Chicoric acid advanced PAQR3 ubiquitination to ameliorate ferroptosis in diabetes nephropathy through the relieving of the interaction between PAQR3 and P110α pathway

ABSTRACT

Purpose

This study aimed to examine the impact of CA on DN and elucidate its underlying molecular mechanisms of inflammation.

Methods

We fed C57BL/6 mice injected with streptozotocin to induce diabetes. In addition, we stimulated NRK-52E cells with 20 mmol/L d-glucose to mimic the diabetic condition.

Results

Our findings demonstrated that CA effectively reduced blood glucose levels, and improved DN in mice models. Additionally, CA reduced kidney injury and inflammation in both mice models and in vitro models. CA decreased high glucose-induced ferroptosis of NRK-52E cells by inducing GSH/GPX4 axis. Conversely, the ferroptosis activator or the PI3K inhibitor reversed positive effects of CA on DN in both mice and in vitro models. CA suppressed PAQR3 expression in DN models to promote PI3K/AKT activity. The PAQR3 activator reduced the positive effects of CA on DN in vitro models. Moreover, CA directly targeted the PAQR3 protein to enhance the ubiquitination of the PAQR3 protein.

Conclusion

Overall, our study has uncovered that CA promotes the ubiquitination of PAQR3, leading to the attenuation of ferroptosis in DN. This effect is achieved through the activation of the PI3K/AKT signaling pathways by disrupting the interaction between PAQR3 and the P110α pathway. These findings highlight the potential of CA as a viable therapeutic option for the prevention of DN and other forms of diabetes.

Graphical abstract

Cichoric acid (CA) extract of Dandelion advanced PAQR3 expression to ameliorate ferroptosis in DN by activating the PI3K/AKT signaling pathways. Importantly, CA advanced PAQR3 ubiquitination to restore PI3K/AKT signaling through the relieving the interaction between PAQR3 and P110α pathway.

KEYWORDS:

• Cichoric acid
• ferroptosis
• diabetes nephropathy
• PAQR3
• ubiquitination

Introduction

Diabetes is a common occurring disease that seriously endangers human health. The prevalence rate of diabetes among adults aged ≥18 years in China is as high as 11.6%, with 114 million patients; In addition, according to the prediction of the World Health Organization, there will be more than 130 million type 2 diabetes patients in China by 2025, which will impose a huge burden on China’s society and economy. With the aging of the population and the aggravation of urbanization, the incidence rate of diabetes has been steadily increasing in China.¹ Although the prevalence of diabetes is increasing year by year, it is still a preventable and curable disease, which has been listed as the key prevention and treatment content in the medical and health system reform of China and Shanghai. The development mode of diabetes is from the normal population to the high-risk population, and then hyperglycemia occurs. Complications may occur in any of these links, which will directly lead to death.² This represents a sixteen-fold increase in prevalence over a span of nearly four decades.³ Hierarchical management, screening and different intervention measures at all stages of diabetes development, especially at high-risk stage and early stage of disease, can not only delay or even reverse the progress of diabetes. Effective health intervention for high-risk groups of diabetes can prevent or delay their progression to diabetes and improve their quality of life.³ With the rapid societal development and changes in dietary habits, has been steadily increasing.^4,5 Previous studies have shown that the body’s micro-inflammatory response plays a significant role in the onset of diabetic nephropathy.^4,6

Diabetic nephropathy (DN) is a common microvascular complication of diabetes, with an incidence rate as high as 20% to 40%. It is also a significant cause of end-stage renal disease.⁷ Under the condition of high blood sugar level for a long time, diabetes patients have proteinuria and the complications of diabetes nephropathy with reduced glomerular filtration rate In China, the occurrence of this disease is increasing year by year, becoming the second factor leading to end-stage renal disease. Patients with diabetes nephropathy are often accompanied by renal function damage, but the disease starts insidiously, progresses slowly, and does not have typical clinical characteristics when it comes to early onset. Therefore, when patients have persistent proteinuria, the disease progress is relatively serious, and patients’ renal function is often irreversibly damaged. Currently, the clinical prevention and treatment methods for DN mainly focus on controlling blood sugar, while also considering the supplementation of vitamins and minerals to alleviate clinical symptoms. However, there is currently no direct and effective treatment method for DN.⁸

Iron death has received widespread attention in recent years. Many studies have found that iron death exists in diabetes and its complications.^9,10 Inhibiting iron death can greatly slow down the occurrence and progress of diabetes and its complications. Traditional Chinese medicine is a unique medical treasure in China.¹¹ It has the advantages of multi-channel, multi-directional, inexpensive, and low adverse reactions. It may be a new direction to treat diabetes and its complications in the future from the perspective of regulating iron death by traditional Chinese medicine¹¹ Fundamentally, Ferroptosis is due to the disorder of the metabolism of lipid oxide in the cell, that is, because the activity of glutathione peroxidase 4 (GPX4) decreases, the lipid oxide cannot be metabolized into non-oxidative toxic substances, and then catalyzed by the way of Fenton reaction under the action of divalent iron ions, resulting in a large amount of iron deposition in the cell, seriously damaging the balance of oxidation and reduction in the cell, attacking biological macromolecules, and triggering cell death.^12,13 Ferroptosis plays a crucial role in kidney disease as well as other multi-system diseases.¹⁴

PI3K/AKT signaling pathway can regulate cell survival, autophagy, apoptosis, oxidative stress, and inflammatory response. Inhibiting its expression can reduce cellular inflammatory response and alleviate renal tissue damage¹⁵. PI3K/Akt pathway can promote Nuclear factor E2-related factor 2 (Nrf2) activation.^16–18 Therefore, activation of Akt/Nrf2/GPX4 pathway holds great significance in inhibiting the occurrence of Ferroptosis.

Progestin and adipo Q acceptor 3 (PAQR3) is a transmembrane protein reported to be located on the Golgi apparatus, and is a member of the PAQR family.^19.20 The deletion of PAQR3 promotes the degradation of Nrf2 protein through the ubiquitination process mediated by Keap1, thus enhancing the sensitivity of cancer cells²¹. In mechanism, the down-regulation of PAQR4 blocked the signal transmission of PI3K/AKT pathway.²²

In clinical practice, dandelion alone or in combination with other herbs is effective in treating diabetes nephropathy.²³ The application of dandelion compatibility in traditional Chinese medicine indicates that it is considered a valuable herb with potential therapeutic effects on this disease.^23–25 Cichoric acid (CA) is a kind of hydroxycinnamic acid, which is an important active ingredient in many natural plants such as dandelion, echinacea, chicory, etc.²⁶ In plants, CA plays a crucial role in protecting against insects, viruses, bacteria, fungi, and nematodes.²⁷ Modern pharmacological studies show that CA has significant biological activities in anti-inflammatory, antioxidant, immune regulation, antibacterial, antiviral, anti-tumor, and other aspects.²⁸ The biological activity of CA is its inhibitory effect on human immunodeficiency virus. With the development and application of molecular biological technology, the inhibitory activity, and mechanism of CA against other viruses have been continuously reported²⁹; Furthermore, CA has also exhibited significant inhibitory effects against various pathogenic bacteria.²⁷ CA is a kind of phenylpropionic acid compound, which mainly exists in Echinacea purpurea and Cichorium chicory. It has antibacterial, antiviral, anti-inflammatory, and immunomodulatory effects. Notably, CA has displayed inhibitory effects against numerous viruses.³⁰ Here, this experiment investigated the effects of CA in DN and its molecular mechanisms of inflammation.

Materials and methods

Animal model, histological examination, and kidneys function

All C57BL/6 mice (5–6 weeks, 18–20 g) were randomly assigned to five groups: sham group (n = 8), model group (n = 8), Low/Med/High CA group (n = 8), ME group (n = 8). DN mice were fed with a high-fat diet (HFD) for 12 weeks, and then injected with STZ (30 mg/kg of streptozotocin; Sigma-Aldrich, St Louis, MO, USA) i.p. for 7 consecutive days. Blood glucose levels were measured using 16.7 mmol/l after one week of the final injection. Low/Med/High CA group were administration with 7.5, 15 or 30 mg/kg/d (dissolved in water) for 6 weeks as literature.³⁰ ME group were administration with 195 mg/kg metformin for 6 weeks. Mice were killed under anesthesia, and kidneys and serum taken for analysis. This study was approved by the Animal Care and Use Committee of Yijishan Hospital of Wannan Medical College (WNWC-AWE-2023008).

Kidney tissue samples were fixed in 4% paraformaldehyde, paraffn-embedded and then sectioned into 5 μM slices for HE, Masson staining, PAS staining. Kidney tissue samples were observed using fluorescence microscope (Zeiss Axio Observer A1, Germany).

Vitro model and treatment with CA

Rat renal tubular epithelial (NRK-52E, Cell Bank of Chinese Academy of Sciences) cells were seeded in culture dish with RPMI 1640 (Gibco) supplemented with 10% FBS (Gibco) under a humidified 5% (v/v) CO₂ atmosphere at 37°C. NRK-52E stimulated with 20 mmol/L d-glucose for DN model. Next, the transfections were performed using Lipofectamine 2000 (Thermo Fisher Scientific). After 48 h of transfection, NRK-52E stimulated with 20 mmol/L d-glucose and CA (5, 10 and 20 μM) as literature³¹ or ME (250 μM of metformin).

Cell counting kit‑8 (CCK8), ELISA assay and quantitative polymerase chain reaction (qPCR)

CCK8, ELISA assay and qPCR were executed as described previously.³² NRK-52E with different Polysaccharide were cultured in a 96-well plate for corresponding time points and then incubated with CCK-8 reagent. LDH activity, GSH, INF-γ, TNF-α, IL-6, and IL-1β levels using Corresponding ELIAS reagent kitS (Nanjing Jiancheng Biological Engineering Institute, Nanjing, China) following the manufacture’s instructions.

qPCR were performed with the ABI Prism 7500 sequence detection system according to the Prime-ScriptTM RT detection kit. Relative levels of the sample mRNA expression were calculated and expressed as 2-^△△Ct.

Flow cytometry for apoptosis

Cells were resuspended with 100 µL binding buffer, through centrifugation at 1000 rpm for 5 min followed by 3 washes with PBS. Cells were stained with propidium iodide (556547, BD Pharmingen, San Diego, USA) for 15 min at room temperature. 200 µl PBS was added to each tube of samples and then these samples were analyzed by FACS flow cytometer (BD Biosciences, San Jose, USA).

Western blotting analysis

Membranes were incubated with PAQR3 (sc -515 831, 1:500, Santa Cruz Biotechnology, Inc), PI3K (4257, 1:2000, Cell Signaling Technology, Inc.), p-PI3K (17366, 1:1000, Cell Signaling Technology, Inc.), Akt (4691, 1:2000, Cell Signaling Technology, Inc.), p-Akt (4060, 1:1000, Cell Signaling Technology, Inc.), GPX4 (ab41787, 1:2000, abcam), GSDMD (ab209845, 1:2000, abcam), and β-Actin (BS6007MH, 1:5000, Bioworld Technology, Inc.) at 4°C overnight. The membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (sc-2004 or sc-2005, 1:5000, Santa Cruz, USA) for 1 h at 37°C after washing with TBST for 15 min. Protein was measured using an enhanced chemiluminescence system (ECL, Beyotime) and analyzed using an Image Lab 3.0 (Bio-Rad Laboratories, Inc.).

PI staining, Calcein/PI stainin, gImmunofluorescence, and electron microscope

PI staining, Calcein/PI staining, