress, and inflammation, and have been shown to lead to proteinuria and histological changes, such as mesangial expansion, glomerular sclerosis, and tubulointerstitial fibrosis (Muskiet et al., 2014). It has been reported that renal fibrosis is final the common pathologic finding observed in the later stages of the disease, and it is characterized by transforming growth factor-β1 (TGF-β1) signalling-mediated ECM accumulation (Li et al., 2014). This, of course, results from the production of fibronectin (FN) and type IV collagen promoted by TGF-β1 (Ueno et al., 2013). Research has shown that the matrix metalloproteinases and tissue inhibitors of the matrix metalloproteinase (MMPs/TIMPs) system have non-neglectable roles in ECM accumulation and deposition (DeCoux et al., 2014; Raffetto and Khalil, 2008). Furthermore, the alterations in the MMPs and TIMPs are usually regulated by TGF-β (Kobayashi et al., 2014). Berberine (BBR; [C20H18NO4]+), a type of isoquinoline alkaloid, is the major active component of Rhizoma coptidis and Cortex phellodendri extraction(Feng et al., 2015). Research has shown that berberine exhibits multiple pharmacological activities, including lowering blood glucose and anti-oxidative stress as well as reducing inflammation, indicating that berberine has general clinical and research value and is a promising therapeutic drug in DN treatment (Ni et al., 2015). Previous studies have demonstrated that in berberine-treated DN rats, the degree of pathological changes was significantly reduced: mesangial matrix accumulation and mesangial expansion were scarcely detected, and the glomerulus atrophy and glomerular sclerosis were also gradually relieved. Berberine could effectively ameliorate renal dysfunction and 3
regulate TGF-β1 expression (Xie et al., 2013). Meanwhile, the elevated TGF-β1 levels induced the secretion of MMP2 and TIMP2, suggesting that there may be some relationship among TGF-β1 expression, ECM accumulation, alteration of the MMPs/TIMPs system and the renoprotective effects of berberine (Sekiuchi et al., 2012). However, the relationships and mechanisms remain incompletely understood. The aim of this study was to investigate the degradation effects of berberine on the ECM, the alterations of its major components, the corresponding changes in its regulatory system, i.e., the MMPs/TIMPs system and the possible molecular mechanisms in a streptozotocin (STZ)-induced diabetic nephropathy rat model.
2. Materials and Methods 2.1 Reagents The berberine was provided by the Anhui Provincial Hospital. STZ and dimethyl sulfoxide (DMSO) were obtained from Sigma Chemical Co. (St. Louis, MO, USA). The anti-type IV collagen (BA2174), anti-FN (BM1871) and anti-TGF-β1 (BA0290) antibodies were obtained from Boster Biological Technology (Boster CO., LTD, China). The anti-MMP2 (bs-0412R), anti-MMP9 (bs-2099R), anti-TIMP1 (bs-0415R) and anti-TIMP2 (bs-0416R) antibodies were purchased from Biosynthesis Biotechnology (Bioss CO., LTD, China). The superSignal west femto kit was purchased from Thermo (Thermo Fisher Scientific, USA). Other chemicals used in these experiments were of analytical grade from commercial sources. 2.2 Animal and experimental protocols A total of 50 mature, Sprague-Dawley rats (male, 180±20 g. Grade II, Certificate No. 028) were purchased from the Animal Department of Anhui Medical University (Hefei, China). Rats were allowed to feed on a standard diet and drink ad libitum and were adapted to the experimental conditions at 20±2◦C, humidity of 60±5% and a 12 h light-dark cycle. After an overnight fast, the rats were administered a single intraperitoneal injection of 35 mg/kg STZ dissolved in a 0.1 mM chilled citrate-phosphate buffer (pH 4.5) and 4
injected immediately within 30 min. Normal control (NC) rats were injected with citrate-phosphate buffer. The development of hyperglycemia (diabetes) in the rats was confirmed by fasting blood glucose (FBG) estimation (≥11.1 mmol/L) 72 h after STZ injection, and only uniformly diabetic rats were included in the DN control (DNC) and drug treatment groups (n=10, each group). Rats in the NC and DNC groups were given an equal volume of the carboxymethyl cellulose (CMC-Na), while the berberine (50, 100, 200 mg/kg) groups were intragastrically administered berberine dissolved in 0.5% CMC-Na at doses of 50, 100 and 200 mg/kg body weight per day, respectively, for 8 weeks starting on the 7th day. Kidney samples were rapidly isolated and frozen in liquid nitrogen or fixed in 10% formaldehyde buffer. Blood was collected, and the serum was extracted after centrifugation and stored at -80◦C until analysis. All experiments were approved by the Ethics Review Committee for Animal Experimentation of Anhui Medical University. 2.3 Measurement of renal function and biochemical markers Animal weight was measured every two weeks from 0 to 8 weeks. Fasting blood glucose was measured every 2 weeks using tail vein blood samples. During the experiment, rats were housed in metabolic cages for 24 h to collect urine for subsequent measurements of urinary total protein (UTP). Blood samples were obtained from the femoral artery and allowed to clot for 30 min at 4◦C, then centrifuged (1268 g, 10 min, 4◦C). The supernatant was collected for measurement of blood urea nitrogen (BUN) and serum creatinine (SCr) by using a Hitachi Model 7600 Series Automatic biochemistry Analyzer. After removing the capsule of the right kidney, the kidneys were weighed. The kidney weight to body weight ratio was calculated. 2.4 Immunohistochemical analyses Immunohistochemical staining for fibronectin, type IV collagen, MMP-2/9 and TIMP-1/2 were performed using a streptavidin-biotin complex (SABV) technique. In brief, the sections were defined, and the endogenous peroxidase activity was quenched by 10 min of incubation with 3% H2O2. The sections were placed in 10 mmol/L citrate buffer, microwaved for 30 min and then cooled to room temperature. 5
Sections were placed in PBS for 10 min, followed by protein-blocking for 15 min. The slides then were incubated for 1 h with primary antibody against anti-MMP2/9, anti-FN, anti-type IV collagen and anti-TIMP1/2 antibody at a 1:100 dilution. A secondary biotinylated antibody was added to the sections for 30 min at 37◦C. For coloration, the slides were incubated at room temperature with a mixture of 0.05% 3, 3-diaminobenzidine (DAB) containing 0.01% H2O2. Finally, areas of positive immunostaining were quantified by computer-based morphometric analysis. The degree of positive staining was determined as the mean of ten different fields on each slide. 2.5 Western blot analyses For protein preparation, the kidney tissues were suspended in RIPA buffer. The protein samples were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electrotransferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA, USA). After blocking with 5% skim milk in Tris-phosphate buffered saline (TPBS) for 2 hours, the membranes were incubated with anti-TGF-β1 (1:200), anti-TIMP1/2 (1:250) and anti-MMP2/9 (1:250) antibodies at 4◦C, with gentle shaking overnight and reblotted with anti-β-actin antibody to verify equal loading of the protein in each lane. After incubation with the appropriate peroxidase-conjugated secondary antibodies, the signals were visualized using the enhanced chemiluminescence Western blot Detection System, in accordance with the manufacturer’s instructions (ImageQuant LAS 4000 mini, GE Healthcare). 2.6 Statistical analyses All experiments were performed at least three times, and the results were reproducible. All values are expressed as the mean ± S.D. Statistical analyses of the data were performed by oneway ANOVA with GraphPad Prism 5.0. Values of P<0.05 were considered to be statistically significant in all cases.
