Natural product Swietenine improve the progression of diabetic nephropathy by inhibiting ferroptosis through activation of the Akt/GSK-3β/Nrf2 signaling pathway

doi:10.21203/rs.3.rs-4375599/v1

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Research Article

Natural product Swietenine improve the progression of diabetic nephropathy by inhibiting ferroptosis through activation of the Akt/GSK-3β/Nrf2 signaling pathway

https://doi.org/10.21203/rs.3.rs-4375599/v1

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Background

Ferroptosis is a newly defined form of iron dependent regulatory cell death distinct from apoptosis, autophagy, and necrosis, characterized by an abnormal increase in intracellular lipid reactive oxygen species. Diabetes nephropathy (DN) is one of the most common complications of diabetes and the most common cause of end-stage renal disease. Recent studies have shown that ferroptosis plays an important role in the occurrence and development of diabetic nephropathy. Swietenine belongs to the limonin class of compounds, which are extracted from the the Swietenia macrophylla King, a plant of the genus Swietenia, family Meliaceae King and have not been artificially synthesized to date. It is a natural product with a variety of pharmacological activities such as anti diabetes, improving inflammation, anti-oxidation, anti-bacterial, anti-tumor, etc. However, it is unclear whether Swietenine can improve diabetes nephropathy by inhibiting the occurrence of ferroptosis in glomerular podocytes (MPC-5) and its potential mechanism.

Objective

This study investigated the natural product Swi through Akt/GSK-3β/Nrf2 signaling pathway inhibits MPC-5 ferroptosis and improves diabetes nephropathy in the process of diabetes.

Method

In vivo, 8-week-old SD rats were induced with STZ/HFD to investigate the effect of Swi on improving DN and resisting ferroptosis. In vitro, the inhibitory effects of Swi on MPC-5 death. By giving verify the activation effect of Akt/GSK-3β/Nrf2 signaling pathway related inhibitors on downstream anti ferroptosis related proteins.

Results

In this study, Swi treatment improved renal injury in DN rats, which was proved by renal function related indexes, histopathological parameters and podocyte damage protein. In addition, Swi inhibited ferroptosis in vivo. Swi improved HG-induced MPC-5 cell viability, inhibited ferroptosis in MPC-5 cells. Swi inhibits ferroptosis by activating the Akt/GSK-3β/Nrf2 signaling pathway, which promotes the expression of downstream anti-ferroptosis related proteins.

Conclusion

Our research findings suggest for the first time that it may be through a new Akt/GSK-3β/Nrf2 dependent ferroptosis regulates the signaling pathway, thereby reducing the level of high glucose induced ferroptosis and improving diabetes nephropathy, which is expected to become a promising candidate drug for the treatment of diabetes

Swietenine

ferroptosis

Diabetic Nephropathy

Akt

GSK-3β

Nrf2

With the continuous improvement of social and economic level and living standards, the number of people suffering from diabetes in the world continues to rise. So far, there are more than 400 million diabetes patients in the world. Long term hyperglycemia can lead to a series of microvascular complications, such as diabetes nephropathy (DN)^[1–2]. DN is one of the most common renal complications in diabetes patients. Its pathological characteristics include podocyte injury and depletion, increased proteinuria and impaired renal function^[3]. The pathogenesis of DN is very complex, involving disturbances in glucose and lipid metabolism, oxidative stress, formation of advanced glycation end products (AGEs), inflammation, and cell death^[4]. At present, it is speculated that renin-angiotensin-aldosterone system (RAAS) such as angiotensin-converting enzyme inhibitors (ACE-I) and angiotensin receptor blockers (ARBs) play a key role in preventing or delaying the progression of DN^[5].

Recent studies have shown that ferroptosis plays a crucial role in the occurrence and development of DN^[6]. Ferroptosis is a newly discovered programmed cell death process, which has gradually entered the field of vision and has become a research hotspot in the field of cell death^[7]. The process of ferroptosis is accompanied by iron dependent accumulation of reactive oxygen species, depletion of glutathione (GSH) causing lipid peroxidation damage, and morphological changes in mitochondria^[8]. The process of ferroptosis can be inhibited by lipophilic antioxidants and iron chelators, and exhibits specific effects on autophagy, apoptosis, and necrosis, hence it is called a novel programmed cell death mode^[9]. The accumulation of iron ions, oxidative stress imbalance caused by depletion of glutathione, and lipid peroxidation are three important mechanisms that induce ferroptosis in podocytes^[10]. The synthesis and reduction of glutathione are the core mechanisms regulating ferroptosis through amino acid metabolism pathways. Insufficient levels of GSH will not metabolize lipid peroxides produced as a result of oxidative stress. The dysfunction of the cystine-glutamate reverse transporter (system xc^-) leads to functional inactivation of Glutathioneperoxidase4 (GPX4), which is involved in inhibiting ferroptosis by promoting the reduction of oxidized glutathione (GSSG) to GSH. However, the inactivation of GPX4 will cause a large number of lipid oxides to be unable to be metabolized by GPX4-catalyzed glutathione reductase reaction, and then lipid oxidation similar to Fenton reaction will occur, generating a large number of reactive oxygen species^[11]. Ferroptosis, characterized by iron dependent lipid peroxidation, provides a new idea for exploring the progress of DN, and then how to improve DN by inhibiting the occurrence of ferroptosis, which still needs to further explore the functional changes and specific molecular mechanisms of diabetes nephropathy.

