Salidroside protect renal function against inflammation and oxidative stress by NF-κB p65/NLRP3 pathway signaling in streptozocin induced diabetic nephropathy mice

doi:10.21203/rs.3.rs-5259964/v1

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Salidroside protect renal function against inflammation and oxidative stress by NF-κB p65/NLRP3 pathway signaling in streptozocin induced diabetic nephropathy mice

https://doi.org/10.21203/rs.3.rs-5259964/v1

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Inflammation and oxidative stress have been reported to be a common product of many pathways that are involved in the pathogenesis of diabetic nephropathy. Salidroside, the major active compound in Rhodiola, provide multiple biological activities and has protective effects for alleviating diabetic renal dysfunction. However, the involved molecular mechanism was still not clarified well. In this study, we intended to explore the protective effects and further mechanism of salidroside in STZ-induced diabetic mice. Biochemical analysis was processed to investigate the anti-inflammatory effects and antioxidative effects in serum and kidney homogenate. Thus, the results showed that salidroside effectively reduced the level of blood glucose and GSP, ameliorated the renal function and kidney fibrosis in STZ-induced DN mice. STZ induced inflammation and oxidative stress in mice, which aggravated renal injury. Salidroside also suppressed the expression of proinflammatory factors (including IL-1, IL-1β, TNF-α) and the decreased MDA level, but increased the level of CAT, GSH-Px and SOD activity in STZ-induced mice. In mechanism, salidroside inhibited the expression of NF-κB p65 and NLRP3 pathway related proteins in vivo. Our findings suggest that salidroside improved renal inflammation and oxidative stress by inhibiting p65 and NLRP3 expression in STZ-induced diabetic mice. Our study provides a new potential treatment on diabetic nephropathy.

salidroside

renal inflammation

oxidative stress

STZ

NF-κB/NLRP3 pathway

Diabetic nephropathy (DN) is the most common cause of end-stage renal disease in worldwide. Based on epidemiological studies, the incidence of diabetes will be expected to increase by over 50% by 2045 compared with 2017 (1, 2). Notably, DN is becoming a huge economic burden, and social problem in China (3). Major hallmarks of DN include accumulation of extracellular matrix (ECM) proteins, such as collagens and mesangial expansion in the kidney glomerular and tubular compartments, which contribute to renal failure in diabetes(4). Pathologically, DN is characterized by oxidative stress, inflammation, metabolic disorders, and fibrosis (5). Substantial evidences have shown that inflammation, pro-inflammatory endothelial activation, and oxidative stress by the metabolism of hyperglycemia and dyslipidemia may have a crucial impact on the development of vascular complications in DN patients or animals (6–9).

Several mechanisms have been implicated in the pathogenesis of DN, including renal inflammation, increased oxidative stress, renal cell apoptosis, and interstitial fibrosis. Accumulated data have emphasized persistent and enhanced inflammation in the pathogenesis of DN, which acts through oxidative stress, transcription factors, and inflammatory cytokines, and finally leads to excessive fibronectin production and extracellular matrix accumulation resulting in acceleration of the pathogenesis of glomerular sclerosis and tubulointerstitial fibrosis(10). In addition, numerous studies clearly indicate that both diabetic state and insulin resistance play a central role in producing oxidative stress. Increases of inflammatory cytokines and reactive oxygen species (ROS) have also been shown in DN. In end-stage renal disease, free glucose activates aldose reductase activity and the polyol pathway, which decreases NADPH/NADP + ratios, the expression of NADPH oxidase 4 is altered, which plays an important role in the production of ROS(11, 12). ROS activate many stress-related pathways and oxidative stress activates inflammation pathways. Inflammatory cytokines, such as tumor necrosis factor-α (TNF-α), interlukin-1b (IL-1β), and IL-6, are reported to occur at higher levels in patients with DN(13, 14). Thus, oxidative stress and inflammation are likely upstream initiators of inflammatory injury of DN. Furthermore, recent studies indicated that the oxidative stress and inflammation cross-talk for positive feedback regulation, there by promoting inflammatory injury in DN(15). the oxidative stress of DN and inflammation are considered as a whole in current research, in which a large amount of active oxygen generated by enhanced oxidative stress caused by a high glucose environment is regarded as a major cause of DN inflammation, and inflammation increases oxidative stress in turn, indicating a significant interaction between them(16).

