Berberine ameliorates diabetic nephropathy by inhibiting TLR4/NF-κB pathway
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Diabetic nephropathy (DN) is the leading cause of end-stage renal failure, contributing to severe morbidity and mortality in diabetic patients. Berberine (BBR) has been well characterized to exert renoprotective effects in DN progression. However, the action mechanism of BBR in DN remains to be fully understood.
The DN rat model was generated by intraperitoneal injection of streptozotocin (STZ, 65 mg/kg body weight) while 30 mM high glucose (HG)-treated podocytes were used as an in vitro DN model. The fasting blood glucose level and ratio of kidney weight to body weight were measured after BBR treatment (50, 100, or 200 mg/kg) in STZ-induced DN rats. The renal injury parameters including 24-h urinary protein, blood urea nitrogen and serum creatinine were assessed. qRT-PCR was performed to detect the transcript amounts of inflammatory factors. The concentrations of inflammatory factors were evaluated by ELISA kits. Western blot analysis was conducted to measure the amounts of TLR4/NF-κB-related proteins. The apoptotic rate of podocytes was analyzed by flow cytometry using Annexin V/propidium iodide.
Berberine reduced renal injury in STZ-induced DN rat model, as evidenced by the decrease in fasting blood glucose, ratio of kidney weight to body weight, 24-h urinary protein, serum creatinine, and blood urine nitrogen. BBR attenuated the systemic and renal cortex inflammatory response and inhibited TLR4/NF-κB pathway in STZ-induced DN rats and HG-induced podocytes. Also, HG-induced apoptosis of podocytes was lowered by BBR administration. Furthermore, blockade of TLR4/NF-κB pathway by resatorvid (TAK-242) or pyrrolidine dithiocarbamate aggravated the inhibitory effect of BBR on HG-induced inflammatory response and apoptosis in podocytes.
Berberine ameliorated DN through relieving STZ-induced renal injury, inflammatory response, and podocyte HG-induced apoptosis via inactivating TLR4/NF-κB pathway.
KeywordsBerberine Streptozotocin Diabetic nephropathy Podocytes TLR4/NF-κB pathway Inflammatory response
transforming growth factor-β1
tumor necrosis factor-α
nuclear factor kappa B
monocyte chemotactic protein-1
enzyme-linked immunosorbent assay
serum dodecyl sulfate-polyacrylamide gels
chemokine (C–C motif) ligand 2
advanced glycation end products-receptor for AGEs
AMP-activated protein kinase
Diabetic nephropathy (DN) is a major complication in patients with either type 1 or type 2 diabetes mellitus and one of the leading causes of end-stage renal failure, contributing to severe morbidity and mortality in diabetic patients . It is characterized by microalbuminuria, glomerular and tubular epithelial hypertrophy, excessive accumulation of extracellular matrix (ECM) protein, thickening of glomerular and tubular basement membranes, which eventually results in the failure of renal function . Accumulating evidence has demonstrated that metabolic and hemodynamic factors, including hyperglycemia, transforming growth factor-β1 (TGF-β1) and angiotensin II are implicated in the development of DN . Among these factors, hyperglycemia is currently regarded as the key initiating factor in the progression of DN . In addition, it is believed that inflammatory processes are also able to accelerate the development and progression of DN . According to the previous studies, DN was accompanied by the increased amount of pro-inflammatory cytokines and chemokines, including tumor necrosis factor-α (TNF-α), interleukin (IL)-1β and IL-6 . Podocytes have been recognized as critical regulators of glomerular injury and reduction in podocytes number mediated by apoptosis are associated with the pathogenesis of DN [7, 8]. Although extensive researches have been done in clarifying the pathogenesis of DN, it still remains a challenging task to develop novel and effective therapeutic strategies in the treatment of DN.