Akt, also known as PKB (protoinkinaseB). It is a serine/threonine specific protein kinase that plays a critical role in various cell growth processes. Under oxidative stress conditions, Akt is activated, leading to phosphorylation of GSK-3β,causing its inactivation^[12–13]. GSK-3β Glycogen Synthase Kinase-3β (GSK-3β) GSK-3β is a widely expressed serine/threonine kinase with multiple biological functions in cells, including regulating metabolism, cell growth, death, and gene transcription. Increasing evidence suggests that GSK-3β It is a key pathway involved in Nrf2 activation^[14–15]. Some studies have shown that nuclear factor erythrocyte type 2 related factor 2 (Nrf2) plays an important role in resisting ferroptosis and improving DN^[16–17]. Under oxidative stress conditions, Nrf2 increases its entry into the nucleus and regulates almost all major regulatory factors through antioxidant response element (ARE) transcription^[18]. Upregulation of Nrf2 can increase the expression of downstream antioxidant enzymes, such as heme oxygenase-1 (HO-1), glutathione peroxidase 4 (GPX4), and quinone oxidoreductase (NQO1). Among them, increased expression of GPX4 can inhibit ferroptosis^[19]. In addition, the cysteine/glutamate transporter SLC7A11 involved in GSH synthesis is also regulated by Nrf2^[20]. Therefore, activating Akt/GSK-3β/Nrf2 to regulate lipid peroxidation and ferroptosis may be a meaningful therapeutic approach and feasible disease intervention strategy for DN, but the specific mechanism of action still needs further exploration.

Swietenine (Swi) belongs to the limonin class compounds, isolated and extracted from large leaf algae, and no artificial synthesis has been found so far. More and more evidence shows that Swi is a natural product with a variety of pharmacological activities, such as anti diabetes, improving inflammation, anti-oxidation, anti-bacterial, anti-tumor, etc. It has been confirmed that Swi can reduce blood sugar, improve the inflammatory response of liver, kidney and pancreas in the process of diabetes, and oxidative stress^[21–23]. This indicates that it may be a potential drug of choice for the treatment of DN.

In this study, Swi improved high glucose induced podocyte ferroptosis and diabetes nephropathy by inhibiting ferroptosis in vivo and in vitro for the first time, The Akt/GSK-3β/Nrf2 signaling pathway is involved in this process. The results of this study provide a new mechanism for Swi mediated resistance to high glucose induced podocyte injury. This indicates that Swi may be a potential candidate drug for the treatment of DN.

Materials

Swi is an extract of S. macrophylla King. The fruit of S. macrophylla King was obtained from Indonesia in January 2016 and identified by Chunping Zhang, an assistant professor in the School of Pharmacy at Xuzhou Medical University.The certificate specimen (No. XZ03092016-68) is stored in the Herbarium, School of Pharmacy, Xuzhou Medical University. The molecular formula and mass of Swi are C₃₂H₄₀O₉ and 568.27.

Cell culture and treatment

The conditionally immortalized mouse MPC-5 podocyte cell line was purchased from Fuheng Biology Company (Shanghai, China) and cultured at RPMI-1640 medium (KeyGEN Bio TECH, China) containing 10% fetal bovine serum (FBS, Evergreen, China), 100 U/ml penicillin (HyClone) and 100µg/ml streptomycin (HyClone) at 37 ℃ under 5% CO².MPC-5 were stimulated with different doses of Swi (0.1, 0.5, 1, 5, 10, 20, and 40 µM) and normal glucose (NG) (11.1 mM) or HG(33.3 mM) for 24 h or 48 h.

Cell experiment grouping

The cell experiment is divided into two parts, the first part of the efficacy test, divided into 6 groups: a NG(11.1 mmol/L glucose) group; a HG (33.3 mmol/L glucose) group; a Swi with low concentration (1 µmol/L) group (HG + Swi_L); a Swi with medium concentration (5 µmol/L) group (HG + Swi_M); and a Swi with high concentration (10 µmol/L) group (HG + Swi_H); a Fer-1 with medium concentration (1 µmol/L) group (HG + Fer-1). In the second part of the mechanism experiment, we added Akt inhibitor MK-2206, GSK-3β inhibitor LY2090314 and Nrf2 inhibitor ML385 (MedChemExpress,USA) and divided the cells into 7 groups: a NG(11.1 mmol/L glucose) group; a HG (33.3 mmol/L glucose) group; a Swi with high concentration (10 µmol/L) group (HG + Swi₁₀); a Fer-1 with medium concentration (1 µmol/L) group (HG + Fer-1); Swi combined with inhibitor group (HG + S₁₀ + MK-2206), (HG + S₁₀ + LY2090314), and (HG + S₁₀ + ML385).

