Inotodiol Ameliorates Oxidative Stress and Apoptosis by Regulating PI3K/Akt/GSK-3β Signaling Pathways in Diabetic Nephropathy

doi:10.21203/rs.3.rs-4008527/v1

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Inotodiol Ameliorates Oxidative Stress and Apoptosis by Regulating PI3K/Akt/GSK-3β Signaling Pathways in Diabetic Nephropathy

https://doi.org/10.21203/rs.3.rs-4008527/v1

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Diabetic nephropathy (DN), a kind of microvascular complication, is a primary cause of end-stage kidney disease worldwide. However, therapeutic drugs for DN treatment are still in lack. Inotodiol (INO), a kind of lanostane triterpenoid isolated from INO that has various biological activities. In this study, we employed db/db mice as the spontaneous DN model in vivo, and high glucose treated MPC5 cells in vitro to elucidate the protective effects and underlying mechanisms of INO in DN. Ratio of right kidney weight/body weight was calculated, and levels of FBG, urine albumin/creatinine (UACR), BUN and Scr were measured. The SOD, CAT, GSH-Px and MDA levels in kidney were detected by using commercial kits. The histopathological changes of renal tissues were assessed by HE, PAS and Masson staining. The intracellular ROS was detected by using fluorescence probe DCHF-DA. Cytotoxicity assay was performed using CCK-8 assay kit. The rate of apoptosis was detected by flow cytometry. The expressions of Bax, Bcl-2, Cytc, Cleaved caspase-3, GSK-3β, pSer-GSK-3β, Akt, p-Akt, Synaptopodin, WT-1, Nrf2, NQO1, Keap1, heme HO-1 were measured by western blot. The expressions of Bax, CytC, WT-1, Synaptopodin, Bcl-2, GSK-3β and pSer9-GSK-3β in renal tissues were measured by immunohistochemistry. Our results showed that INO treatment reduced the FBG, BUN, Scr and UACR levels in db/db mice. Moreover, INO increased the expressions of Synaptopodin and WT-1 proteins. Besides, INO treatment also mitigated kidney histopathological changes, reduces kidney oxidative stress as reflected by reduced levels of Keap-1, NOX4 and MDA, but increased levels of kidney antioxidants SOD, CAT, GSH-Px, Nrf2, NQO1 and HO-1. Additionally, kidney apoptosis decreased as reflected by decreased protein levels of Cytc, Bax and Cleaved caspase-3 while its anti-apoptosis Bcl-2 protein levels increased. Mechanistically, INO inhibited GSK-3β activity by activating the PI3K/Akt signaling pathway, increased the level of anti-apoptosis, decreased level of oxidative stress and reduced podocyte injury in vivo and in vitro. Collectively, these results indicated that INO protected against DN through ameliorating oxidative stress and apoptosis via the PI3K/Akt/GSK-3β pathway.

Diabetic nephropathy

Inotodiol

Oxidative stress

Apoptosis

Podocyte

PI3K/Akt/GSK-3β pathway

Diabetic nephropathy (DN), occurs in up to 50% of those living with diabetes, is a major cause of end-stage kidney disease (ESKD) that requires treatment with dialysis or kidney transplantation, and is associated with significantly increased cardiovascular morbidity and mortality ¹. The pathological characteristic of DN includes glomerular hypertrophy, mesangial matrix deposition, basement membrane thickening, tubulointerstitial fibrosis and atrophy ². Clinically, DN is featured by progressive albuminuria, subsequent to destruction of the glomerular filtration barrier by diabetic insults ³. The current standard of therapy for DN involves stringent blood pressure control via blockade of the renin-angiotensin system and control of hyperglycemia. Despite these strategies, DN is still seen to progress relentlessly. A pressing need for novel therapeutic agents has fueled endless basic science research projects and clinical trials in the quest for a more specific therapy ⁴.

The pathogenesis of DN is complicated, and oxidative stress is considered as the core of DN pathogenesis. High glucose can lead to increased production of reactive oxygen species (ROS), resulting in enhanced oxidative stress response ⁵. Nuclear factor erythroid 2-related factor 2 (Nrf2) is an important transcription factor for the body to resist oxidative stress. Inhibition of Nrf2-mediated activities of antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) aggravated kidney injury ⁶. In parallel to increased oxidative stress, inflammation, cell apoptosis and tissue fibrosis drive the relentless progressive loss of kidney function ⁷.

Glomerular podocytes, the key structural components of the barrier, plays a key role in controlling glomerular permselectivity ⁸. Indeed, as podocytes have limited ability to repair or regenerate, podocyte injury and loss have been shown as strong predictors of progressive DN, directly correlating with the magnitude of proteinuria ⁹. It has been found that various factors, such as glycolipid toxicity, angiotensin, and glycosylation end products, induce podocyte apoptosis through different pro-apoptotic pathways, leading to the occurrence of DN ¹⁰.

Phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) pathway mainly acts in regulation of cell proliferation, differentiation and apoptosis. Upon an extracellular stimulus, PI3K phosphorylates phosphatidylinositol4, 5-bisphosphate (PIP2) to generate phosphatidylinositol-3,4,5-triphosphate (PIP3). The generated PIP3 further induces Akt plasma membrane translocation and activates Akt under the catalysis of phosphoinositide-dependent kinase-1 (PDK1) ¹¹. Glycogen synthase kinase-3β (GSK-3β) is the downstream target of Akt. Activated Akt phosphorylates GSK-3β and blocks its activity to modulate various cellular functions ¹². Hence, targeting the PI3K/AKT/GSK-3β signaling pathway may be a new strategy for DN treatment.

Natural products with biological activity was a promising therapeutic approach towards preventing or arresting the progression of DN. INO, a kind of lanostane triterpenoid isolated from Inonotus obliquus, is a natural compound with many biological activities, including antitumor, antioxidant, anti-allergy, and hypoglycemic activity (Tian et al., 2022). Research demonstrated that 2α-hydroxy-inotodiol could effectively protect SH-SY5Y cells against H₂O₂-induced oxidative stress and apoptosis via the Nrf2 and BDNF/TrkB/ERK/CREB signaling pathway ¹³. Nevertheless, there are few reports about the effect of INO on DN. In this research, 8-week-old db/db mice with middle stage of DN in vivo and MPC5 cells treated with high glucose in vitro were used as the research model to unveil the effect and its underlying mechanisms of INO in DN development ¹⁴.

2.1 Chemicals and reagents

INO lyophilized powder was prepared into uniform suspension, before administration in vitro, the solution was diluted in medium. As for in vivo experimental studies, INO was dissolved in a 1% sodium carboxymethyl cellulose (CMC-Na, 9004-32-4, Sinopharm Chemical Reagent Co., Ltd, China) solution. Metformin (Met) with 98% purity was purchased from Sino-American Shanghai Squibb Pharmaceuticals Ltd (Shanghai, China).

2.2 Extraction of INO

After drying to constant weight, Inonotus obliquus were crushed into powder with a particle size of 40 meshes and then soaked in isopropyl alcohol for 0.5 h before being loaded into the chromatographic column. After eluting with isopropyl alcohol for 2 h, the extract was concentrated under reduced pressure at 50°C with 1/2 volume of the eluent. The upper petroleum ether phase was collected after extracting with petroleum ether for three times for 30 minutes. The dried powder of petroleum ether was recovered under reduced pressure at 60°C and loaded into a C18 modified silica gel packed chromatographic column then eluted with a gradient alcohol solution for 2h. The 100% ethanol elution peak was recovered under reduced pressure at 60°C, and then the concentrated solution was freeze-driedat − 80°C to obtain INO lyophilized powder.

2.3 Animals and treatment

In our research, C57BLKS/db (db/db) mice were used as the spontaneous DN model. Specific-pathogen-free grade male C57BLKS/J db/m and db/db mice (six-weeks-old) were purchased from Jiangsu Cavens Biotechnology Co., Ltd. The db/db mice were randomly divided into four groups as follows: the normal control group (db/m), the DN group (db/db), the low-dose (10 mg/kg/d) INO treatment group, the high-dose (20 mg/kg/d) INO treatment group, and the mice treated with Met (250 mg/kg/d) were as the positive control group. The dosage of INO was according to previous report ¹⁵. A 1% CMC-Na solution was employed for both untreated db/db mice and db/m mice. The mice were housed in a controlled environment with temperature of 23 ± 2℃, humidity of 60%±2% and 12 h light/dark cycle. Urine samples were collected one day before the end of the experiment. The mice were sacrificed after 8 weeks, blood samples were collected and bilateral kidneys were removed rapidly.

2.4 Cell culture

MPC5 cells were procured from Fenghui Biology Co., Ltd (Hunan, China) and cultured in low-glucose Dulbecco’s Modified Eagle’s Medium (DMEM) (Gibco, San Diego, CA, USA) containing 10% fetal bovine serum (FBS) (HyClone, USA) and 10 U/ML interferon-γ (IFN-γ) at 33°C for proliferation. Then the cells were differentiated without IFN-γ at 37°C for 14 days. For further study, the MPC5 cells were stimulated with high glucose (HG) (40 Mm glucose) (G8510, Solarbio, Beijing, China) and mannitol (MG) (34.5 Mm mannitol + 5.5 Mm glucose) (SG8510, Solarbio, Beijing, China) containing 10% FBS with or without incubation of INO for 24 h. The MPC5 cells were divided into the normal control (NC, 11.1 Mm) group, the HG (40 Mm) group, the HG + INO (10 Μm) group, the HG + TDZD-8 (5Μm) group, and the HG + INO + TDZD-8 group.

2.5 CCK-8 assay

MPC5 cells were seeded in 96 well microplates at a density of 5000 cells/well. Cells were cultured for 24 h and serum starved, then treated with HG (40 Mm) and different concentrations of INO. After exposed to INO for 48 h, cytotoxicity assay was performed using CCK-8 assay kit (Beyotime, Nantong, China). The absorbance value at 450 nm was read using a 96-well plate reader (BioTek, Winooski, VT, USA) and the OD450 nm absorbance was proportional to the viability of the cells.

2.6 Intracellular ROS Detection

The MPC5 cells were cultured in 6-well chambered cover glass and treated as indicated above. The cells were then washed three times with phosphate buffered saline (PBS). Next, the cells were incubated with 10 Mm fluorescence probe DCHF-DA in PBS at 37°C for 30 min and washed in order to remove the residual probes. The level of intracellular ROS was detected by a fluorescent microscope (Olympus BX63, Japan).

2.7 Biochemical measurement

FBG levels were measured by ACCU-CHEK Guide (929, Luoshi, Shanghai, China). BUN, Scr and UACR levels were measured using commercial kits from Nanjing Jiancheng Bioengineering Institute (Nanjing, China) according to the manufacturer’s instructions.

2.8 Determination of Oxidative Stress Indicators

The SOD, CAT, GSH-Px and MDA levels in kidney were detected by using commercial kits obtained from Beyotime Biotechnology (China) according to the manufacturer’s instructions. Corresponding protein concentrations were measured by employing BCA protein quantitative kit (Beyotime, Nantong, China).

2.9 Histopathological staining of kidney tissues

The histology of kidney tissues were assessed by HE, PAS and Masson staining. The kidney tissues were perfused with saline, then fixed in 4% paraformaldehyde for 48 h, embedded in paraffin, and sliced (thickness: 3µm). After routine dewaxing of kidney paraffin sections, HE, PAS and Masson staining was performed according to the manufacturer’s instructions. Positive cells were morphometrically quantified with Image J software.

2.10 Immunofluorescence analysis

MPC5 cells were digested into suspension and inoculated in a 12-well plate covered with small round glass dishes. After adhering, cells were subjected to various treatments and kept up culturing for 48 h. After abandoning the medium, cells were washed with PBS for 3 times, then fixed with ice methanol for 20 min at 4℃. The cells were subsequently incubating in QuickBlock Blocking Buffer for 10–15 min at room temperature and then incubated with NOX4 (Proteintech, 14347-1-AP, 1:50) and Synaptopodin (Proteintech, 21064-1-AP, 1:200) primary antibodies for 4℃ overnight. The next day, washed the cells with PBS for 3 times and then incubated with CoraLite488-conjugated Goat Anti-Rabbit IgG (H + L) (Proteintech, China) for 1 h at room temperature in the dark. The cell nucleus were stained with DAPI (C1005, Beyotime, Nantong, China) for 3 min. Images were obtained by Olympus BX43F fluorescence microscope.

2.11 Immunohistochemistry analysis

The expressions of Bax, Cytc, Bcl-2, WT-1, Synaptopodin, GSK-3β and pSer9-GSK-3β in kidney tissues were measured by immunohistochemistry (IHC). Kidney tissues were fixed with 4% paraformaldehyde, embedded in paraffin, and cut into 3 µm thick sections. Kidney paraffin sections were dewaxed, rehydrated, antigen repaired and blocked with goat serum for 20 min at 37℃. Then the sections were incubated with primary antibodies including Bax (CST, 2772S, 1:200), Cytc (Proteintech, 10993-1-AP, 1:100), Bcl-2 (Abcam, Ab182858, 1:500), WT-1 (CST, 83535S, 1:50), Synaptopodin (Proteintech, 21064-1-AP, 1:100), GSK-3β (CST, 12456S, 1:200) and pSer9-GSK-3β (CST, 5558T, 1:200) over night at 4℃. After washing with PBS, the sections were incubated with corresponding HRP-labeled antibody at 37℃ for 1 h. Subsequently, the sections were stained with DAB (Proteintech, China) for 2 min and then re-stained with hematoxylin. Finally, the sections were observed under stereomicroscope (Leica, Germany). Image J software was used to measure the positive staining area. The integral optical density (IOD) of positive staining area were measured using Image-ProPlus 6.0 software (Media Cybernetics, Silver Spring, MD, USA).

2.12 Flow cytometry

MPC5 cells were washed with PBS three times and harvested by trypsin digestion. After incubation with 10 µL Annexin V-FITC and 5 µL propidium iodide in the dark, the cells were analyzed using a BD FACSVerse flow cytometer (BD Bioscience, San Jose, CA, USA). The rate of apoptosis was expressed by Annexin V/PI+− (early apoptosis) and Annexin V/PI (late apoptosis) status++.