Berberine (BBR), an isoquinoline alkaloid isolated from Coptidis rhizome, Cortex phellodendri, and Berberis vulgaris, has been extensively used for long as an oriental medicine to treat gastroenteritis and secretory diarrhea [9, 10, 11, 12]. Increasing evidence has exhibited that BBR has a wide range of pharmacological activities, for instance, anti-oxidant stress, anti-inflammatory, anti-tumor, anti-microbial and anti-fibrosis effects, suggesting the clinical and research value of BBR . Furthermore, BBR has been well characterized to exert renoprotective effects in DN progression [14, 15]. However, the underlying mechanism of the renoprotective effect of BBR on DN remains to be further explored.
Toll-like receptors (TLRs) are a conserved family of pattern recognition receptors in the innate immune system that activate downstream inflammatory signaling pathways in response to exogenous microbial pathogens . Activation TLRs signal was associated with the activation of nuclear factor kappa B (NF-κB) activity and consequent increased release of pro-inflammatory cytokines and chemokines such as IL-6, monocyte chemotactic protein-1 (MCP-1), and IL-1β, in turn initiating local inflammation and leukocyte accumulation [17, 18]. NF-κB, a downstream effector of TLR4 signaling pathway, is a ubiquitous and important nuclear transcription factor which mediates several inflammatory processes . There is striking evidence that activation of NF-κB plays a critical role in renal inflammation and fibrosis of the progression of DN . Among all TLRs, TLR4 has been reported to be implicated in the pathogenesis of acute kidney injury, chronic kidney diseases, and the occurrence of DN .
In the present study, we used streptozotocin (STZ)-induced in vivo model of DN and high glucose (HG)-induced podocytes as an in vitro model to investigate the protective effect of BBR on DN and its possible molecular basis. Our study demonstrated that BBR could reduce streptozotocin (STZ)-induced renal injury and inflammatory response, and podocyte apoptosis by inhibiting the TLR4/NF-κB pathway.
Animal models and treatment protocols
The animal study was approved by the Experimental Animal Ethical Committee of Huaihe Hospital of Henan University and performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Male Sprague–Dawley rats (weighing 250 ± 20 g) were purchased from Shanghai Science Academy Animal Center (Shanghai, China) and housed in the standard laboratory conditions. All rats were allowed free access to food and water ad libitum at a controlled temperature room with a constant 12-h light/dark cycle. After a week of adaptive feeding, these rats were randomly divided into 5 groups (n = 10/group): NC group, DN group, DN + BBR (50 mg/kg), DN + BBR (100 mg/kg), and DN + BBR (200 mg/kg). Rats in DN group, DN + BBR (50 mg/kg), DN + BBR (100 mg/kg), and DN + BBR (200 mg/kg) were intraperitoneally injected with 65 mg/kg streptozotocin (STZ, Sigma-Aldrich, St. Louis, MO, USA) dissolved in a 0.1 mM chilled citrate–phosphate buffer (pH 4.5) to induce diabetes . The rats in NC group were injected with chilled citrate–phosphate buffer (0.1 mM, pH 4.4). Control rats in DN group received an equal amount of citrate–phosphate buffer alone by intraperitoneal injection. When the fasting blood glucose levels from tail vein of STZ-induce diabetic rats were over 16.7 mM at 5 days after STZ injection, these rats were considered as diabetes. One week later, the diabetic rats in DN + BBR (50 mg/kg), DN + BBR (100 mg/kg), and DN + BBR (200 mg/kg) were orally treated with BBR dissolved in 0.5% carboxymethyl cellulose at a dose of 50, 100 or 200 mg/kg every day, respectively. The volume of 0.5% carboxymethyl cellulose to dissolve the different doses of BBR (50, 100, and 200 mg/kg) was 4, 2, and 1 ml, respectively. Meanwhile, the rats in NC group and DN group were gavaged with the same volume of 0.5% carboxymethyl cellulose. The fasting blood glucose and body weight were measured every 2 weeks for 8 weeks. The rats were sacrificed at 8 weeks after BBR treatment. Blood samples were collected, and the serum was separated by centrifugation and stored at − 80 °C until analysis. Meanwhile, right kidney samples were rapidly excised, weighted, and stored at − 80 °C until analysis. The ratio of kidney weight to body weight was considered as kidney weight index.