Mitogen-activated protein kinases (MAPKs; mainly include ERK1/2 and p38) are involved in both normal renal physiology and in the pathology of various forms of kidney injury, including renal fibrosis(17). Activated MAPK is thought to be the physiologic activator of the nuclear transcription factor nuclear factor κB (NF-κB). Activation of the NF-κB pathway is associated with expression of inflammatory cytokines and transcription of NF-κB is reported to be enhanced in the development of DN. NF-κB participates in processes that lead to kidney damage and renal interstitial fibrosis(18). In diabetes, activated NF-κB translocates into the nucleus and triggers the excessive expression of its downstream target gene TGF-β1, causing ECM accumulation and renal fibrosis(19). Besides, several studies reported that urate and lipid accumulated in DM patients can activate NLRP3 inflammasome and cause inflammatory reaction, which results in kidney injury(20). A new study further proposes that inhibiting the expression and activation of NLRP3 inflammasome in DN patients can improve the renal function of patients(21). This indicates that NLRP3 inflammasome not only participates in the morbidity of DN, but also may be closely related to further progression of DN.

Rhodiolae Radix, have been widely used in Traditional Chinese Medicine and has been reported to have a very good effectiveness in the control of blood glucose(22). Salidroside, 2-(4-hydroxyphenyl)ethyl-β-D-glucopyranoside, a compound with a phenol glycoside chemical structure, is isolated from the Rhodiolae Radix as one of its main active ingredients and is thought responsible for all the documented pharmacological effects of the medicinal plant. Previous studies demonstrated that salidroside has important bioactivities including anti-fatigue, anti-inflammatory, anticancer, antioxidant(23). Besides, other studies indicated that salidroside has a significant effect on glucose, fat and amino acid metabolism(24). Salidroside could stimulate glucose uptake in differentiated L6 rat skeletal myoblast cells by activating AMP-activated protein kinase(25), and alleviates high glucose-induced oxidative stress and extracellular matrix accumulation in rat glomerular mesangial cells(26). However, few studies have examined the hypoglycemic activity of salidroside and the precise mechanisms are unknown. The present study was undertaken to evaluate the effects of salidroside on diabetes-induced oxidative stress in DN mice. In this study, we aimed to determine the protective effects of salidroside on the development of renal inflammation and oxidative stress in STZ induced diabetic mice. Salidroside prevents damage by inhibiting inflammatory process by regulating NF-κB and exerts a marked renoprotective effect. A better understanding of the salidroside molecule and its cardioprotective effects may lead to more advanced strategies for treating vascular complications associated with DN.

2.1 Reagents

Streptozocin was purchased from Sigma Chemical (St. Louis, MD, USA, No. 14110807, purity ≧ 99%). Salidroside was purchased from the Weikeqi biological technology Co.,Ltd.(CAS 10338-51-9; Molecular formula:C14H20O7; Molecular weight: 300.3; Purity: 98.20%, Sichuan, China). The BCA protein assay kit was purchased from Thermo Fisher (Pittsburgh, PA). Oxidative stress biomarkers were using detection kits (SOD: A00-1; MDA: A003-1; CAT: A007-1; GSH-Px: A005, Jiancheng Bioengineering Ltd., Nanjing, China). ELISA assay kit was obtained from Myhalic biotechnology co. LTD (Wuhan China).