3. Results 3.1 Berberine ameliorates the abnormalities of renal function and biochemical markers in DN rats 6
The significant increase in blood glucose was detected after the induction of diabetes by streptozocin (P<0.01). Administration of berberine (50 mg/kg) did not reduce blood glucose levels, and berberine (100, 200 mg/kg) had undistinguishable hypoglycaemic effects short-term treatment (0-4 week), suggesting that low dosage and short-term treatment are not likely to produce a satisfactory outcome. Berberine (100, 200 mg/kg) was proved to be satisfactory by continuous treatment compared with the DNC group, which suggested that it may be a long-term method to reduce blood sugar (Fig 1A). Diabetic rats had a lower body weight than the NC rats from the 2nd week onwards, while berberine treatment had no effect on decreasing the body weight in the therapeutic process (Fig 1B). The ratio of kidney weight to body weight in the diabetic rats was significantly increased after streptozocin-injection compared with the NC group. Different dosages of berberine could effectively decrease the ratio of kidney weight to body weight, suggesting that there is indeed a potential role for improving the kidney weight for berberine under conditions of diabetic nephropathy (Fig 1C). The urinary total protein (UTP), blood urea nitrogen (BUN) and serum creatinine (SCr) levels of the DNC group were significantly higher than those of the NC rats after the streptozocin injection. Berberine (100, 200 mg/kg) treated diabetic nephropathy rats exhibited a remarkable decrease in the UTP, BUN, and SCr levels. There were no differences in the UTP level between berberine (50 mg/kg) and untreated streptozocin-induced diabetic nephropathy rats (Fig 1D-F). 3.2 Immunohistochemical staining for type IV collagen, fibronectin, MMP2/9 and TIMP1/2 We next examined whether berberine inhibits the increase in type IV collagen and fibronectin. Semi-quantitative analysis exhibited a significant rise in the expression of type IV collagen and fibronectin in the DNC group (P<0.01). Berberine treatment could significantly reduce the expression levels of type IV collagen and fibronectin in a dose-dependent manner (Fig 2-3). The immunohistochemical staining showed a decreased expression level of MMP2 and an increased pattern of TIMP-2 distribution 7
in the DNC group (Fig 4A). The expression level of TIMP-1 was elevated, and the distribution of MMP-9 was similar to the pattern shown by its specific inhibitor, TIMP-1, compared with the NC group (Fig 5A). Understandably, the semi-quantitative analysis showed that the ratio of MMP-2/TIMP-2 was decreased in the DNC group (Fig 4B), which was consistent with the ratio of MMP-9/TIMP-1 (Fig 5B). It is worth noting that berberine treatment could reverse the abnormal changes and significantly up-regulate the ratio of MMP-2/TIMP-2 and MMP-9/TIMP-1, which suggested that berberine might prove to be of great protective effect against diabetic nephropathy, acting by reducing ECM accumulation and accelerating the degradation process. 3.3 Western blot analysis of the expression of TGF-β1, MMP2/9 and TIMP1/2 When compared with the NC rats, increased TGF-β1 expression was observed in the diabetic rats. Berberine (100, 200 mg/kg) treatment could significantly decrease the expression of TGF-β1. In contrast, no significant changes in decreasing this protein were noted in the berberine (50 mg/kg) treatment group (Fig 6). Western blot analysis also showed the relatively lower expression levels of MMP-2, while higher expression levels of TIMP-2 were observed compared with the NC group. The ratio of MMP-2/TIMP-2 was also decreased subsequently (Fig 7). Furthermore, in our experiment, TIMP-1 expression was elevated in the kidney tissues of untreated rats compared to the NC group. The expression pattern of MMP-9 was similar to that of its specific inhibitor, TIMP-1. It should be noted, however, that the increasing amplitude was different. That is to say, the increasing magnitude of TIMP-1 was higher than that of MMP-9, producing a decreased MMP-9/TIMP-1 ratio (Fig 8). Compared with the DNC group, the DN rats treated with berberine (50 mg/kg) showed a slight effect on ameliorating the symptoms above. However, the berberine (100, 200 mg/kg) treatment could significantly up-regulate the ratio of MMP-2/TIMP-2. Moreover, berberine (100, 200 mg/kg) treatment could also significantly increase the expression level of MMP-9 and help restore the damaged balance of MMP-9/TIMP-1 (Fig 7-8). 8
4. Discussion The study presented here evaluated the renoprotective role of berberine in diabetic nephropathy. According to the experiment results, fasting blood glucose, body weight were higher in the diabetic rats than in the NC rats. In the DNC group, the levels of hematologic traits and urine biochemical parameters, including UTP, BUN and Scr, were remarkably increased after STZ injection. These data suggested that the rat model of DN was successfully established (Betz and Conway, 2014). After treatment with berberine, the physiological indicators in the cases showed varying degrees of improvement (Zhao et al., 2014). The data above also suggested that berberine could not only reduce the blood glucose and urine total protein excretion except for the body weight but also improve the renal pathological changes, such as ECM accumulation, glomerulus atrophy and glomerular sclerosis, thus preventing the progression of DN (Wang et al., 2013). These changes indicated that berberine had desirable protective effects on the renal structure and physiological functions of DN rats. DN is one of the most common diabetic microvascular complications. It is characterized by increased ECM deposition and reduced matrix degradation. The ECM is mainly composed of type IV collagen and fibronectin. In essence, the maintenance of the ECM is the result of the dynamic balance between ECM protein synthesis and degradation (Brown et al., 2015). However, this balance is disturbed under the diabetic state, resulting in alterations in the main pathological processes, such as abnormalities in the renal structures and functions, glomerular sclerosis and renal fibrosis (Simonson, 2007). One encouraging note is that berberine treatment could decrease the expression levels of type IV collagen and fibronectin. This seems to be a possible justification for the hypothesis that berberine exerts its renoprotective role by inhibiting ECM accumulation, which indicates that berberine has wide research potential. The MMPs/TIMPs system is the most important enzyme degrading system of proteins in the diabetic kidney. The expression levels and activities of the system directly impact the ECM balance. MMPs exist in the kidney tissue as zymogens and are activated by plasmin; at the same time, they remain under the influence of TIMPs 9