At the end of the experiment, the animals were maintained in metabolic cages for 24 h to harvest urine for assessing 24-h urinary protein with an enzyme-linked immunosorbent assay (ELISA) kit (Runyu Biotechnology Co., Shanghai, China). The fasting blood glucose level was determined based on the glucose oxidase-catalyzed reaction (chemistry analyzer; Auto Analyzer Quik-Lab, Ames, Spain). To assess renal function, blood urea nitrogen and serum creatinine in the serum of blood samples were measured using an automatic biochemistry analyzer (Hitachi, Tokyo, Japan).
Determination of IL-1β, IL-6, and MCP-1 levels
Renal corticals were homogenized and centrifuged at 9000×g for 30 min at 4 °C. The levels of proinflammatory cytokines in kidney homogenate and serum, including IL-1β, IL-6, and MCP-1, were determined using commercially acquired ELISA kits (Abcam Inc., Cambridge, MA, USA).
Cell culture and treatment
Conditionally immortalized mouse podocytes were purchased from Yubo Bio-Technique Co. Ltd (Shanghai, China) and cultured in RPMI 1640 medium (Hyclone, Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS; Hyclone), 100 U/ml penicillin/streptomycin, 5.6 mM glucose (Dingguo Changsheng Biotechnology Co., Ltd., Beijing, China) and 10 U/ml recombinant mouse interferon-γ (IFNγ; Pepro Technology, Rocky Hill, NJ, USA) at 33 °C in a 5% CO2 humidified incubator. To investigate the effect of BBR on DN, podocytes were pre-treated with 30 mM high glucose (HG) for 24 h prior to treatment with BBR at a dose of 10, 30 or 90 μM for 24 h. In some experiment, podocytes were pre-treated with 30 mM HG in the presence of TLR4 antagonist resatorvid (TAK-242, 1 μΜ; ApexBio, Houston, TX, USA), NF-κB inhibitor pyrrolidine dithiocarbamate (PDTC; 50 μM; Sigma), or combined with NF-κB activator phorbol myristate acetate (PMA, 100 ng/ml; Sigma), followed by treated with 30 μM BBR for 24 h.
Quantitative real-time PCR (qRT-PCR)
Total RNA was extracted from treated podocytes with TRIzol reagent (Invitrogen Invitrogen, Carlsbad, CA, USA) and quantified by NanoDrop 2000/2000c spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). Complementary DNA (cDNA) was synthesized from 1 µg total RNA by reverse transcription using a high capacity cDNA reverse transcription kit (TaKaRa, Tokyo, Japan). qPCR analysis of interleukin (IL)-1β, IL-6, and MCP-1 mRNA was performed with SYBR Premix ExTaq II kit (TaKaRa) and specific primers on an Applied Biosystems 7900 Real-Time PCR system (Applied Biosystems, Foster City, CA, USA). The relative quantification of mRNA levels was calculated based on the 2−ΔΔCt method and normalized to GAPDH. The primers were as follows: GAPDH, forward: 5′-CAG TGC CAG CCT CGT CTA T-3′, reverse: 3′-AGG GGC CAT CCA CAG TCT TC-5′; IL-1β, forward: GTG ATG TTC CCA TTA GAC AGC, reverse: CTT TCA TCA CAC AGG ACA GG; IL-6, forward: 5′-ATG AAC TCC TTC TCC ACA AGC GC-3′, reverse: 5′-GAA GAG CCC TCA GGC TGG ACT G-3′; MCP-1, forward: 5′-TCA GCC AGA TGC AGT TAA CGC-3′, reverse: 5′-TGA TCC TCT TGT AGC TCT CCA GC-3′.