2.2 Animal experiments

C57BL/6J male mice (8 weeks, weighed 20 ± 2g) from the Laboratory Animal Center of Southern Medical University (Guangzhou, China) were used. All animal experiments were approved by the Animal Care Committee of Southern Medical University. The mice were acclimated for 7 days prior to dosing, during which time they had free access to food and water ad libitum and were fed under specific conditions at 25 ± 2°C and with a humidity of 55 ± 5%, lights on 12 h every day. A diabetic mouse model was induced by daily intraperitoneal injection of streptozocin (STZ) (Sigma-Aldrich, St. Louis,MO, USA; 70 mg/kg body weight) in sodium citrate buffer (0.1 M, pH 4.5) and immediately injected within 30 min of preparation for 3 days. The control mice were injected intraperitoneally with saline solution. After STZ injection for 3 days, the diabetic state was assessed by measuring blood glucose (GLU) levels after fasting overnight. Mice with random blood glucose levels in excess of 16.7 mmol/L was considered to diabetic mice. Diabetic mice were divided randomly into 2 groups: the DM group (untreated diabetic mice) served as the diabetic control and only received vehicle, whereas the salidroside group treated diabetic mice was treated with 100mg/kg oral once daily for 8 weeks. The mice were sacrificed under ether anesthesia at the end of the experiment.

2.3 Measurement of body weight, blood glucose and renal index

Body weights of mice were measured at 2-week intervals. Blood glucose was detected in two weeks intervals by One Touch Ultra blood glucose monitoring system (Life Scan Inc., Milpitas, CA, USA) by blood sampling from the tail vein. Renal index (RI) was ratio of kidney weight (KW) and body weight (BW) were recorded.

2.4 Biochemical assay

Serum was obtained from the blood by centrifugation (12,000 g, 4°C) for 15min. Serum levels of GSP, creatinine (SCr) and urea nitrogen (BUN) were measured using automatic biochemical analyzer (ACE, USA). Serum TNF-α, IL-1β and IL-6 were determined using commercially available ELISA assay kits (Nanjing Jiangcheng Bioengineering Institute, Nanjing, China).

2.5 Histopathological examination

The renal tissues were fixed in 4% buffered fparaformaldehyde, embedded in paraffin and sectioned 4-µm thickness were stained with hematoxylin-eosin (H&E) and trichromeperiodic acidschiff stain (PAS) according to standard procedure for histological examination under a light microscopy.

2.6 Oxidative stress biomarker’s measurements

Serum and kidney tissues were collected for oxidative stress analysis. Then, SOD, GSH-Px and CAT activity and the MDA level were measured using detection kits (SOD: A00-1; MDA: A003-1; CAT: A007-1; GSH-Px: A005, Jiancheng Bioengineering Ltd., Nanjing, China) according to the manufacturer’s instructions.

2.7 Immunocytochemical staining

The sections were deparaffinized and rehydrated, and endogenous peroxidase activity was blocked by incubating the samples with 3% H₂O₂ for 15 minutes in the dark. Antigen retrieval was performed by heating in a pressure cooker in citrate buffer (Saiguo, Guangzhou, China) for 10 minutes. The samples were then incubated at room temperature for 40 min and washed again with PBS for 3 times, then blocked with 5% BSA for 30 min at room temperature. Immediately after that, the sections were incubated with primary antibodies, including anti-1L-1β (1:200,Affinity, USA), IL-6 (1:200,Affinity, USA), TGF-β (1:200,Affinity, USA),anti-p65 (1:100, Affinity, USA) and anti-NLRP3 (1:100, Affinity, USA) at 4°C overnight. Then, the tissues were washed again with PBS 3 times and incubated with a biotin-conjugated secondary antibody for 40 min at 37°C, then a 3,3'-diaminobenzidine (DAB) color development kit (Gene Tech, GK500705, Shanghai, China) was performed following manufacturer’s instructions. Ultimately the sections were counterstained with hematoxylin and imaged under a microscope.

2.8 Immunofluorescence

Tissues were washed with PBS for 3 times and blocked with 5% BSA at room temperature for 40 min. After being washed with PBS, the cells were incubated with antibodies against NF-κB p65 (1:200, AF5006, Affinity, USA) and NLRP3 (1:250, Affinity, USA) at 4°C overnight. Then, the cells were washed with PBS and incubated with a fluorescent secondary antibody (DyLight 594, E032420, EarthOx, USA; DyLight 488, A23220, Abbkine, AmyJet) in the dark for 60 min, followed by staining with DAPI for 10 min and washing with PBS 2 times. Afterwards, a fluorescence microscope was used to capture images.