(Giannandrea and Parks, 2014). In physiological conditions, the MMPs and TIMPs were in the equilibrium condition as being adjusted by the homeostasis to control the quantity of activated MMPs within the allowable range, so that they could help maintain a stable ECM (Tan and Liu, 2012). However, this balance is also shifted under diabetic conditions, leading to abnormal ECM accumulation and degradation (Han et al., 2006). As important components of the MMPs/TIMPs regulation system, MMP-2 and MMP-9 play key roles in the development of DN (Solini et al., 2011). Despite stimulating new insight into the MMPs/TIMPs and a profitable decade of medical research attention, the regulation mechanisms of MMPs/TIMPs and the effects of berberine on this system remain enigmatic (Catania et al., 2007). Indeed, we note that changes in MMP2 and MMP9 under diabetic state are opposite, motivating us to explore the special role of TIMPs in our research. Therefore, we found that the ratios of MMP2/TIMP2 and MMP9/TIMP1 were similar, and both were reduced under the diabetic nephropathy state. After treatment, we found that berberine reversed the abnormal changes of MMP2 and MMP9, while reducing the expression of TIMP1 and TIMP2 and elevating the ratio of MMP2/TIMP2 and MMP9/TIMP1. These changes alleviate the inhibitory effects of TIMP1 and TIMP2 and enhance the degradation effects of MMP2 and MMP9, eventually ameliorating the accumulated ECM and renal fibrosis (Rysz et al., 2007). Because berberine treatment was observed to regulate the MMPs/TIMPs system in the present study, we conclude that berberine could inhibit ECM accumulation by regulating the MMPs/TIMPs system in the kidneys of DN rats. This suggests that berberine is a promising therapeutic drug for DN treatment. The TGF-β related signalling pathway is a well-known mediator in DN progression, mediating fibrosis by stimulating the synthesis of ECM molecules and by decreasing ECM degradation (Deshpande et al., 2013). A variety of molecular mediators and intracellular signalling pathways associated with DN, such as the hyperglycemia, angiotensin II, and oxidative stress pathways, are connected and uniformly stimulate the TGF-β related signalling pathway (Ni et al., 2015). In addition, TGF-β1 has also been recognized as a powerful multifunctional cytokine that plays an important role in 10
regulating the MMPs/TIMPs system (Zhang et al., 2015). In accordance with these findings, our present study showed that the expression of TGF-β1 was increased remarkably (P<0.01) in diabetic rats, in conjunction with the abnormal changes in the MMPs/TIMPs system, suggesting that the aforementioned viewpoints are correct. When the expression level of TGF-β1 decreased, the abnormal ratio of the MMPs/TIMPs system returned to normal after the berberine treatment. These data were consistent with the results for the MMPs/TIMPs system and the ECM component proteins from immunochemistry. It seems that berberine could regulate the ratio of the MMPs/TIMPs system and increase the quantity and activity of the MMPs by inhibiting the expression and production of TGF-β1. This issue may be resolved through the study of tissue-specific TGF-β1 deficient rats in a follow-up experiment. The aforementioned changes indicated that berberine could decrease the expression levels of TGF-β1 and gradually increase the ratios of MMP-2/TIMP-2 and MMP-9/TIMP-1. The increased MMP-2/TIMP-2 and MMP-9/TIMP-1 ratios will then ameliorate the role of TIMP1/2 on the inhibitory effects of MMP9/2. Furthermore, berberine can also increase the expression and activities of MMP2 and MMP9, which leads to decreased ECM deposition and increased ECM degradation, thus ameliorating the structural damage and functional disorders of the kidney, such as mesangial expansion, glomerulus atrophy and glomerular sclerosis, and preventing the progression of DN (Yang et al., 2014). Recent research from our group has suggested that berberine could exert its renoprotective effects by regulating the Gαi-AC-cAMP signalling pathway. The alteration in the Gαi-AC-cAMP signalling pathway produced decreased levels of TGF-β1, which contributed to the renal protective effects under identical experimental conditions (Tang et al., 2013). We therefore assumed that berberine regulates the Gαi-AC-cAMP signalling pathway to elevate the levels of cAMP. The increased cAMP acts as a second messenger and is intimately linked with the suppression of TGF-β1. The decreased levels of TGF-β1 then up-regulate the ratio of MMPs/TIMPs to enhance the degradation effects of the MMPs. Next, MMP2 and MMP9 reduced the ECM accumulation by inhibiting the production of type IV collagen and fibronectin, thus playing pivotal roles in 11
ameliorating the mesangial expansion, glomerulus atrophy and glomerulosclerosis. In further trials, more controlled experiments are needed to confirm the proposed mechanisms.
5. Conclusions In conclusion, our data demonstrated that berberine has a renoprotective effect on diabetic nephropathy that might be associated with changes in the extracellular matrix through the regulation of the MMPs/TIMPs system in streptozocin-induced diabetic neophropathy rats. These results suggest that berberine has a therapeutic role in the prevention of diabetic nephropathy and is a promising anti-diabetic nephropathy drug.
Acknowledgments This work was supported by the National Natural Science Foundation of China (No.81102864), the Anhui Provincial Natural Science Foundation (China, No. 1508085MH179).
Conflict of interest There are no competing financial interests in relation to the work.
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Figure legends Fig 1. Berberine ameliorates abnormalities in the renal functions and biochemical markers of DN rats The DN rat model was induced using streptozotocin via a single intraperitoneal injection at a dose of 35 mg/kg after the rats were provided with a high sugar/ lipid diet for 8 weeks. Berberine (50, 100, 200 mg/kg/day) was given intragastrically to the diabetic nephropathy rats from the first week to last week after injection. The fasting blood glucose (FBG) levels (A), body weight(B), kidney weight-to-body weight ratio(C), urinary total protein (UTP) excretion(D), blood urea nitrogen (BUN) and serum creatinine (SCr) levels (E) were determined. **P<0.01 versus the NC group; #P<0.05 and ##P<0.01 compared with the DNC group.
Fig 2. Immunohistochemical staining for the expression of type IV collagen in the kidneys of the DN rats. A. Representative immunohistochemical staining analyses of the expression of type IV collagen in the streptozotocin-induced diabetic nephropathy rats. B. Quantification 15
of immunohistochemical staining analysis of type IV collagen in the streptozotocin-induced diabetic nephropathy rats. All data were normalized to the NC group value and are represented as the mean±S.D. ##
P<0.01 versus the NC group; **P<0.01 versus the DNC group.