Cell viability assay

MPC-5 cells were cultured in 96-well plates at a density of 5000 per well.Cell viability was detected using the Cell Counting Kit-8 assay kit (Meilunbio, Dalian, China), following the manufacturer’s instructions. Cells were plated into 96-well culture plate for 24 h. After the corresponding treatments, CCK-8 detection reagent was added and incubated for 2 h. The absorbance was measured at 450 nm using a microplate reader.

Renal function measurement

Urea nitrogen, urinary protein, serum creatinine and serum urea acid were detected using a commercial kit (Jiancheng, Nanjing, China) with reference to the manufacturer's protocol.

Iron content measurement

The content of ferrous (Fe²⁺) was determined by an iron assay kit (Elabscience, China).Samples of treated cells or rat kidney tissue were collected and homogenized in iron assay buffers.Then, a total of 10 µL of supernatant in each sample was incubated with an iron probe (90µL) at 37 ℃ for 30min.The absorbance of the sample was detected at 593 nM.

GSH and MDA content measurement

GSH kit (Nanjing Jiancheng, China) and MDA assay kit (Nanjing Jiancheng, China) were determined according to the manufacturer's protocol.

Lipid reactive oxygen species measurement

The MPC-5 cells were intervened with different methods, subse-quently, 10 µM BODIPY™ 581/591 C11 (Thermo Fisher Scientific, USA) was administered to the cells for 1 h. The cells were washed twice with PBS. Images were captured with a confocal microscope.

Transmission electron microscopy

Sampling and fixation: Determine the sampling site of fresh tissue, minimize mechanical damage such as pulling, contusion and extrusion, tissue volume generally does not exceed 1mm×1mm×1mm, and quickly put into electron microscope fixation solution at 4 ℃ for 2–4 h.0.1M phosphate buffer PB (PH7.4) was rinsed 3 times for 15min each time. After fixation: 1% osmic acid·0.1M phosphate buffer PB (PH7.4) was fixed at room temperature (20 ℃) for 2h.0.1M phosphate buffer PB (PH7.4) was rinsed 3 times for 15min each time. Dehydration: 50%-70%-80%-90%-95%-100%-100% alcohol-100%-100% acetone-100% acetone ascending dehydration, 15min each time. Penetration: Acetone: 812 embedding agent = 1:12-4h, acetone: 812 embedding agent = 1:2 Overnight penetration, pure 812 embedding agent 5-8h, pour pure 812 embedding agent into the embedding plate, insert the sample into the embedding plate, embedding: 60℃ oven polymerization for 48h.Section: Ultra-thin microtome slices 60-80nm ultra-thin sections. Staining: Uranium lead double staining (2% uranium acetate saturated alcohol solution, lead citrate, each staining 15min), slices dried overnight at room temperature. The images were analyzed by transmission electron microscope.

Animal experiments

All animal ethics are approved by the Animal Ethics Committee of Xuzhou Medical University(No.202303T011).SD male rats were purchased from Xuzhou Medical University((SPF grade, 6–7 weeks, 180–200 g).After 1 week of adaptive feeding, 5 rats were selected as a control group and fed a normal diet throughout the period.In addition, the rats were fed HFD for 3 weeks( XTHF45,Xietong Bio., Nanjing, China).HFD-fed rats were injected intraperitoneally with 35 mg/kg STZ after fasting overnight to construct a DN rat model(STZ, 104G021, Solarbio, Beijing, China; 30 mg/kg, STZ was dissolved in 0.1mol/L sodium citrate buffer, pH = 4.5).The blood glucose level after streptozotocin injection was measured by glucose meter.After 72 hours, rats with FBG exceeding 11.1mM persisted for 3 days and were considered diabetic rats.DN rats were randomly divided into 4 groups: DN group and Swi low-dose and high-dose treatment groups (25 mg/kg,50 mg/kg) and Fer-1 positive drug group (2.5 mg/kg) and fed HFD continuously. The dosing group was treated by gavage administration, and both untreated DN and normal rats were treated with 1% CMC-Na solution by gavage control. FBG was measured every 2 weeks throughout the experiment.After 8 weeks, the rats were killed and their kidneys, urine and blood were collected for further experiments.