2.9 Western blotting

Kidney tissyes were lysed in RIPA buffer. The protein concentration was determined by a BCA protein assay kit (Thermo Fisher Scientific, USA). The protein samples were loaded equally onto 12% SDS polyacrylamide gels by electrophoresis (at constant pressure, 60 V for 40 min, 120 V for 65 min), transferred to PVDF membranes (Millipore, Billerica, MA, USA) under a constant current of 350 mA for 120 min, blocked with 5% BSA in 1× TBST for 1 h and incubated with the primary antibodies at dilutions according to the manufacturer’s recommendations. Next, the membranes were incubated with a species-matched HRP-conjugated secondary antibody (1:5000, Affinity, USA) for 1 h and washed with 1× TBST 3 times (10 min each time). Finally, the blots were visualized with enhanced chemiluminescence (ECL) HRP substrate reagent (Immobilon Western, WBKLS0500, Millipore, USA) using a CCD system (ImageStation 2000 MM, Kodak, NY, USA).

2.10 Statistical analysis

All statistical analyses were performed by SPSS 22.0 software (SPSS, Chicago, USA) and GraphPad Prism Software 5.0 (CA, US). Data are presented as the mean ± SD and were analyzed by one-way ANOVA. A post hoc analysis of Bonferroni's Test was performed when ANOVA found significant interactions between variables. A p-value < 0.05 was considered statistically significant.

3.1 Salidroside mitigates hyperglycemia and protected renal function in STZ-induced diabetic mice.

The effects of salidroside on metabolic disorders in diabetic mice was shown in Fig. 1. Compared with the normal control mice, the STZ-induced mice exhibited a significant increase in FBG levels (more than 16.7 mmol·L − 1), which was an indicator of T2DM. However, salidroside administration for 8 weeks led to a remarkable reduction in FBG levels in diabetic mice (Fig. 1A). In addition, serum GSP levels were significantly increased in diabetic mice compared with normal mice (Fig. 1B, p < 0.01). However, changes of GSP levels in diabetic mice were counteracted after 100mg/kg salidroside treatment (Fig. 1B, p < 0.05). Salidroside administration also increased the STZ-induced decrease in body weight and kidney weight levels (Fig. 1C-1D). Besides, compared to control group, levels of serum creatinine and BUN in diabetic mice were increased, and salidroside administration reduced the level of serum creatinine and BUN. These results suggested salidroside may play important role in hypoglycemic and renal protective effects.

3.2 Salidroside alleviates renal injury in STZ-induced diabetic mice.

Histological observations of renal were performed to support the biochemical evidences.Kidney histology by H&E staining showed clearly visible nephropathy by STZ, as evidenced by an increase in the mesangial expansion and severely damaged in the glomerular membrane and tubules (Fig. 2A). The PAS staining results showed that glomerular extracellular matrix (ECM) deposition was significantly increased by STZ, which was greatly reduced in the presence of salidroside (Fig. 2B). In addition, the levels of TGF-β in kidney of mice was evaluated. Salidroside treatment significantly reduced the occurrence of renal lesions and inhibited renal fibrosis caused by STZ induced DN mice (Fig. 2C).

3.3 Salidroside ameliorates inflammation in STZ-induced diabetic mice.

Next, we examined the effect of salidroside on pro-inflammatory factor expression in STZ-induced diabetic mice. Compared to control mice, the levels of serum TNF-α, IL-6 and IL-1β were increased in diabetic mice, and salidroside treatment decreased the level of serum TNF-α, IL-6 and IL-1β (vs. control group, ^*p < 0.05, ^**p < 0.01, Fig. 3A). The results of immunocytochemical staining showed the similar results to that determined by Elisa (Fig. 3B). These results suggest that inflammation plays important role in diabetic and salidroside reduces the expression of pro-inflammatory factor in STZ-induced diabetic mice.