Fig 3. Immunohistochemical staining for the expression levels of fibronectin in the kidneys of the DN rats. A. Representative immunohistochemical staining analyses of the expression of fibronectin in streptozotocin-induced diabetic nephropathy rats. B. Quantification of immunohistochemical staining analysis of fibronectin in the streptozotocin-induced diabetic nephropathy rats. All data were normalized to the NC group value and are represented as the mean±S.D. ##
P<0.01 versus the NC group; *P<0.05 and **P<0.01 versus the DNC group.
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Fig 4. Immunohistochemical staining for the expression levels of MMP-2 and TIMP-2 in the kidneys of the DN rats. A. Representative immunohistochemical staining analysis of the expression of MMP-2 and TIMP-2 in the kidneys of diabetic nephropathy rats. B. Quantification of immunohistochemical staining analysis of MMP-2 and TIMP-2 in streptozotocin-induced diabetic nephropathy rats. The expression levels of MMP-2 were significantly decreased in the DNC rats compared to the NC group rats. In contrast, the TIMP-2 protein levels in the DNC group were significantly increased compared to those of the NC rats. A low concentration of berberine (50 mg/kg) could slightly improve the above phenomena (P<0.05), while a higher concentration of berberine (100, 200 mg/kg) could significantly change the levels of MMP-2 and TIMP-2 and the ratio of MMP-2 and TIMP-2 (P<0.01). All data were normalized to the NC group value and are represented as n=5, mean±S.D. **P<0.01 versus the NC group; #P<0.05 and ##P<0.01 compared with the DNC group.
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Fig 5. Immunohistochemical staining for the expression of MMP-9 and TIMP-1 in the kidneys of the DN rats. A. Representative immunohistochemical staining analysis of the expression of MMP-9 and TIMP-1 in the kidneys of diabetic nephropathy rats. B. Quantification of immunohistochemical staining analysis of MMP-9 and TIMP-1 in the streptozotocin-induced diabetic nephropathy rats. The expression levels of the TIMP-1 protein in the DNC rats were significantly elevated compared to those of the NC group. Furthermore, the pattern of MMP-9 distribution is similar to the pattern shown by its specific inhibitor, TIMP-1. Berberine (50 mg/kg) cannot effectively depress the expression of MMP9 and TIMP1, while the berberine (100, 200 mg/kg) treatment groups could significantly down-regulate the levels of MMP-9 and TIMP-1 and the ratio of MMP-2 and TIMP-2. All data were normalized to the NC group value and are represented as n=5, mean±S.D. **P<0.01 versus the NC group; #P<0.05 and ##P<0.01 compared with the DNC group.
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Fig 6. The expression levels of TGF-β1 in the kidneys of the DN rats as detected by Western blot analysis. A. Representative Western blot analysis of the expression of TGF-β1 in the streptozotocin-induced diabetic nephropathy rats. B. Quantification of the Western blot analysis of TGF-β1 in streptozotocin-induced diabetic nephropathy rats. The expression level of TGF-β1 was normalized to that of β-actin. All data were normalized to the NC group value and are represented as mean±S.D. ##
P<0.01 versus the NC group; **P<0.01 compared with the DNC group.
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Fig 7. Expression levels of the MMP-2 and TIMP-2 proteins in the kidneys of the DN rats detected by Western blot. A. Representative Western blot analysis of the expression levels of MMP-2 and TIMP-2 in streptozotocin-induced diabetic nephropathy rats. B. Quantification of the Western blot analysis of MMP-2 and TIMP-2 in the streptozotocin-induced diabetic nephropathy rats. The expression levels of MMP-2 and TIMP-2 were normalized to those of β-actin. All data were normalized to the NC group value and are represented as the mean±S.D. **P<0.01 versus the NC group; ##P<0.01 compared with the DNC group.
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Fig 8. Expression levels of MMP-9 and TIMP-1 in the kidneys of DN rats as detected by Western blot analysis. A. Representative western-blot analyses of the expression levels of MMP-9 and TIMP-1 in the streptozotocin-induced diabetic nephropathy rats. B. Quantification of the Western blot analyses of MMP-9 and TIMP-1 in the streptozotocin-induced diabetic nephropathy rats. The expression levels of MMP-9 and TIMP-1 were normalized to those of β-actin. All data were normalized to the NC group value and are represented as mean±S.D. **P<0.01 versus the NC group; #P<0.05 and ##P<0.01 compared with the DNC group.
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Graphical Abstract
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PII: DOI: Reference:
S0014-2999(15)30168-0 http://dx.doi.org/10.1016/j.ejphar.2015.07.040 EJP70139
To appear in: European Journal of Pharmacology Received date: 24 May 2015 Revised date: 13 July 2015 Accepted date: 16 July 2015 Cite this article as: Wei-Jian Ni, Hai-Hua Ding, Hong Zhou, Yuan-Ye Qiu and Li-Qin Tang, Renoprotective effects of berberine through regulation of the MMPs/TIMPs system in streptozocin-induced diabetic nephropathy in rats, European Journal of Pharmacology, http://dx.doi.org/10.1016/j.ejphar.2015.07.040 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Renoprotective effects of berberine through regulation of the MMPs/TIMPs system in streptozocin-induced diabetic nephropathy in rats1 Wei-Jian Ni1, Hai-Hua Ding1, Hong Zhou2, Yuan-Ye Qiu1, Li-Qin Tang1* Author names and affiliations: W.-J. Ni 1
Affiliated Anhui Provincial Hospital; Anhui Medical University, Anhui Province,
People’s Republic of China. E-mail: [email protected] H.-H. Ding 1
Affiliated Anhui Provincial Hospital; Anhui Medical University, Anhui Province,
People’s Republic of China. E-mail: [email protected] H. Zhou, 2
Anhui Provincial Cancer Hospital, Anhui Province, People’s Republic of China.