Western blot analysis
Kidney homogenate and podocytes were collected and lysed in cell lysis buffer (Beyotime, Haimen, China) with protease inhibitor cocktail and phosphatase inhibitor (both from Sigma-Aldrich) for protein extraction. Equal amount of protein lysates (30 μg) were separated by 10% serum dodecyl sulfate-polyacrylamide gels (SDS-PAGE) and electrotransferred onto nitrocellulose (NC) membranes (Millipore, Billerica, MA, USA). After being blocked with 5% non-fat dry milk in PBS for 1 h, the membranes were probed with the primary antibodies against TLR4, phosphorylated-p65 (p-p65), p65, p-IκBα, IκBα, Cleaved Caspase-3, Bcl-2 and β-actin (all from Santa Cruz Biotechnology, Santa Cruz, CA) at 4 °C overnight, followed by incubated with a horseradish peroxidase-conjugated secondary antibody (Invitrogen) for 2 h at room temperature. Peroxidase-labeled protein bands were detected by enhanced chemiluminescence reagents (Millipore) and the protein intensity was quantified with Image-Pro Plus 6.0 software (Media Cybernetics, Rockville, MD, USA).
Podocytes were double stained with FITC-Annexin V and propidium iodide (PI) from a FITC Annexin V Apoptosis Detection Kit I (BD Biosciences, San Jose, CA, USA). The apoptotic rats were analyzed using a FACScan flow cytometer (BD Biosciences).
Data are displayed as mean ± standard deviation (SD). Statistical analysis was performed with GraphPad Prism 5 software (GraphPad Software Inc., San Diego, CA, USA). Comparison among experimental groups was performed using unpaired two-tailed Student’s t test and analysis of variance (ANOVA), with a value of P < 0.05 being considered as statistically significant.
BBR ameliorated renal injury in STZ-induced DN rat model
BBR attenuated the systemic and renal cortex inflammatory response in STZ-induced DN rats
BBR inhibited the activation of TLR4/NF-κB pathway in STZ-induced DN rat model
BBR decreased HG-induced apoptosis of podocytes
BBR weakened HG-induced inflammatory response in podocytes
BBR blocked the activation of TLR4/NF-κB pathway in HG-induced podocytes
BBR treatment inhibited HG-induced inflammatory response and apoptosis in podocytes by blocking TLR4/NF-κB pathway
PDTC treatment aggravated the inhibitory effect of BBR on HG-induced inflammatory response and apoptosis in podocytes
The current study evaluated the renoprotective effects and molecular mechanism of BBR on the pathogenesis of DN in STZ-induced DN rat model and HG-induced DN podocytes model. In the present study, we provided strong evidence that BBR not only attenuated renal dysfunction and inflammatory response, but also suppressed TLR4/NF-κB pathway in STZ-induced DN rats and HG-induced podocytes. Furthermore, mechanistic analysis demonstrated that blockade of TLR4/NF-κB pathway by TAK-242 or PDTC exacerbated the inhibitory effect of BBR on HG-induced inflammatory response and apoptosis in podocytes. Therefore, our study suggested that BBR ameliorated DN by inhibiting TLR4/NF-κB pathway.
As has been well described, BBR has a wide range of pharmacological activities and has been demonstrated to exert protective effects against DN progression by ameliorating a variety of pathological changes . More recently, it was demonstrated that BBR displayed a tendency to ameliorate renal dysfunction and renal inflammation, improve glucose and lipid metabolism disorders, and decrease fasting blood glucose in DN rats [23, 24]. Moreover, BBR could inhibit renal fibrosis and inhibited renal tubular epithelial-to-mesenchymal transition of DN [25, 26]. Additionally, BBR inhibits high glucose-induced glomerular mesangial cell proliferation and ECM accumulation, and attenuates tubulointerstitial fibrosis in DN, which suggests that BBR can be used as a further potential therapeutic drug for DN [27, 28]. Additionally, it was previously demonstrated that the doses of BBR that can be administrated in patients with type 2 diabetes and diabetic rats are 300 mg three times a day and 100 mg/kg, respectively [29, 30]. In the present study, we demonstrated that BBR challenge at 100 or 200 mg/kg significantly reduced renal injury in STZ-induced DN rat model, as evidenced by a decrease in fasting blood glucose, ratio of kidney weight to body weight, 24-h urinary protein, serum creatinine, and blood urine nitrogen, while BBR at a low dose of 50 mg/kg had no significant effect on these parameters associated with kidney function. Moreover, we found that BBR also weakened STZ-induced inflammatory factors production in DN rats, and inhibited HG-induced apoptosis and inflammatory response in podocytes, confirming the renoprotective effect of BBR in DN.