3.4 Salidroside ameliorates oxidative stress in STZ-induced diabetic mice.

The results revealed that a decrease in SOD, CAT and GSH-Px activity, accompanied with an increase in MDA content in STZ induced mice, whereas salidroside administration reversed this phenomenon both in serum and kidney tissue (vs. STZ group, ^#p < 0.05, ^##p < 0.01, Fig. 4A-4B). These results proved that salidroside provided a protective effect against oxidative stress in STZ-induced diabetic mice.

3.5 Salidroside reduces nuclear protein NF-κB p65 and NLRP3 in diabetic mice.

Finally, we examined the expression of AMPK, NF-κB p65 and NLRP3 and the related key proteins by immunocytochemical staining.The results showed that salidroside inhibited the NF-κB p65 and NLRP3 signaling pathways (^**p < 0.01, Fig. 6A-6B). In addition, the similar results were shown that activation of p65 and NLRP3 in STZ-induced mice by immunocytochemical staining and immunofluorescence (vs. the control group, Fig. 6A-6B). However, salidroside effectively regulated the expression of these proteins in STZ-induced diabetic mice. These results suggest that salidroside ameliorates renal inflammation and oxidative by regulating the NF-κB p65/NLRP3 signaling pathway in STZ-induced diabetic mice.

Compelling evidence suggests that hyperglycemia is a major contributing factor in the deterioration of the kidney and the development of DN. The occurrence of DN is predominantly mediated by high glucose levels and micro-vascular hemodynamic changes and is further mediated by a variety of inflammatory cytokines in diabetic kidneys, which activate the inflammatory pathway(27). Inflammation and oxidative stress also plays a pivotal role in the pathogenesis of DN, emphasizing the potential therapeutic effect of its blockade for improving kidney function during diabetes.Currently, the strategy of DN treatment is to control blood sugar, reduce urinary protein, lower blood pressure, and control hyperlipidemia(28). Although good glycemic control may be the best prevention of DN, it develops in spite of treatment of diabetes. Inhibitors of oxidative stress and inflammation should provide useful targets for therapy.

It has been shown that salidroside has multiple activities such as anti-inflammation, anti-oxidative stress, anti-cancer, etc. Salidroside protected against MPP(+) -induced Parkinson's disease in PC12 cells by inhibiting inflammation, oxidative stress and cell apoptosis(29). Previous study found that salidroside could attenuate body weight gain and improve glucose homeostasis in obese mice by repressing inflammation in white adipose tissues and enhanced leptin signaling transduction in the hypothalamus(30). Another study showed that salidroside reduces high-glucose-induced cell apoptosis and oxidative stress via upregulating Heme Oxygenase-1 (HO-1) expression in mice podocyte(31). However, In this study, we intend to study the effect of salidroside on renal protection in STZ induced diabetic mice in vivo. Thus, the present study evaluated the nephroprotective effect of salidroside (100mg/kg d) in STZ-induced DN mice. Diabetes was induced by injecting STZ (70 mg/kg) i.p. for 3 days. Biochemical parameters, including fasting blood glucose, creatinine, BUN in the serum, and albumin in the urine, were determined in STZ-induced DN mice. Moreover, the level of inflammatory mediators in the serum and oxidative stress parameters both in the serum and kidney tissue homogenate was assessed in STZ-induced DN mice. Expressions of NF-κB p65 and NLRP3 in the tissue homogenate were estimated by IHC staining. Our study found that salidroside protect renal function against inflammation and oxidative stress injury in STZ induce diabetic mice, partially by supressing NF-κB p65/NLRP3 signaling pathway.