E-mail: [email protected] Y.-Y. Qiu 1
Affiliated Anhui Provincial Hospital; Anhui Medical University, Anhui Province,
People’s Republic of China. E-mail: [email protected] L.-Q. Tang 1
Affiliated Anhui Provincial Hospital; Anhui Medical University, Anhui Province,
People’s Republic of China. E-mail: [email protected] *Address for correspondence: L.-Q.Tang, Affiliated Anhui Provincial Hospital; Anhui Medical University, 17# Lujiang Road, Hefei 230001, Anhui Province, People’s Republic of China. E-mail: [email protected]
Tel/Fax: +86551-62283378-809
1
Acknowledgments
This work was supported by the National Natural Science Foundation of China (No.81102864), the Anhui Provincial Natural Science Foundation (China, No. 1508085MH179). 1
Abstract Berberine has proven protective effects on diabetic nephropathy, but the mechanism for its effects has not been comprehensively established. Hence, we aimed to explore the renoprotective mechanism of berberine on the accumulation of extracellular matrix, alterations of its major components and corresponding changes in the regulatory system, including the matrix metalloproteinases/tissue inhibitor of matrix metalloproteinases (MMPs/TIMPs) system, in diabetic nephropathy rats. In the experiments, diabetic nephropathy rats were treated with berberine (0, 50, 100, 200 mg/kg) respectively. The protein levels of transforming growth factor-β1 were then detected by Western blot, while fibronectin and type IV collagen levels were assessed using immunohistochemistry. Changes in the MMP2/9 and TIMP1/2 levels were detected using two forms simultaneously. In addition, we also measured the characteristics and biochemical indicators of the diabetic nephropathy rats. The results showed that berberine could ameliorate the fasting blood glucose, and the majority of biochemical and renal function parameters, but did not have an effect on body weight. Immunohistochemistry and Western blot examination revealed a significant increase in the MMP9 and TIMP1/2 levels, with an obvious decrease in MMP2 expression in the diabetic nephropathy rats. Berberine (100 and 200 mg/kg) could significantly improve the abnormal changes in the MMPs/TIMPs system. Meanwhile, reductions in the transforming growth factor-β1, fibronectin and type IV collagen expression levels were observed in the berberine treatment groups. Therefore, the renoprotective effects of berberine on diabetic nephropathy might be associated with changes in the extracellular matrix through the regulation of the MMPs/TIMPs system in the rat kidney.
Keywords: Diabetic nephropathy; berberine; MMPs/TIMPs system; ECM; renal fibrosis
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1. Introduction Diabetic nephropathy (DN), the major cause of end-stage renal disease (ESRD) worldwide, is one of the most common and severe chronic complications of diabetes mellitus (DM) (Sidaway, 2014). It is characterized by proteinuria, mesangial expansion, glomerular basement membrane (GBM) thickening, persistent fibrosis and a gradual decrease in renal function, which result from increasing extracellular matrix (ECM) deposition at the glomerular level (Kato and Natarajan, 2014). A number of factors are important for the development of DN, including hyperglycemia, hypertension, oxidative stress, and inflammation, and have been shown to lead to proteinuria and histological changes, such as mesangial expansion, glomerular sclerosis, and tubulointerstitial fibrosis (Muskiet et al., 2014). It has been reported that renal fibrosis is final the common pathologic finding observed in the later stages of the disease, and it is characterized by transforming growth factor-β1 (TGF-β1) signalling-mediated ECM accumulation (Li et al., 2014). This, of course, results from the production of fibronectin (FN) and type IV collagen promoted by TGF-β1 (Ueno et al., 2013). Research has shown that the matrix metalloproteinases and tissue inhibitors of the matrix metalloproteinase (MMPs/TIMPs) system have non-neglectable roles in ECM accumulation and deposition (DeCoux et al., 2014; Raffetto and Khalil, 2008). Furthermore, the alterations in the MMPs and TIMPs are usually regulated by TGF-β (Kobayashi et al., 2014). Berberine (BBR; [C20H18NO4]+), a type of isoquinoline alkaloid, is the major active component of Rhizoma coptidis and Cortex phellodendri extraction(Feng et al., 2015). Research has shown that berberine exhibits multiple pharmacological activities, including lowering blood glucose and anti-oxidative stress as well as reducing inflammation, indicating that berberine has general clinical and research value and is a promising therapeutic drug in DN treatment (Ni et al., 2015). Previous studies have demonstrated that in berberine-treated DN rats, the degree of pathological changes was significantly reduced: mesangial matrix accumulation and mesangial expansion were scarcely detected, and the glomerulus atrophy and glomerular sclerosis were also gradually relieved. Berberine could effectively ameliorate renal dysfunction and 3
regulate TGF-β1 expression (Xie et al., 2013). Meanwhile, the elevated TGF-β1 levels induced the secretion of MMP2 and TIMP2, suggesting that there may be some relationship among TGF-β1 expression, ECM accumulation, alteration of the MMPs/TIMPs system and the renoprotective effects of berberine (Sekiuchi et al., 2012). However, the relationships and mechanisms remain incompletely understood. The aim of this study was to investigate the degradation effects of berberine on the ECM, the alterations of its major components, the corresponding changes in its regulatory system, i.e., the MMPs/TIMPs system and the possible molecular mechanisms in a streptozotocin (STZ)-induced diabetic nephropathy rat model.
2. Materials and Methods 2.1 Reagents The berberine was provided by the Anhui Provincial Hospital. STZ and dimethyl sulfoxide (DMSO) were obtained from Sigma Chemical Co. (St. Louis, MO, USA). The anti-type IV collagen (BA2174), anti-FN (BM1871) and anti-TGF-β1 (BA0290) antibodies were obtained from Boster Biological Technology (Boster CO., LTD, China). The anti-MMP2 (bs-0412R), anti-MMP9 (bs-2099R), anti-TIMP1 (bs-0415R) and anti-TIMP2 (bs-0416R) antibodies were purchased from Biosynthesis Biotechnology (Bioss CO., LTD, China). The superSignal west femto kit was purchased from Thermo (Thermo Fisher Scientific, USA). Other chemicals used in these experiments were of analytical grade from commercial sources. 2.2 Animal and experimental protocols A total of 50 mature, Sprague-Dawley rats (male, 180±20 g. Grade II, Certificate No. 028) were purchased from the Animal Department of Anhui Medical University (Hefei, China). Rats were allowed to feed on a standard diet and drink ad libitum and were adapted to the experimental conditions at 20±2◦C, humidity of 60±5% and a 12 h light-dark cycle. After an overnight fast, the rats were administered a single intraperitoneal injection of 35 mg/kg STZ dissolved in a 0.1 mM chilled citrate-phosphate buffer (pH 4.5) and 4