There is increasing evidence supporting that inflammation exerts important roles in the pathogenesis of DN . TLR4/NF-κB pathway appears to play a critical role in the pathogenesis with a variety of inflammatory conditions . The proinflammatory role of TLR4/NF-κB pathway has been demonstrated to be involved in the progression of diabetes and diabetic complication [33, 34]. Upon stimulation, activation of TLR4 pathway subsequently activated the NF-κB pathway and triggered NF-κB-dependent inflammatory response, which might ultimately aggravate renal dysfunction in acute and chronic kidney diseases [35, 36]. In podocytes and tubular epithelial cells, exposure to high glucose promoted TLR4 activation, resulting in NF-κB activation and consequent inflammatory and fibrogenic responses . Silencing of TLR4 with small interfering RNA attenuated high glucose-induced IκB/NF-κB activation and inhibited downstream inflammatory cytokines IL-6 and chemokine (C–C motif) ligand 2 (CCL-2) in human proximal tubular epithelial cells . Consistently, the present study demonstrated that BBR treatment blocked HG-induced activation of TLR4/NF-κB pathway in both STZ-induced DN rats and HG-treated podocytes. It was previously reported that BBR inhibited the NF-κB activation and fibronectin (FN) accumulation by inhibiting RhoA/ROCK signaling in diabetic rat kidneys and high glucose-induced glomerular mesangial cells . In addition, BBR could improve insulin resistance of skeletal muscle through inhibiting the active of the TLR4/IκBβ/NF-κB signaling pathway . BBR inhibited IL-1β-induced nitric oxide (NO) production in primary mixed glia and BV-2 cells via inactivation of TLR4/adapter protein myeloid differentiation factor 88 (MyD88)/NF-κB signaling . Besides, the anti-bacterial effects of BBR were mediated by acting as a high-affinity LPS antagonist and blocking the LPS/TLR4 signaling . Moreover, BBR repressed LPS-induced cell proliferation and FN expression in rat mesangial cells through impeding the activation of NF-κB signaling pathway and protein expression of its downstream inflammatory mediators . Our study further revealed that inhibition of TLR4/NF-κB pathway by TAK-242 or PDTC suppressed the HG-induced inflammatory response and apoptosis in podocytes. Moreover, combination with BBR treatment and inhibition of TLR4/NF-κB pathway exacerbated the inhibitory effect of BBR on HG-induced inflammatory response and apoptosis in podocytes, indicating that BBR ameliorated DN by inhibiting the TLR4/NF-κB pathway. Additionally, many other signaling pathways, such as advanced glycation end products-receptor for AGEs (AGEs-RAGE)  and AMP-activated protein kinase (AMPK) , have been elucidated to be implicated in the renoprotective effects induced by BBR.
In conclusion, our study demonstrated that BBR ameliorated DN by attenuating renal injury, inflammatory response and podocytes apoptosis. Mechanistic analysis revealed that the renoprotective effect of BBR on DN depended on inhibition of TLR4/NF-κB pathway. The present study contributed to a better understanding of the mechanism underlying BBR involved in DN and provided new evidence for the potential application of BBR in the treatment of DN.
This work was designed and conceived by LZ, JH and RY. The experiments were carried out by LX and WP. The manuscript was prepared by LZ and JH. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
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The animal study was approved by the Experimental Animal Ethical Committee of Huaihe Hospital of Henan University and performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.
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