2.13 Western blot

Total protein samples of kidney tissues and MPC5 cells were extracted by RIPA lysis buffer containing 1% protease inhibitors (BOSTER, China) and 1% phosphatase inhibitors (MedChemExpress, USA). The protein levels were measured by BCA assay (Beyotime, China). Equal amounts of protein were separated by 12% SDS-PAGE gels. Then the proteins were transferred to NC membrane (BOSTER, China) and blocked with 5% fat-free milk for 70 min at room temperature. Subsequently, the membranes were incubated with primary antibodies at 4℃ overnight including Bax (CST, 2772S 1:1000), Bcl-2 (Abcam, Ab182858, 1:1000), Cytc (Proteintech, 10993-1-AP, 1:1000), Cleaved caspase-3 (CST, 9664S, 1:1000), GSK-3β (CST, 12456S, 1:1000), pSer-GSK-3β (CST, 5558T, 1:1000), Akt (CST, 4685S, 1:1000), p-Akt (CST, 4060S, 1:1000), Synaptopodin (Proteintech, 21064-1-AP, 1:1000), WT-1 (CST, 83535S, 1:1000), NQO1(Abcam, ab34173, 1:1000), Keap-1 (CST, 8047S, 1:1000), HO-1 (CST, 43966S, 1:1000), Nrf2 (Proteintech, 16396-1-AP, 1:1000) and β-actin (Abcam, ab8227, 1:1000). Corresponding Anti-rabbit IgG HRP-linked antibody (CST,7074S,1:3000) were incubated for 2h at room temperature. Target proteins were detected by an ECL system. β-actin was used as an internal reference and Image J software was used to analyse the grey values of the bands.

2.14 Statistical analysis

Statistical analysis was performed using the GraphPad Prism version 8.0 (GraphPad Software Inc., USA). Values were expressed as the mean ± standard deviation (SD). Two groups of the data were compared using the two-tailed Student’s t test. Data from more than two groups were compared using one-way analysis of variance (ANOVA) followed by Turkey’s post-test. P values were adjusted for multiple comparisons when appropriate. P < 0.05 was considered to indicate statistical significance.

3.1 INO Alleviated Morphological Changes of Kidney and Improved Renal Function

In Fig. 1A, HE and PAS staining showed significant morphological changes in db/db mice, including glomerular hypertrophy, glomerular mesangial matrix accumulation, glomerular basement membrane thickening and tubular basement membrane thickening, nevertheless INO and Met treatment alleviated these changes (P < 0.05) (Fig. 1B). Masson staining showed that glomerular and tubular-interstitial fibrosis occurred in db/db mice, however, the degree of fibrosis was relieved after administration with INO and Met (P < 0.05) (Fig. 1A and C). These data indicated that INO improved morphological changes of kidney in db/db mice. As shown in Fig. 1D and E, the FBG levels and kidney weight of the mice were measured. The FBG levels and the ratio of kidney weight/body weight of db/db mice were significantly increased when compared with those of db/m mice (P < 0.05). Compared with db/db mice, INO and Met treatment decreased FBG levels and reduced ratio of kidney weight/ body weight (P < 0.05). At the same time, INO and Met markedly reduced BUN (Fig. 1F), Scr (Fig. 1G), UACR (Fig. 1H) levels in db/db mice (P < 0.05), suggesting that INO improved kidney function in db/db mice.

3.2 INO Mitigated Podocyte Injury in Vivo and in Vitro

Podocyte was the last barrier of glomerular filtration membrane. A variety of injury factors such as immunity, inflammation, poison, infection, metabolism, environment and genetic background could directly or indirectly affect podocyte injury, resulting in proteinuria and glomerular fibrosis. In our research, the protein expressions of WT-1 and Synaptopodin, markers of glomerular podocytes, were examined by western blot. In db/db mice, protein levels of WT-1 and Synaptopodin were significantly decreased (P < 0.05) (Fig. 2A and B). The same result of Synaptopodin expression was confirmed by IHC analysis (Fig. 2C and D). However, these changes were reversed by INO and Met treatment, which indicated that INO lessened the glomerular podocyte injury in DN. In vitro experiments, podocytes were cultured in medium with a high concentration of glucose (40 mmol/L) and then treated with INO at different concentrations (1, 2.5, 5, 10, 20, 40, 80 and 160 umol/L) for 24 h (P < 0.05) (Fig. 2E). We finally selected 10 umol/L as the experiment concentration of INO due to the CCK-8 assay to evaluate the effect of INO treatment on podocyte injury (Fig. 2F). Notably, the expression of podocyte-specific markers WT-1 and Synaptopodin were markedly rescued by INO treatment in MPC5 cells. (P < 0.05) (Fig. 2G and H). Our results indicated that INO could mitigate podocyte injury in vivo and in vitro experiments.