injected immediately within 30 min. Normal control (NC) rats were injected with citrate-phosphate buffer. The development of hyperglycemia (diabetes) in the rats was confirmed by fasting blood glucose (FBG) estimation (≥11.1 mmol/L) 72 h after STZ injection, and only uniformly diabetic rats were included in the DN control (DNC) and drug treatment groups (n=10, each group). Rats in the NC and DNC groups were given an equal volume of the carboxymethyl cellulose (CMC-Na), while the berberine (50, 100, 200 mg/kg) groups were intragastrically administered berberine dissolved in 0.5% CMC-Na at doses of 50, 100 and 200 mg/kg body weight per day, respectively, for 8 weeks starting on the 7th day. Kidney samples were rapidly isolated and frozen in liquid nitrogen or fixed in 10% formaldehyde buffer. Blood was collected, and the serum was extracted after centrifugation and stored at -80◦C until analysis. All experiments were approved by the Ethics Review Committee for Animal Experimentation of Anhui Medical University. 2.3 Measurement of renal function and biochemical markers Animal weight was measured every two weeks from 0 to 8 weeks. Fasting blood glucose was measured every 2 weeks using tail vein blood samples. During the experiment, rats were housed in metabolic cages for 24 h to collect urine for subsequent measurements of urinary total protein (UTP). Blood samples were obtained from the femoral artery and allowed to clot for 30 min at 4◦C, then centrifuged (1268 g, 10 min, 4◦C). The supernatant was collected for measurement of blood urea nitrogen (BUN) and serum creatinine (SCr) by using a Hitachi Model 7600 Series Automatic biochemistry Analyzer. After removing the capsule of the right kidney, the kidneys were weighed. The kidney weight to body weight ratio was calculated. 2.4 Immunohistochemical analyses Immunohistochemical staining for fibronectin, type IV collagen, MMP-2/9 and TIMP-1/2 were performed using a streptavidin-biotin complex (SABV) technique. In brief, the sections were defined, and the endogenous peroxidase activity was quenched by 10 min of incubation with 3% H2O2. The sections were placed in 10 mmol/L citrate buffer, microwaved for 30 min and then cooled to room temperature. 5
Sections were placed in PBS for 10 min, followed by protein-blocking for 15 min. The slides then were incubated for 1 h with primary antibody against anti-MMP2/9, anti-FN, anti-type IV collagen and anti-TIMP1/2 antibody at a 1:100 dilution. A secondary biotinylated antibody was added to the sections for 30 min at 37◦C. For coloration, the slides were incubated at room temperature with a mixture of 0.05% 3, 3-diaminobenzidine (DAB) containing 0.01% H2O2. Finally, areas of positive immunostaining were quantified by computer-based morphometric analysis. The degree of positive staining was determined as the mean of ten different fields on each slide. 2.5 Western blot analyses For protein preparation, the kidney tissues were suspended in RIPA buffer. The protein samples were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electrotransferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA, USA). After blocking with 5% skim milk in Tris-phosphate buffered saline (TPBS) for 2 hours, the membranes were incubated with anti-TGF-β1 (1:200), anti-TIMP1/2 (1:250) and anti-MMP2/9 (1:250) antibodies at 4◦C, with gentle shaking overnight and reblotted with anti-β-actin antibody to verify equal loading of the protein in each lane. After incubation with the appropriate peroxidase-conjugated secondary antibodies, the signals were visualized using the enhanced chemiluminescence Western blot Detection System, in accordance with the manufacturer’s instructions (ImageQuant LAS 4000 mini, GE Healthcare). 2.6 Statistical analyses All experiments were performed at least three times, and the results were reproducible. All values are expressed as the mean ± S.D. Statistical analyses of the data were performed by oneway ANOVA with GraphPad Prism 5.0. Values of P<0.05 were considered to be statistically significant in all cases.
3. Results 3.1 Berberine ameliorates the abnormalities of renal function and biochemical markers in DN rats 6
The significant increase in blood glucose was detected after the induction of diabetes by streptozocin (P<0.01). Administration of berberine (50 mg/kg) did not reduce blood glucose levels, and berberine (100, 200 mg/kg) had undistinguishable hypoglycaemic effects short-term treatment (0-4 week), suggesting that low dosage and short-term treatment are not likely to produce a satisfactory outcome. Berberine (100, 200 mg/kg) was proved to be satisfactory by continuous treatment compared with the DNC group, which suggested that it may be a long-term method to reduce blood sugar (Fig 1A). Diabetic rats had a lower body weight than the NC rats from the 2nd week onwards, while berberine treatment had no effect on decreasing the body weight in the therapeutic process (Fig 1B). The ratio of kidney weight to body weight in the diabetic rats was significantly increased after streptozocin-injection compared with the NC group. Different dosages of berberine could effectively decrease the ratio of kidney weight to body weight, suggesting that there is indeed a potential role for improving the kidney weight for berberine under conditions of diabetic nephropathy (Fig 1C). The urinary total protein (UTP), blood urea nitrogen (BUN) and serum creatinine (SCr) levels of the DNC group were significantly higher than those of the NC rats after the streptozocin injection. Berberine (100, 200 mg/kg) treated diabetic nephropathy rats exhibited a remarkable decrease in the UTP, BUN, and SCr levels. There were no differences in the UTP level between berberine (50 mg/kg) and untreated streptozocin-induced diabetic nephropathy rats (Fig 1D-F). 3.2 Immunohistochemical staining for type IV collagen, fibronectin, MMP2/9 and TIMP1/2 We next examined whether berberine inhibits the increase in type IV collagen and fibronectin. Semi-quantitative analysis exhibited a significant rise in the expression of type IV collagen and fibronectin in the DNC group (P<0.01). Berberine treatment could significantly reduce the expression levels of type IV collagen and fibronectin in a dose-dependent manner (Fig 2-3). The immunohistochemical staining showed a decreased expression level of MMP2 and an increased pattern of TIMP-2 distribution 7
in the DNC group (Fig 4A). The expression level of TIMP-1 was elevated, and the distribution of MMP-9 was similar to the pattern shown by its specific inhibitor, TIMP-1, compared with the NC group (Fig 5A). Understandably, the semi-quantitative analysis showed that the ratio of MMP-2/TIMP-2 was decreased in the DNC group (Fig 4B), which was consistent with the ratio of MMP-9/TIMP-1 (Fig 5B). It is worth noting that berberine treatment could reverse the abnormal changes and significantly up-regulate the ratio of MMP-2/TIMP-2 and MMP-9/TIMP-1, which suggested that berberine might prove to be of great protective effect against diabetic nephropathy, acting by reducing ECM accumulation and accelerating the degradation process. 3.3 Western blot analysis of the expression of TGF-β1, MMP2/9 and TIMP1/2 When compared with the NC rats, increased TGF-β1 expression was observed in the diabetic rats. Berberine (100, 200 mg/kg) treatment could significantly decrease the expression of TGF-β1. In contrast, no significant changes in decreasing this protein were noted in the berberine (50 mg/kg) treatment group (Fig 6). Western blot analysis also showed the relatively lower expression levels of MMP-2, while higher expression levels of TIMP-2 were observed compared with the NC group. The ratio of MMP-2/TIMP-2 was also decreased subsequently (Fig 7). Furthermore, in our experiment, TIMP-1 expression was elevated in the kidney tissues of untreated rats compared to the NC group. The expression pattern of MMP-9 was similar to that of its specific inhibitor, TIMP-1. It should be noted, however, that the increasing amplitude was different. That is to say, the increasing magnitude of TIMP-1 was higher than that of MMP-9, producing a decreased MMP-9/TIMP-1 ratio (Fig 8). Compared with the DNC group, the DN rats treated with berberine (50 mg/kg) showed a slight effect on ameliorating the symptoms above. However, the berberine (100, 200 mg/kg) treatment could significantly up-regulate the ratio of MMP-2/TIMP-2. Moreover, berberine (100, 200 mg/kg) treatment could also significantly increase the expression level of MMP-9 and help restore the damaged balance of MMP-9/TIMP-1 (Fig 7-8). 8
4. Discussion The study presented here evaluated the renoprotective role of berberine in diabetic nephropathy. According to the experiment results, fasting blood glucose, body weight were higher in the diabetic rats than in the NC rats. In the DNC group, the levels of hematologic traits and urine biochemical parameters, including UTP, BUN and Scr, were remarkably increased after STZ injection. These data suggested that the rat model of DN was successfully established (Betz and Conway, 2014). After treatment with berberine, the physiological indicators in the cases showed varying degrees of improvement (Zhao et al., 2014). The data above also suggested that berberine could not only reduce the blood glucose and urine total protein excretion except for the body weight but also improve the renal pathological changes, such as ECM accumulation, glomerulus atrophy and glomerular sclerosis, thus preventing the progression of DN (Wang et al., 2013). These changes indicated that berberine had desirable protective effects on the renal structure and physiological functions of DN rats. DN is one of the most common diabetic microvascular complications. It is characterized by increased ECM deposition and reduced matrix degradation. The ECM is mainly composed of type IV collagen and fibronectin. In essence, the maintenance of the ECM is the result of the dynamic balance between ECM protein synthesis and degradation (Brown et al., 2015). However, this balance is disturbed under the diabetic state, resulting in alterations in the main pathological processes, such as abnormalities in the renal structures and functions, glomerular sclerosis and renal fibrosis (Simonson, 2007). One encouraging note is that berberine treatment could decrease the expression levels of type IV collagen and fibronectin. This seems to be a possible justification for the hypothesis that berberine exerts its renoprotective role by inhibiting ECM accumulation, which indicates that berberine has wide research potential. The MMPs/TIMPs system is the most important enzyme degrading system of proteins in the diabetic kidney. The expression levels and activities of the system directly impact the ECM balance. MMPs exist in the kidney tissue as zymogens and are activated by plasmin; at the same time, they remain under the influence of TIMPs 9
(Giannandrea and Parks, 2014). In physiological conditions, the MMPs and TIMPs were in the equilibrium condition as being adjusted by the homeostasis to control the quantity of activated MMPs within the allowable range, so that they could help maintain a stable ECM (Tan and Liu, 2012). However, this balance is also shifted under diabetic conditions, leading to abnormal ECM accumulation and degradation (Han et al., 2006). As important components of the MMPs/TIMPs regulation system, MMP-2 and MMP-9 play key roles in the development of DN (Solini et al., 2011). Despite stimulating new insight into the MMPs/TIMPs and a profitable decade of medical research attention, the regulation mechanisms of MMPs/TIMPs and the effects of berberine on this system remain enigmatic (Catania et al., 2007). Indeed, we note that changes in MMP2 and MMP9 under diabetic state are opposite, motivating us to explore the special role of TIMPs in our research. Therefore, we found that the ratios of MMP2/TIMP2 and MMP9/TIMP1 were similar, and both were reduced under the diabetic nephropathy state. After treatment, we found that berberine reversed the abnormal changes of MMP2 and MMP9, while reducing the expression of TIMP1 and TIMP2 and elevating the ratio of MMP2/TIMP2 and MMP9/TIMP1. These changes alleviate the inhibitory effects of TIMP1 and TIMP2 and enhance the degradation effects of MMP2 and MMP9, eventually ameliorating the accumulated ECM and renal fibrosis (Rysz et al., 2007). Because berberine treatment was observed to regulate the MMPs/TIMPs system in the present study, we conclude that berberine could inhibit ECM accumulation by regulating the MMPs/TIMPs system in the kidneys of DN rats. This suggests that berberine is a promising therapeutic drug for DN treatment. The TGF-β related signalling pathway is a well-known mediator in DN progression, mediating fibrosis by stimulating the synthesis of ECM molecules and by decreasing ECM degradation (Deshpande et al., 2013). A variety of molecular mediators and intracellular signalling pathways associated with DN, such as the hyperglycemia, angiotensin II, and oxidative stress pathways, are connected and uniformly stimulate the TGF-β related signalling pathway (Ni et al., 2015). In addition, TGF-β1 has also been recognized as a powerful multifunctional cytokine that plays an important role in 10
regulating the MMPs/TIMPs system (Zhang et al., 2015). In accordance with these findings, our present study showed that the expression of TGF-β1 was increased remarkably (P<0.01) in diabetic rats, in conjunction with the abnormal changes in the MMPs/TIMPs system, suggesting that the aforementioned viewpoints are correct. When the expression level of TGF-β1 decreased, the abnormal ratio of the MMPs/TIMPs system returned to normal after the berberine treatment. These data were consistent with the results for the MMPs/TIMPs system and the ECM component proteins from immunochemistry. It seems that berberine could regulate the ratio of the MMPs/TIMPs system and increase the quantity and activity of the MMPs by inhibiting the expression and production of TGF-β1. This issue may be resolved through the study of tissue-specific TGF-β1 deficient rats in a follow-up experiment. The aforementioned changes indicated that berberine could decrease the expression levels of TGF-β1 and gradually increase the ratios of MMP-2/TIMP-2 and MMP-9/TIMP-1. The increased MMP-2/TIMP-2 and MMP-9/TIMP-1 ratios will then ameliorate the role of TIMP1/2 on the inhibitory effects of MMP9/2. Furthermore, berberine can also increase the expression and activities of MMP2 and MMP9, which leads to decreased ECM deposition and increased ECM degradation, thus ameliorating the structural damage and functional disorders of the kidney, such as mesangial expansion, glomerulus atrophy and glomerular sclerosis, and preventing the progression of DN (Yang et al., 2014). Recent research from our group has suggested that berberine could exert its renoprotective effects by regulating the Gαi-AC-cAMP signalling pathway. The alteration in the Gαi-AC-cAMP signalling pathway produced decreased levels of TGF-β1, which contributed to the renal protective effects under identical experimental conditions (Tang et al., 2013). We therefore assumed that berberine regulates the Gαi-AC-cAMP signalling pathway to elevate the levels of cAMP. The increased cAMP acts as a second messenger and is intimately linked with the suppression of TGF-β1. The decreased levels of TGF-β1 then up-regulate the ratio of MMPs/TIMPs to enhance the degradation effects of the MMPs. Next, MMP2 and MMP9 reduced the ECM accumulation by inhibiting the production of type IV collagen and fibronectin, thus playing pivotal roles in 11
ameliorating the mesangial expansion, glomerulus atrophy and glomerulosclerosis. In further trials, more controlled experiments are needed to confirm the proposed mechanisms.
5. Conclusions In conclusion, our data demonstrated that berberine has a renoprotective effect on diabetic nephropathy that might be associated with changes in the extracellular matrix through the regulation of the MMPs/TIMPs system in streptozocin-induced diabetic neophropathy rats. These results suggest that berberine has a therapeutic role in the prevention of diabetic nephropathy and is a promising anti-diabetic nephropathy drug.
Acknowledgments This work was supported by the National Natural Science Foundation of China (No.81102864), the Anhui Provincial Natural Science Foundation (China, No. 1508085MH179).
Conflict of interest There are no competing financial interests in relation to the work.
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Figure legends Fig 1. Berberine ameliorates abnormalities in the renal functions and biochemical markers of DN rats The DN rat model was induced using streptozotocin via a single intraperitoneal injection at a dose of 35 mg/kg after the rats were provided with a high sugar/ lipid diet for 8 weeks. Berberine (50, 100, 200 mg/kg/day) was given intragastrically to the diabetic nephropathy rats from the first week to last week after injection. The fasting blood glucose (FBG) levels (A), body weight(B), kidney weight-to-body weight ratio(C), urinary total protein (UTP) excretion(D), blood urea nitrogen (BUN) and serum creatinine (SCr) levels (E) were determined. **P<0.01 versus the NC group; #P<0.05 and ##P<0.01 compared with the DNC group.
Fig 2. Immunohistochemical staining for the expression of type IV collagen in the kidneys of the DN rats. A. Representative immunohistochemical staining analyses of the expression of type IV collagen in the streptozotocin-induced diabetic nephropathy rats. B. Quantification 15
of immunohistochemical staining analysis of type IV collagen in the streptozotocin-induced diabetic nephropathy rats. All data were normalized to the NC group value and are represented as the mean±S.D. ##
P<0.01 versus the NC group; **P<0.01 versus the DNC group.
Fig 3. Immunohistochemical staining for the expression levels of fibronectin in the kidneys of the DN rats. A. Representative immunohistochemical staining analyses of the expression of fibronectin in streptozotocin-induced diabetic nephropathy rats. B. Quantification of immunohistochemical staining analysis of fibronectin in the streptozotocin-induced diabetic nephropathy rats. All data were normalized to the NC group value and are represented as the mean±S.D. ##
P<0.01 versus the NC group; *P<0.05 and **P<0.01 versus the DNC group.
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Fig 4. Immunohistochemical staining for the expression levels of MMP-2 and TIMP-2 in the kidneys of the DN rats. A. Representative immunohistochemical staining analysis of the expression of MMP-2 and TIMP-2 in the kidneys of diabetic nephropathy rats. B. Quantification of immunohistochemical staining analysis of MMP-2 and TIMP-2 in streptozotocin-induced diabetic nephropathy rats. The expression levels of MMP-2 were significantly decreased in the DNC rats compared to the NC group rats. In contrast, the TIMP-2 protein levels in the DNC group were significantly increased compared to those of the NC rats. A low concentration of berberine (50 mg/kg) could slightly improve the above phenomena (P<0.05), while a higher concentration of berberine (100, 200 mg/kg) could significantly change the levels of MMP-2 and TIMP-2 and the ratio of MMP-2 and TIMP-2 (P<0.01). All data were normalized to the NC group value and are represented as n=5, mean±S.D. **P<0.01 versus the NC group; #P<0.05 and ##P<0.01 compared with the DNC group.
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Fig 5. Immunohistochemical staining for the expression of MMP-9 and TIMP-1 in the kidneys of the DN rats. A. Representative immunohistochemical staining analysis of the expression of MMP-9 and TIMP-1 in the kidneys of diabetic nephropathy rats. B. Quantification of immunohistochemical staining analysis of MMP-9 and TIMP-1 in the streptozotocin-induced diabetic nephropathy rats. The expression levels of the TIMP-1 protein in the DNC rats were significantly elevated compared to those of the NC group. Furthermore, the pattern of MMP-9 distribution is similar to the pattern shown by its specific inhibitor, TIMP-1. Berberine (50 mg/kg) cannot effectively depress the expression of MMP9 and TIMP1, while the berberine (100, 200 mg/kg) treatment groups could significantly down-regulate the levels of MMP-9 and TIMP-1 and the ratio of MMP-2 and TIMP-2. All data were normalized to the NC group value and are represented as n=5, mean±S.D. **P<0.01 versus the NC group; #P<0.05 and ##P<0.01 compared with the DNC group.
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Fig 6. The expression levels of TGF-β1 in the kidneys of the DN rats as detected by Western blot analysis. A. Representative Western blot analysis of the expression of TGF-β1 in the streptozotocin-induced diabetic nephropathy rats. B. Quantification of the Western blot analysis of TGF-β1 in streptozotocin-induced diabetic nephropathy rats. The expression level of TGF-β1 was normalized to that of β-actin. All data were normalized to the NC group value and are represented as mean±S.D. ##
P<0.01 versus the NC group; **P<0.01 compared with the DNC group.
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Fig 7. Expression levels of the MMP-2 and TIMP-2 proteins in the kidneys of the DN rats detected by Western blot. A. Representative Western blot analysis of the expression levels of MMP-2 and TIMP-2 in streptozotocin-induced diabetic nephropathy rats. B. Quantification of the Western blot analysis of MMP-2 and TIMP-2 in the streptozotocin-induced diabetic nephropathy rats. The expression levels of MMP-2 and TIMP-2 were normalized to those of β-actin. All data were normalized to the NC group value and are represented as the mean±S.D. **P<0.01 versus the NC group; ##P<0.01 compared with the DNC group.
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Fig 8. Expression levels of MMP-9 and TIMP-1 in the kidneys of DN rats as detected by Western blot analysis. A. Representative western-blot analyses of the expression levels of MMP-9 and TIMP-1 in the streptozotocin-induced diabetic nephropathy rats. B. Quantification of the Western blot analyses of MMP-9 and TIMP-1 in the streptozotocin-induced diabetic nephropathy rats. The expression levels of MMP-9 and TIMP-1 were normalized to those of β-actin. All data were normalized to the NC group value and are represented as mean±S.D. **P<0.01 versus the NC group; #P<0.05 and ##P<0.01 compared with the DNC group.
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Graphical Abstract
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