Histological analysis

Kidney specimens fixed in 4% paraformaldehyde were dehydrated, cleared, and embedded in paraffin wax for preparation of subsequent 4µm sections and staining with hematoxylin and Ession (H&E), Periodic acid-Schiff (PAS) and Masson.

Immunofluorescence staining (IF)

The Immunohistochemistry study was performed as previously described. Following dewaxing and hydration, the sections were washed three times in PBS. In the second, there were blotted with 10% goat serum for 15 min at room temperature. On the third, slices were incubated with GPX4 for 12 h at 4 ℃. After three washes, they were instilled with the secondary antibody (Alexa Fluor 594 IgG) for 2h at room temperature. In the end, slices were visualized with diaminobenzidine for 5 min and photographed under fluorescence microscope.

Western blotting analysis

A nuclear extract kit was used to separate nuclear and cytoplasmic proteins of kidney tissues, according to the manufacturer’s protocol. Kidney general proteins were extracted with lysis buffer plus RIPA(Beyotime, China), 1% phenylmethylsulfonyl fluoride (Beyotime, China), and 1% phosphatase inhibitors(Solarbio, China). The protein concentration was determined using a BCA kit (Beyotime, China). Subsequently, 50 µg of protein samples was separated by electrophoresis through a 10% sodium dodecyl sulfate-polyacrylamide gel. The samples were then transferred to a polyvinylidene difluoride membrane, which was blocked in BSA for 1 h at room temperature. The membrane was then incubated with primary antibodies, including anti-GPX4(Huaan Biotechnology, China,1 :1000), anti-SLC7A11(Huaan Biotechnology, China,1 :1000), anti-Nrf2 (1 : 1000), anti-PTGS2(Huaan Biotechnology, China,1 :1000), anti-podocin(Huaan Biotechnology, China,1 :1000), anti-p-Akt(Huaan Biotechnology, China,1 :1000), anti-Akt (1 : 1000), anti-p-GSK3β(Huaan Biotechnology, China,1 :1000), anti-GSK3β (Huaan Biotechnology, China,1 :1000)anti-GAPDH (Huaan Biotechnology, China,1 :1000), anti-β-actin(Huaan Biotechnology, China,1 :1000)antibodies overnight at 4°C. Rabbit Ig antibody (Vicmed, China, 1:10000) was subjected as the secondary antibody. Membranes were developed using ECL substrate for detection.

Statistical analysis

Statistical analyses of all experimental data were performed using SPSS 16.0 software, and the results were expressed as mean ± SEM (mean standard error). Statistical differences in outcomes were determined using a one-way ANOVA and independent sample T-test. A p-value of less than 0.05 was considered statistically significant.

Swi treatment improved renal function and pathological changes in DN rats

In order to verify the improvement effect of Swi on DN in vivo, 8-week old SD rats were used to induce type 2 DN model by STZ combined with high-fat diet, and Swi was administered by gavage every day. Although Swi treatment did not have a significant effect on mouse weight(Fig. 1. A), there was a certain degree of improvement in blood sugar in the treatment group(Fig. 1. B). The renal function test results showed that the levels of urea nitrogen, creatinine, uric acid, and urine protein in the model group were significantly higher than those in the normal group. Compared with the modeling group, administration of Swi and Fer-1 significantly reduced the levels of urea nitrogen, creatinine, uric acid, and urinary protein in DN rats(Fig. 1. C-F). HE, PAS and MASSON staining were used to observe the effect of Swi on renal histopathology in type 2 diabetic rats(Fig. 1. G). HE staining showed that the mesangial matrix of DN rats was hyperplasia, more epithelial cells of renal tubules were shed, and some inflammatory cells were found in the renal interstitium. However, after treatment with Swi or Fer-1, the degree of mesangial stroma proliferation was reduced, and the degree of epithelial cell shedding and brush margin of renal tubules was decreased. PAS was used for glycogen staining. In the model group, there was more fibrin deposition in the mesangial matrix, more fibrin deposition in the basement membrane, and obvious renal interstitial fiber tissue hyperplasia. After treatment with Swi or Fer-1, it was found that there was less fibrin deposition in the mesangial matrix, less fibrin deposition in the basement membrane, and less interstitial fiber tissue hyperplasia. MASSON was used to stain collagen fibers, and the results showed that the degree of fibrosis was more severe in the model group, and the degree of fibrosis was reduced after Swi or Fer-1 treatment. Further detection of changes in podocin levels, a biomarker of glomerular injury in renal tissue, was performed using protein blotting(Fig. 1. H-I). The results showed that compared with the normal group, the podocin protein levels in the renal tissue of the model group rats were significantly reduced, while compared with the control group, the podocin levels in the renal tissue of rats treated with Swi and Fer-1 were significantly improved. These data indicate that Swi treatment improves renal function and pathological changes in DN rats.