3.3 INO Administration Alleviated Oxidative Stress by Activating Nrf2 Signaling Pathways in Vivo and in Vitro

To evaluate the effects of INO on oxidative stress in DN, we detected the levels of ROS and NOX4 in Fig. 3A and B. The results revealed that the increased levels of intracellular ROS induced by HG treatment measured were inhibited after INO treatment (Fig. 3A), which demonstrated that INO alleviated HG-induced cell ROS level. Meanwhile, INO treatment lessened the increased expression of NOX4 in HG group in MPC5 cells (Fig. 3B). In addition, the activities of three major antioxidant enzymes including SOD, CAT and GSH-Px decreased significantly in db/db mice, after INO and Met administration, the activities of them increased greatly. Whereas the level of MDA was significantly reduced in INO treatment group (P < 0.05) (Fig. 3C–F). These results demonstrated that INO administration alleviated oxidative stress injury via enhancing the contents and activities of antioxidant enzymes. Subsequently, we explored the potential mechanism of INO in regulating of oxidative stress on DN. Nrf2 is an important transcription factor regulating cellular oxidative stress response, and is also a central regulator of intracellular redox homeostasis. By inducing and regulating the expressions of a series of antioxidant proteins, Nrf2 can reduce cell damage caused by ROS, which keep cells in a stable state and maintain the redox dynamic balance of the body. In this research, western blot results showed the protein expression levels of Nrf2, NQO1 and HO-1 were lower in db/db mice kidneys when compared to db/m mice. Meanwhile, the protein expression levels of Keap-1 was relatively higher in the kidney corpuscles of the untreated db/db mice when compared to db/m mice (P < 0.05) (Fig. 3G and H). Following INO treatment, Nrf2, NQO1 and HO-1 protein expressions were increased and the Keap-1 protein expression in the kidney was decreased significantly (P < 0.05) (Fig. 3G and H). Furthermore, in vitro experiments, western blot analysis showed that a significantly lower Nrf2, but a significantly higher Keap-1 (Fig. 3I and J) protein levels were observed in HG induced MPC5 cells injury when compared to NC group. However, after treatment with INO, Nrf2 protein levels were increased while Keap-1 protein levels were decreased when compared to HG group. (P < 0.05) (Fig. 3I and J). Our results demonstrated that INO possessed strong antioxidant capacity against oxidative stress damage caused by DN by activating Nrf2 signaling pathways in vivo and in vitro.

3.4 INO Inhibited Apoptosis of Kidney Cells in Vivo and in Vitro

As shown in Fig. 4A and B, the expressions of pro-apoptotic proteins Cytc, Cleaved caspase-3 and Bax were significantly increased in db/db group and this phenomenon was inhibited after INO treatment (P < 0.05). The expression of anti-apoptosis protein Bcl-2 decreased greatly in db/db group, but INO treatment restore its expression (P < 0.05). Similar result was demonstrated by immunohistochemistry (P < 0.05) (Fig. 4C and D). The results of immunohistochemistry and western blot showed that pro-apoptotic factor Cytc, Cleaved caspase-3 and Bax was upregulated in db/db group, whereas its expression was inhibited after INO treatment. Meanwhile, INO treatment caused a decrease in the protein levels of Bax, Cleaved caspase-3 and an increase in Bcl-2 protein levels in MPC5 cells (P < 0.05) (Fig. 4E and F). Consistent with the above results, flow cytometry data of PI/Annexin V-stained MPC5 cells showed that INO reduced HG-induced cell apoptosis (Fig. 4G). In sum, our data suggested that INO protected against DN through inhibiting apoptosis of kidney cells.

3.5 INO Attenuated DN by Modulating the PI3K/Akt/GSK-3β pathway

Recent studies have shown that the PI3K/Akt/GSK-3β signaling pathway is involved in the progression of DN and apoptosis of kidney cells. As shown in Fig. 5A and B, western blot results indicated that the ratio of pSer9-GSK-3β/ GSK-3β and p-Akt/ Akt in db/db mice were decreased when compared to normal mice, whereas both high and low doses of INO treatment reversed the decreased ratio of pSer9-GSK-3β/ GSK-3β and p-Akt/ Akt (P < 0.05) (Fig. 5A and B). The protein expression of pSer9-GSK-3β in kidney cortex of mice were examined by IHC as well. There was no apparent change in kidney expression of total GSK-3β with different treatments of db/db mice (P < 0.05) (Fig. 5C and D), the results were consistent with those of western blot. We also explored the effect of PI3K/Akt/GSK-3β signaling pathway in MPC5 cells in vitro. Similarly, INO treatment upregulated the decreased ratio of pSer9-GSK-3β/ GSK-3β and p-Akt/ Akt induced by high glucose (P < 0.05) (Fig. 5E and F). Furthermore, as shown in Fig. 5G, INO treatment increased the levels of Synaptopodin in HG-induced MPC5 cells, while this elevated effect was inhibited following the treatment of TDZD-8 which was the inhibitor of GSK-3β. These findings indicated that INO could attenuate DN-induced podocyte injury by modulating the activity of the PI3K/Akt/GSK-3β signaling pathway.