Streptozotocin (STZ), an antibiotic produced by Streptomyces achromogenes, is frequently used to induce a DM model with multiple low doses in experimental animals by damaging insulin-producing b cells of the pancreas and causing hyperglycemia in mamma. In our study, we used a STZ 70mg/kg a day injection to build diabetic mice model in which STZ damages pancreatic b cells, resulting in hypoinsulinemia and hyperglycemia. After that fasting blood glucose was measured, 16.7 mmol/L was considered as 83% mice was susseful. DM leads to disorders related to glucose and fatty acids metabolism. The characteristic symptoms of DM include an increased desire for food associated with body weight loss and heased due to the decrease in glucose metabolism and increase in fat metabolism(32) (Rossmeisl et al., 2003).According to our results, treatment with salidroside ameliorated these parameters which were able to decrease the level of fasting blood glucose(Fig. 1a) anf GSP in serum(Fig. 1b), and improve body weight(Fig. 1c) and kidney weight(Fig. 1d) andat the end of the intervention period, indicating a regulatory effect on these metabolic disorders. Besides, several biochemical parameters, including creatinine and urea level in the serum and microalbumin urea in urine, are markers of DN(33).Our data also showed that the levels of creatinine and BUN were altered in the DN group and that treatment with salidrosde attenuated the altered levels of these in the serum STZ-induced DN mice(Fig. 1e-Fig. 1f).

It is believed that DN is one kind of chronic inflammation. Inflammatory cytokines, such as tumor necrosis factor-α (TNF-α), interlukin-1β (IL-1β), and IL-6, are reported to occur at higher levels in patients with DN(34). Previous haveshown that during the development of DN, inflammation is obvious, renal tissue is accompanied with the infiltration of various inflammatory cells, including mononuclear macrophages, and significant increase in the concentration of various pro-inflammatory cytokines can often be detected in the tissues and circulating blood(35). In the present study, the degree of inflammation activation was evaluated by the level of inflammatory mediators including IL-6, IL-1β, TNF-α both in serum and kidney tissue(Fig. 3a-3b). We found that treatment with SAL significantly down-regulated gene expressional levels of these inflammatory parameters, suggesting that the STZ-induced inflammatory reactions were suppressed by SAL. Besides, the PAS staining results showed that glomerular extracellular matrix (ECM) deposition wassignificantly increased by STZ, which was greatly reduced in the presence of salidroside (Fig. 2B). Kidney histology by H&E staining also showed clearly visible nephropathy by STZ (Fig. 2A). Salidroside could partially reduce these pathological changes (Fig. 2A-2B). These data clearly indicate that salidroside plays a beneficialrole against diabetic nephropathy in STZ-treated mice.

STZ also has toxic effects on generation of ROS causing oxidative damage on the kidney for evaluating the therapeutic potential of antidiabetics(36). Oxidative stress is generally accepted as a likely causative factor in the development of insulin resistance and complications of diabetes (Giacco and Brownlee, 2010). Previous studies have shown that ginsenoside Re and fermented red ginseng extracts significantly enhance SOD activity and reduce MDA level in diabetic mice (Kim et al., 2011; Liu et al., 2012). Oxidative stress in diabetes is also associated with a reduction in antioxidant capacity, which can compound the deleterious effects of free radicals. The antioxidant enzymes such as SOD and MDA play a major role in scavenging toxic free radicals. The activity changes of these enzymes can reflect the degree of diabetic effect (Shanmugam et al., 2011). Results showed that the activities of SOD, GSH-Px and CAT were significantly increased, the MDA contents remarkably reduced in liver and kidney homogenate in STZ-induced diabetic Kunming mice(37). Our study also demonstrated that the level of SOD, GSH-Px and CAT were significantly increased, the MDA contents remarkably reduced both in serum and kidney homogenate in STZ-induced mice, while treatment with saliroside ameliorated the phenomena (Fig. 4A-4B).

Studies using rodents indicate that increases in oxidative stress and inflammatory process could be responsible for developing DN. The inflammatory cytokines IL-6, IL-1β, and TNF-α were reported to be involved in the development of DN by stimulating the NF-κB signaling pathway(38). In DN, inflammation occurs by activating the NF-κB signaling pathway and by enhanced oxidative stress (39). Following activation, NF-κB translocates to the nucleus,stimulating rapidly the subsequent transcription of genes such as endothelin-1 (ET-1), IL-6, and TNF-α that promote the development of DN. NF-κB is mainly represented by the p65/p50 heterodimeric complex and this complex is retained in the cytoplasm in an inactive form bound to an additional inhibitory subunit – IκBα(40). During activation, the inhibitory subunit IκBα is rapidly phosphorylated at Ser32 and Ser36 by IKKα/β and subsequently ubiquitinated and degraded by the 26S proteasome complex. Once released, free NF-κB translocates to the nucleus and activates the transcription of various inflammatory gene products. Our present study revealed that saldroside attenuates the altered level of inflammatory cytokines in the serum of DN mice and reduced the expression of NF-κB protein the tissue homogenate of DN mice compared to the negative control group.