Swi inhibits oxidative stress and ferroptosis in the kidneys of DN rats

Research has confirmed that ferroptosis plays a crucial role in DN rats^[24]. We

found that endogenous antioxidant GSH levels decreased in DN rats, but recovered after treatment with Swi(Fig. 2. A), which is consistent with the finding of Swi inhibiting MDA levels in DN rats(Fig. 2. B). Subsequently, we detected ferrous content in kidney tissue of rats and found that Swi and Fer-1 reduced the upregulation of ferrous content in DN rats(Fig. 2. C). Studies have shown that excessive intake of iron during the process of ferroptosis can lead to iron overload, disrupt reduction homeostasis, and further induce oxidative stress and mitochondrial damage^[25]. Mitochondrial damage is the first special morphological change in the process of ferroptosis^[26]. Therefore, we evaluated the effect of Swi on mitochondrial damage in the glomeruli of DN rats. The observation results of transmission electron microscopy showed that there were changes in the subcellular structure of glomerular mitochondria in the normal group, modeling group, and after administration. The results showed that there was a certain degree of difference among the five groups of cells. The modeling group had the most severe mitochondrial damage, with moderate to severe swelling, membrane damage, more matrix dissolution, and cristae rupture and disappearance; The low-dose group showed secondary damage, with mild to moderate swelling, a small amount of shallow and dissolved matrix, and some mitochondrial membranes were damaged; The high-dose group showed slight swelling of mitochondria, slightly blurred membrane structure, slightly uneven distribution of matrix within the membrane, and disordered arrangement of cristae; The structure of most mitochondria in the normal group and positive drug group is still intact, without significant swelling. The bilayer membrane structure is intact, the matrix distribution is uniform, and the cristae are arranged relatively neatly(Fig. 2. D). Then, we investigated whether the protective effect of Swi on renal oxidative stress in DN rats is related to ferroptosis. SLC7A11 and GPX4 are central regulatory factors of ferroptosis^[27]. We used Western blotting to analyze changes in the ferroptosis resistant proteins SLC7A11 and GPX4 in kidney tissue.Compared to the normal group, the expression of SLC7A11 and GPX4 was significantly reduced in the kidneys of DN rats. After treatment with Swi and Fer-1, both showed significant recovery(Fig. 2E-G). IHC staining showed a decrease in GPX4 positive staining in the DN group compared to the NC-treated kidneys, and an increase in GPX4 positive staining after Swi and Fer-1 administration. These data suggest that Swi treatment improves oxidative stress and ferroptosis in DN rats.

3.3 Swi significantly improves high glucose induced MPC-5 death

To study the protective effect of Swi on DN in vitro, we first simulate the hyperglycemia of diabetic patients, exposed MPC-5 to a high glucose environment of 33.3 (mM), studied the inhibitory effect of different concentrations of Swi on the death of MPC-5 stimulated by high glucose, treated podocytes stimulated by high glucose with different concentrations of Swi, and set the set concentrations of Swi to 0.1, 0.5, 1.0, 5.0, 10, 20, and 40 µM, respectively. The processing time is 24 and 48 hours respectively. The CCK-8 measurement results indicate that MPC-5 ranges from 0.1 to 10 µM showed a significant improvement effect, and we found that MPC-5 cell activity peaked at 10 µM concentration of Swi The vitality of cells has reached its peak. Therefore, the low, medium, and high dose administration concentrations of Swi for podocytes were selected as 1, 5, and 10 µM, respectively. The administration time was selected as 24 hours for further research(Fig. 3. A-B). In addition, we also conducted a concentration screening of the ferroptosis inhibitor ferrostatin-1 (Fer-1) on the improvement of MPC-5 under the same high glucose concentration and time stimulation. The results showed that at a concentration of 1 µM, the improvement in cell viability is most significant. Therefore, we choose 1 µM represents the administration concentration of the positive drug(Fig. 3. C). In summary, our results indicate that Swi can significantly improve MPC-5 death induced by high glucose.

3.4 Swi protects MPC-5 cells from oxidative stress and ferroptosis induced by high glucose

We further confirmed the protective effect of Swi on in vitro oxidative stress and anti-ferroptosis, and first evaluated its antioxidant effect^[28]. In high glucose stimulated MPC-5, the decrease in GSH content and the increase in MDA content were observed, while treatment with Swi or Fer-1 significantly inhibited the depletion of GSH levels and the increase in MDA content(Fig. 3. D-E). The accumulation of ferrous content is one of the markers of ferroptosis. We found that Swi or Fer-1 treatments down-regulated HG-induced increases in Fe²⁺ levels(Fig. 3. F). Due to the accumulation of lipid peroxidation being an important factor leading to ferroptosis, we used C11-BODIPY581/591 probes to detect lipid peroxidation; Green fluorescence indicates oxidation, while red fluorescence indicates non oxidation. Confocal microscopy images showed that compared with the control group, HG induced ROS accumulation, while cells treated with Swi or Fer-1 reduced lipid peroxidation(Fig. 3. G). To further determine whether Swi can protect high glucose induced MPC-5 from the effects of ferroptosis, we used Western blotting to determine the expression of SLC7A11 and GPX4 in MPC-5. The results showed that HG led to a decrease in the expression of SLC7A11 and GPX4, while Swi or Fer-1 treatment upregulated their expression(Fig. 4. A-C). Next, we evaluated the expression of ferroptosis marker protein PTGS2. HG caused an increase in PTGS2 expression, while Swi or Fer-1 treatment downregulated its expression(Fig. 4. D-E). In summary, our research findings indicate that Swi can alleviate the occurrence of high glucose induced MPC-5 oxidative stress and ferroptosis.