DN is a complication of diabetes mellitus and a major cause of chronic kidney failure. It is characterised by the accumulation of large amounts of extracellular matrix in the glomerular and tubulointerstitial areas of the kidney and the thickening of the intrakidney vascular system, which gradually becomes hyalinized ¹⁶. In DN clinical practice, conventional strategies such as blockade of the renin-angiotensin system, controlling blood pressure, blood sugar and weight may not provide effective relief. There is an urgent need for substantial research efforts to develop new therapeutic approaches ¹⁷. A large number of studies have shown that the active ingredients in natural herbs, which have a protective effect against kidney injury, are expected to be an alternative strategy for DN treatment because of their multi-targeted effects. INO has a wide range of biological activities and affects the oxidative stress, apoptosis, inflammatory response, and other aspects ¹⁸. Nevertheless, there were few research were conducted on the effect and mechanism of INO on DN. The db/db mice model is an internationally recognized animal model of diabetes and the pathogenesis of DN in db/db mice is similar to that in human ¹⁹. In our research, after the administration of INO, levels of FBG, right kidney weight/ body weight, BUN, Scr, and UACR were decreased. Structural lesions, including glomerular hypertrophy, thickened glomerular basement membrane, increased extracellular matrix deposition and glomerulosclerosis, are involved in progressive proteinuria in DN ²⁰. Our results indicated that INO administration attenuated glomerular injury, including glomerular mesangial cell proliferation and kidney fibrosis, in db/db mice. These results proved the kidney protective effect of INO.

Oxidative stress is an important pathogenic mechanism in DN. Increased ROS production or inadequate antioxidant systems can lead to ROS accumulation and oxidative stress. In kidney cells, oxidative stress has a variety of deleterious effects, including lipid peroxidation, DNA damage, protein modification, activation of pro-inflammatory and pro-fibrotic pathways, and apoptosis ²¹. Zhao et al demonstrated that INO attenuates the damage caused by CCL₄-induced oxidative stress in mice ²². This was also confirmed in our study. Persistent hyperglycaemia resulted in downregulation of SOD, GSH-Px and CAT levels and upregulation of MDA levels, all of which were significantly alleviated in INO treatment group. In diabetic patients, the main cause of ROS production is mediated by various NADPH oxidases (NOX), but the presence of abnormalities in the antioxidant system and mitochondrial function may also contribute to its occurrence ²³. NOX4, also known as kidney oxidase, is found in the kidney vascular system, glomerular thylakoid cells, tubular cells and podocytes ²⁴. In the current study, INO treatment significantly inhibited ROS levels reduced NOX4 expression. Similarly, Song et al demonstrated that ROS can accelerate the disease development of DN by treating human kidney-2 (HK-2) cells with HG to establish a cellular model of DN, knocking down NOX4 intracellularly and alleviating HG-induced cell damage by inhibiting AMPK/mTOR signaling in HK-2 cells ²⁵. Nrf2 is a key transcription factor that regulates oxidative stress and is capable of regulating both oxidative stress damage in type 2 DN ^26,27. Due to prolonged oxidative stress, Nrf2 dissociates from Nrf2-Keap1 complex in the cytoplasm resulting in decreased levels of the cytoplasmic Nrf2 while free cytoplasmic Keap-1 levels increase. Following its dissociation from this complex, Nrf2 enters the nucleus to bind to antioxidant response element which resulted in increases the levels of NQO1 and HO-1. However, due to its utilization to counteract the increase in ROS and other pro-oxidants, the levels of these anti-oxidative enzymes markedly decrease in diabetes ²⁸. The ability of INO to maintain high levels of cytoplasmic Nrf2 indicate that a lesser amount of Nrf2 is translocated into the nucleus in view of the diminishing oxidative stress level in kidney in db/db mice following INO treatment. Besides, INO treatment also resulted in downregulation of Keap1, indicating lesser dissociation of Nrf2-Keap-1 complexes as a result of reduced oxidative stress. In the meantime, the upregulation of NQO1 and HO-1 following INO treatment would also help to convert the ROS to inert species. Our findings reveal that INO could alleviate the levels of oxidative stress though Nrf2-Keap-1 signaling pathway.