The NOD-like receptor pyrin 3 (NLRP3) inflammasome is an intracellular platform that recruits the adaptor molecule-apoptosis-associated speck-like protein (ASC) by pyrin domain, and then ASC hydrolyzes procaspase-1, and finally, active caspase-1 cleaves pro-IL-1β into its mature form in response to “danger” signals(41) .NLRP3 inflammasome signaling molecules and inflammatory cytokine IL-1β release from GMCs were induced by high glucose(42),Previous to the currently observed hyperglycemia-associated inflammasome activation in podocytes, and in mice with hyperhomocy-steinanemia or diet-induced obesity and glomerular endothelial cells, suggesting that inflammasome activation in various kidney cells may contribute to diabetic nephropathy(43, 44). Nlrp3-inflammasome activation, accomanied with increasing level of IL-1β, IL-1β cleavage, IL-18 and caspase1 were observed in diabetic nephropathy mice(45). Our present study revealed that the expression of Nlrp3-inflammasome, IL-1β cleavage, IL-18 and caspase1 was up-regulated in STZ in diabetic nephropathy mic. But salidroside attenuates the level of Nlrp3-inflammasome, IL-1β cleavage, IL-18 and caspase1 in the kidney tissue homogenate of DN mice compared to DN group (Fig. 5).

In conclusion, our study indicated that salidroside can palliate renal inflammation and oxidative stress in STZ induced diabetic nephropathy mice, at least partially through suppressing the activation of the NF-κB p65/NLRP3 pathway. Consequently, our study further provide idea of salidroside about the anti-inflammatory effect andalso its immunomodulatory effects. Salidroside may be one of potential target therapy for DN patients.

diabetic nephropathy

IHC

immunohistochemistry

Competing interests

The authors declare that they have no competing interests.

Funding

This study was supported in part by the Project of National Natural Science Foundation of China (Program No. 82104795), Project of Guangzhou Municipal Science and Technology Bureau ( No.202201011438), and Project Guangdong Provincial Administration of Traditional Chinese Medicine (No. 20221255).

Author Contribution

Rong Hu designed the study and Mingqing Wang analysed the data, Rong Hu and Lingyu Liu carried out all of the experiment and collected the results. Other authors provided ideas and helped performing this experiment. Rong Hu wrote the manuscript. All authors read and approved the final manuscript.

Acknowledgments

Not applicable.

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No competing interests reported.

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Salidroside protect renal function against inflammation and oxidative stress by NF-κB p65/NLRP3 pathway signaling in streptozocin induced diabetic nephropathy mice

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Abstract

Figures

1. Introduction

2. Materials and methods

2.1 Reagents

2.2 Animal experiments

2.3 Measurement of body weight, blood glucose and renal index

2.4 Biochemical assay

2.5 Histopathological examination

2.6 Oxidative stress biomarker’s measurements

2.7 Immunocytochemical staining

2.8 Immunofluorescence

2.9 Western blotting

2.10 Statistical analysis

3. Results

3.1 Salidroside mitigates hyperglycemia and protected renal function in STZ-induced diabetic mice.

3.2 Salidroside alleviates renal injury in STZ-induced diabetic mice.

3.3 Salidroside ameliorates inflammation in STZ-induced diabetic mice.

3.4 Salidroside ameliorates oxidative stress in STZ-induced diabetic mice.

3.5 Salidroside reduces nuclear protein NF-κB p65 and NLRP3 in diabetic mice.

4. Discussion

Abbreviations

Declarations

Competing interests

Funding

Author Contribution

Acknowledgments

References

Additional Declarations

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