3.5 Network pharmacology-based prediction of the target of Swi on DN

We further explored the protection mechanism of Swi in reducing DN. In our study, we found through network pharmacology target prediction of Swi and DN that they intersect with a total of 61 identical targets(Fig. 5. A). Through KEGG enrichment pathway analysis, we found that the PI3K-Akt signaling pathway has a higher enrichment index than other signaling pathways(Fig. 5. B). Therefore, molecular docking of Swi and Akt was performed, and the results showed a binding energy of 6.4 kcal/mol, indicating a good binding effect(Fig. 5. C-D). (meaningful when binding energy<-5 kcal/mol).

3.6 Swi inhibiting ferroptosis through activation of the Akt/GSK-3β/Nrf2 pathway.

To explore the mechanism of the upregulation of Nrf2 activity by Swi, the level of Akt and GSK-3β, upstream proteins of Nrf2, and their phosphorylated levels were examined using western blotting.As shown in the figure, compared with the NG group, the ratio of phosphorylation level to general level in the HG group decreased. Compared with the HG group, the ratio of phosphorylation levels to general levels in the Swi and Fer-1 groups increased(Fig. 6. A-H). Therefore, it can be seen that the Akt/GSK-3β/Nrf2 signaling pathway can be activated after Swi treatment.

To further confirm whether the protective effect of Swi against ferroptosis in HG-induced MPC-5 cells involved activation of Akt/GSK-3β/Nrf2 signaling, we used pathword-specific inhibitors for cell administration and protein blotting showed phosphorylation of Akt and GSK-3β after respective pathway inhibitors were administered the nuclear shift of Nrf2 was significantly reduced(Fig. 7. A-F). And consistent with previous research results, as shown in figure G, SLC7A11 and GPX4 proteins were significantly reduced under HG stimulation. Swi reverses the effect of HG and increases its anti ferroptosis effect. Pathway related inhibitors are given to block Swi's effect and downregulate the levels of SLC7A11 and GPX4 proteins(Fig. 7. G-I). From this, It can be concluded that Swi can play a role in preventing ferroptosis by activating the Akt/GSK-3β/Nrf2 signaling pathway.

Diabetes and its complications have brought considerable burden to public health. According to statistics, 30% of diabetes patients are affected by diabetes nephropathy (DN), and the incidence rate of the disease is increasing every year, so it has caused considerable burden to public health^[29]. DN is one of the most serious microvascular complications in type 2 diabetes. It is characterized by acute and transient renal dysfunction and even death. Many studies show that inflammation, oxidative stress, hyperglycemia, hyperlipidemia, advanced glycation end products (AGEs) and other pathological factors can lead to the formation and aggravation of DN, usually without adequate treatment, It will further develop into chronic kidney disease (CKD). Although several preventive measures can delay the onset and progress of diabetes nephropathy, the incidence rate related to this disease is still high, and new treatment methods are needed^[30–31]. At present, the scope of drug treatment for DN is very narrow, mainly based on the application of ACEIs and ARBs to block RASS and control blood sugar. Other drugs are used to supplement and enhance this RASS blockade or blood glucose control, such as SGLT2 inhibitors, GLP-1 agonists, mineralocorticoid receptor antagonists, and endothelin antagonists^[32]. Therefore, there is an urgent need to develop new treatment methods for DN. Research has found that the pathological and physiological mechanisms of DN are very complex. In its occurrence and development process, there is a comprehensive effect of multiple factors, in the past, cell necrosis or apoptosis. It is considered to be a direct factor affecting renal injury in diabetes^[33–35]. However, with the discovery of ferroptosis, it has aroused widespread concern. Many studies have shown that the accumulation of oxygen free radicals (ROS) and iron overload are important determinants of DN in diabetes. SLC7A11 and GPX4 are key antioxidant systems that reduce oxidative stress and are also central regulatory factors that resist the occurrence of ferroptosis^[36–37]. The death form of ferroptosis is essentially caused by an iron dependent reactive oxygen species ROS and lipid peroxidation^[38]. In recent years, an increasing number of studies have shown that ferroptosis plays an important role in the occurrence and development of DN^[39]. Inhibiting the occurrence of ferroptosis may be a potential therapeutic target for DN.

At present, many studies have shown that inhibiting the occurrence of ferroptosis plays a crucial role in the treatment and improvement of DN. Glabridin, a bioactive component in liquorice, plays a role in improving DN by regulating VEGF/Akt/ERK signaling pathway to inhibit ferroptosis^[40]. Calycosin inhibits HG induced ferroptosis by reducing lipid ROS and free iron input in HK-2 cells, and plays a protective role in DN^[41]. NAC maintains mitochondrial redox homeostasis and inhibits ferroptosis by activating SIRT3-SOD2/Gpx4 pathway, thus leading to DN NAC maintains mitochondrial REDOX homeostasis by activating SIRT3-SOD2/Gpx4 pathway and inhibits ferroptosis, thereby improving diabetic nephropathy^[42]. Umbelliferone inhibits ferroptosis and delays the progress of DN by activating Nrf-2/HO-1 pathway^[43]. Swi, a natural product, has been proved to be one of the few medicinal plants with anti diabetes effect^[44]. Swi’s recent research includes the treatment of various diseases caused by inflammation and oxidative stress^[45]. According to reports, Swi can be accessed through NF-κB/NLRP3/Caspase-1 signaling pathway reduces inflammation and oxidative stress and improves DN^[46]. In HepG2 cells induced by H₂O₂, Swi has antioxidant capacity through AKT/Nrf2/HO-1 signaling pathway, significantly inhibits Nrf2 nuclear translocation and HO-1 expression, reduces inflammatory response, and protects the liver of type 2 diabetes mice^[47].In this study, we showed for the first time that Swietenine significantly improved STZ/HFD-induced diabetic nephropathy and HG-induced MPC-5 cell death in vivo and in vitro by inhibiting the occurrence of oxidative stress and ferroptosis. Meanwhile, our research has found that the possible mechanism of these effects is that Swi activates through activation the Akt/GSK-3β/Nrf2 signaling pathway improves ferroptosis in DN while promoting antioxidant stress. In summary, these data provide scientific basis for the future clinical search for drugs to prevent DN.

The STZ/HFD induced DN model and HG induced podocytes have been widely studied in DN research. Firstly, in vivo, we found that Swi treatment of STZ/HFD induced diabetic rats resulted in a certain degree of improvement in the occurrence of elevated blood sugar, uric acid, creatinine, BUN, and urinary albumin excretion rate. It also reduced glomerular hypertrophy, increased mesangial matrix, and fibrin deposition in pathological parameters. In addition, the increase in podocin levels, a marker of glomerular injury, also reflects Swi protective effect on DN. Meanwhile, in vitro, HG stimulation can cause podocyte damage, while Swi significantly enhances cell viability. Considering that ferroptosis plays an important role in the occurrence and development of DN^[48], and ferroptosis is a regulatory cell death pathway with unique morphological, biochemical, and genetic characteristics^[49]. Targeting upstream regulatory factors of iron removal cascade reactions, including dysregulated iron levels and ROS production. When oxidative and antioxidant imbalances occur within cells, one possible reason may be the Fenton reaction between the overloaded divalent iron ions and hydrogen peroxide, that is, Fe²⁺ can induce oxidative stress^[50]. Generate hydroxyl radicals with stronger oxidative capacity, increase intracellular ROS levels, and ROS is the ultimate executor of cell ferroptosis^[51]. As is well known, HG stimulation can lead to an abnormal increase in reactive oxygen species (ROS), leading to abnormal lipid peroxidation^[52]. GSH and MDA are recognized as representatives of oxidative stress levels and are also biomarkers of ferroptosis. MDA derived from GSH and lipid peroxidase can lead to ferroptosis due to the depletion of GSH and the accumulation of lipid peroxides, and MDA is the final product of ferroptosis^[53]. At the same time, during the process of ferroptosis, special mitochondrial morphological changes will first occur, including mitochondrial membrane wrinkling, reduction or disappearance of cristae, increased membrane density, outer membrane rupture, and no chromatin condensation^[54]. In our study, it was noted that Swi can inhibit the production of MDA, Fe²⁺ and lipid ROS, increase the activity of GSH and improve the damage of glomerular mitochondria, and then Swi has a protective effect on HG-induced MPC-5 and STZ/HDF-induced DN rats.

As is well known, the main antioxidant in cells is glutathione. The Xc⁻ system is a reverse transporter of cysteine and glutamate in the cell membrane, with SLC7A11 being the main subunit at play. The system Xc⁻ transports glutamate out of cells in a 1:1 ratio, while also transporting cysteine into cells, which is then reduced to cysteine and participates in GSH biosynthesis^[55]. Inhibition of cysteine synthesis and GSH levels can directly affect the activity of GPX4, leading to increased ROS production and accumulation of lipid peroxidation induced ferroptosis^[56]. Some studies have shown that inhibiting GPX4 ubiquitination can reduce oxidative stress and ferroptosis, thereby improving DN^[57]. Zhang S, et al. proved that the renal protection function of Vitexin in HK-2 cells stimulated by HG and in DN induced by HFD/STZ was achieved by reducing GPX4 mediated ferroptosis, and expounded the important role of GSH/GPX4 axis in preventing lipid oxidation induced DN^[58]. These studies indicate that the SLC7A11/GPX4 axis plays an important role in maintaining lipid peroxidation homeostasis and regulating ferroptosis.

We further explored the protective mechanism of Swi in mitigating DN. In our study, we found through network pharmacology target prediction of Swi and DN that there are a total of 61 target intersections between the two. However, through KEGG enrichment pathway analysis, we found that the PI3K-Akt signaling pathway has a higher enrichment index than other signaling pathways, and the binding energy of Swi and Akt molecular docking results is -6.4 kcal/mol, indicating a good binding effect. It is meaningful when the binding energy is less than − 5. The nuclear factor red blood cell 2 related factor 2 (Nrf2) plays an important role in anti ferroptosis and is a key transcription factor in the body's antioxidant stress response. Promote cell survival and maintain cellular redox homeostasis^[59–61]. Therefore, we assume that Swi may phosphorylate Akt/GSK-3β. Furthermore, Nrf2 is activated to maintain its transcriptional function in the nucleus, and further acts on the downstream gene glutathione pathway of Nrf2 to inhibit ferroptosis, thereby alleviating kidney injury. We first observed that under HG stimulation,the Akt/GSK-3β/Nrf2 signaling pathway phosphorylated protein was significantly inhibited, and after administration, The expression level of Akt/GSK-3β/Nrf2 signaling pathway phosphorylation has significantly improved, indicating that Swi can significantly activate Akt/GSK-3β/Nrf2 signaling pathway. Subsequently, we used related inhibitors of the Akt/GSK-3β/Nrf2 signaling pathway lead to phosphorylation of Akt/GSK-3β, respectively the expression of nuclear input with Nrf2 is reduced. Then we observed that SLC7A11 and GPX4 were significantly expressed in the normal group, and with the intervention of Swi and Fer-1, SLC7A11 and GPX4 were restored. In the inhibitor group, the expression of SLC7A11 and GPX4 did not recover under the intervention of Swi, indicating that Swi may pass through the Nrf2 signaling pathway inhibits ferroptosis in podocytes. However, we did not further explore Swi and the specific binding sites of the Akt/GSK-3β/Nrf2 signaling pathway need to be elucidated through other experiments.

This finding suggests that Swi may serve as a potential therapeutic strategy for the management of DN, especially in cases where iron metabolism disorders are closely related to oxidative stress, providing new possibilities for the clinical treatment of DN. However, further research still needs to clarify its molecular mechanisms and potential side effects.

Credit authorship contribution statement

Mengyuan Lu conceived and designed the research project. Jingyu Duan, Shuang Liu and Wenhao Deng carried out the study. Feilong Pei drafted the manuscript. Yaowu Liu and Chunping Zhang revised the manuscript. Chunping Zhang supervised the experimentators. All authors have read and approved the final manuscript for publication. All authors have read and approved the final manuscript for publication.

All data were generated in-house, and no paper mill was used. All authors agree to be accountable for all aspects of work ensuring integrity and accuracy. All data were generated in-house, and no paper mill was used. All authors agree to be accountable for all aspects of work ensuring integrity and accuracy.

Conflict of interest

The authors confirm that there are no conflicts of interest.

Orcid

Chunping Zhang https://orcid.org/0009-0000-1646-828X

Funding

This research was supported by the Natural Science Foundation of Jiangsu Province (BK20181154); the National Natural Science Foundation of China (31300222).

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No competing interests reported.

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Natural product Swietenine improve the progression of diabetic nephropathy by inhibiting ferroptosis through activation of the Akt/GSK-3β/Nrf2 signaling pathway

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Abstract

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Introduction

Materials and methods

Materials

Cell culture and treatment

Cell experiment grouping

Cell viability assay

Renal function measurement

Iron content measurement

GSH and MDA content measurement

Lipid reactive oxygen species measurement

Transmission electron microscopy

Animal experiments

Histological analysis

Immunofluorescence staining (IF)

Western blotting analysis

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Results

Swi treatment improved renal function and pathological changes in DN rats

Swi inhibits oxidative stress and ferroptosis in the kidneys of DN rats

3.3 Swi significantly improves high glucose induced MPC-5 death

3.4 Swi protects MPC-5 cells from oxidative stress and ferroptosis induced by high glucose

3.5 Network pharmacology-based prediction of the target of Swi on DN

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