In DN development, apoptosis also occurs in epithelial cells in the proximal ileal tubule, leading to tubular atrophy, which leads to tubular cell depletion and subsequent formation of tubular glomeruli associated with loss of kidney function, cell apoptosis leading to glomerular injury and foot cell depletion, which is associated with proteinuria and structural glomerular damage in DN ²⁹. Cytc is a pro-apoptotic signal under stress conditions, and when Cytc is released from the mitochondria into the cytoplasm, it binds to apoptotic protease activator 1 (Apaf-1) to form an apoptosome, which in turn leads to the activation of cystathione-9 and the downstream cysteine activation of the aspartyl cascade ^30,31. Mtitochdrial ROS (mtROS) can activate inflammatory vesicles and trigger an inflammatory response, causing Cytc to be released from the mitochondria into the cytoplasm, leading to apoptosis ³². We found that the expressions of Cleaved casepase-3, Cytc, Bcl-2 and Bax was significantly increased in db/db mice, and the IHC results also showed a decrease in the protein contents of Cytc, Bcl-2 and Bax in db/db mice. The expressions of Cleaved caspase-3 increased and the Bcl-2/ Bax ratio decreased in the high glucose environment, indicating that the level of apoptosis was higher in the model group, while the expression of Cleaved casepase-3 decreased and the Bcl-2/ Bax ratio increased in the INO treatment groups compared to the DN group, indicating that INO treatment could inhibit apoptosis. In addition, the number of late apoptotic cells was significantly increased in the HG group compared to the NC group by flow cytometry, and the number of late apoptotic cells was reduced after INO intervention. These results suggest that INO showed protective effect on DN by inhibiting apoptosis.

Podocytes are a major component of the glomerular filtration barrier. podocyte homeostasis is critical for maintaining the structural and functional integrity of the glomerular filtration barrier ⁸. Injury and loss of podocytes lead to albuminuria, a hallmark of diabetic nephropathy and a predictor for the progression of kidney diseases. As podocytes have limited ability to repair and/or regenerate, the extent of podocyte injury is a major prognostic determinant in diabetic nephropathy and other common causes of end-stage kidney disease ⁹. Studies have shown that the progression of various glomerular diseases is related to the abnormal changes of podocytes. There are a variety of specific markers on glomerular podocytes, such as Synaptopodin, WT-1, nephrin and C3b receptors. Changes in the content, structure and function of any of these markers can lead to podocyte abnormalities. Wilm’s tumor-1 (WT-1) plays a key role in epithelial differentiation. During kidney development, WT-1 and Synaptopodin are expressed in mesenchyme and glomerular visceral epithelium and it generates the conversion of these cells into mature podocytes. The continuous expression of WT-1 and Synaptopodin in terminally differentiated podocytes is essential for maintenance of podocyte function ³³. In the current study, WT-1 and Synaptopodin protein expressions in the kidney of db/db mice were increased significantly after INO treatment, which indicated that INO protected against glomerular podocytes injury. This result is in accordance with that of another study in which administered wogonin, a flavonoid derived from the root of Scutellaria baicalensis Georgi, significantly ameliorated the expression of nephrin and WT-1 in mice and in cells ³⁴.

PI3K is a key regulatory molecule of several signaling pathways involved in the regulation of cell growth, metabolism and apoptosis throughout the process ³⁵. P-Akt is a downstream protein of the PI3K signaling pathway and plays an important role in cell growth and apoptosis ³⁶. GSK-3β is a Ser/Thr kinase involved in a variety of metabolic processes that include glycogen metabolism, Wnt signaling and sensitization to apoptosis ³⁷. Interest in GSK-3β expanded greatly with the realization that it also acts as a convergence point for multiple cell signaling pathways involved in inflammation, immunomodulation, embryogenesis, tissue injury, repair and regeneration ^38,39. GSK-3 exists as two isoforms, GSK-3α and GSK-3β. In different organ tissues, the two isoforms are differentially expressed. In the kidney cortex, GSK-3β is primarily expressed and located to glomeruli and proximal tubular cells ^40,41. In murine kidneys, GSK-3β was predominantly expressed in glomeruli and distributed intensely in podocytes ⁴². Numerous studies have established a correlation between GSK-3β and type 2 diabetes, with high GSK-3β expression causing insulin resistance and the PI3K/Akt signaling pathway altering its phenotype and causing podocyte damage, thereby contributing to the development of DN ⁴³. Akt is activated by phosphorylation and activated Akt (p-Akt) inversely regulates GSK-3β ⁴⁴. To verify the potential mechanism of INO on DN, westerm blot and IHC staining were used to detect the distribution and protein expressions of PI3K/Akt/GSK-3β signaling pathway. Our research demonstrated that following INO treatment, activated p-Akt inhibited GSK-3β expression by upregulating the level of Ser9 phosphorylation at the site of GSK-3β and suppressing GSK-3β protein expression in vivo and in vitro experiments. In our study, cell intervention with a GSK-3β inhibitor in high glucose-stimulated MPC5 cells resulted in alleviation of cellular damage. Besides, PI3K/Akt/GSK-3β signaling pathway is closely associated with a variety of kidney diseases and is key to Nrf2 inactivation and oxidative stress. Zhou S et al suggest the GSK-3β regulated Nrf2 antioxidant response as a novel therapeutic target for protecting podocytes and treating proteinuric glomerulopathies ⁴¹. This is also demonstrated in our study. Meanwhile, activated GSK-3β stimulated the opening of the mitochondrial permeability transfer pore by decreasing the level of Bcl-2 and increasing the level of Bax and Cleaved caspase-3, thereby contributing to the release of Cytc from the mitochondria. These results suggest that INO may improve DN by mediating the PI3K/Akt/GSK-3β signaling pathway.

In this study, we unveil the protective effect and the molecular mechanism of INO on DN through in vivo and in vitro experiments. We identified that INO administration can alleviate diabetes-induced kidney dysfunction, which may be attributed to the beneficial effect on endothelial cells through reducing oxidative stress and apoptosis, attenuating high glucose-induced podocyte injury. Mechanistically, these pharmacological properties of INO were through upregulating the activity of the PI3K/Akt pathway, inhibiting the activation of GSK-3β and reducing the levels of Bax, Cytc and Cleaved caspase-3, increasing the antioxidant levels of Nrf2, NQO1 and HO-1. INO counteracts the DN-induced inhibition of the PI3K/Akt/GSK-3β pathway, alleviates oxidative stress and ultimately suppresses kidney cell apoptosis. Taken together, our study suggests that INO may act as a novel therapeutic strategy to DN.

FBG fasting blood glucose

BUN blood urine nitrogen

Scr serum creatinine

SOD superoxide dismutase

CAT catalase

GSH-Px glutathione peroxidase

MDA malondialdehyde

HE hematoxylin and eosin

PAS periodic acid Schiff

ROS reactive oxygen species

CCK-8 counting Kit-8

Bcl-2 B-cell lymphoma 2

Cytc Cytochrome C

GSK-3β glycogen synthase kinase-3β

WT-1 Wilms tumor 1

Nrf2 nuclear factor erythroid 2-related factor 2

NQO1 NAD(P)H quinone oxidoreductase1

Keap-1 kelch-like ECH-associated protein 1

HO-1 heme oxygenase-1

DN Diabetic nephropathy

INO Inotodiol

MPC5 mouse podocyte clone 5

HK-2 human kidney-2

Met Metformin

ESKD end-stage kidney disease

PI3K Phosphoinositide 3-kinase

mtROS Mtitochdrial ROS

FBS fetal bovine serum

HG high glucose

MG mannitol

NC normal control

IHC immunohistochemistry

NOX NADPH oxidases

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.

Ethical Declarations

Ethics approval and consent to participate

All the animal experiments were approved by the guidelines of the Ethics Committee of Shanxi Provincial People's Hospital(Protocol number 2022237).

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Funding

This work was supported by Shanxi Provincial Key Laboratory Project (grant number 201805D111020), Key Laboratory construction Project of Shanxi Provincial Health Commission (grant number 2020SYS01), Shanxi Provincial Key Medical Scientific Research Project Key Research Project (grant number 2020XM02), Fundamental Research Program of Shanxi Province (No.201901D211522) and Local science and technology development funds projects guided by central government (No.YDZJSX2021C027).

Author contributions

Lingling Tian: Conceptualization, Methodology, Resources, Software, Data curation and Writing – original draft. Qi Duan: Supervision, Validation and Writing – review & editing. Rongshan Li: Validation, Project administration. Yafeng Li: Supervision, Funding acquisition and Writing – review & editing.

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No competing interests reported.

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Inotodiol Ameliorates Oxidative Stress and Apoptosis by Regulating PI3K/Akt/GSK-3β Signaling Pathways in Diabetic Nephropathy

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Abstract

Figures

1 Introduction

2 Materials and methods

2.1 Chemicals and reagents

2.2 Extraction of INO

2.3 Animals and treatment

2.4 Cell culture

2.5 CCK-8 assay

2.6 Intracellular ROS Detection

2.7 Biochemical measurement

2.8 Determination of Oxidative Stress Indicators

2.9 Histopathological staining of kidney tissues

2.10 Immunofluorescence analysis

2.11 Immunohistochemistry analysis

2.12 Flow cytometry

2.13 Western blot

2.14 Statistical analysis

3 Results

3.1 INO Alleviated Morphological Changes of Kidney and Improved Renal Function

3.2 INO Mitigated Podocyte Injury in Vivo and in Vitro

3.3 INO Administration Alleviated Oxidative Stress by Activating Nrf2 Signaling Pathways in Vivo and in Vitro

3.4 INO Inhibited Apoptosis of Kidney Cells in Vivo and in Vitro

3.5 INO Attenuated DN by Modulating the PI3K/Akt/GSK-3β pathway

4 Discussion

5 Conclusion

Abbreviations

Declarations

References

Additional Declarations

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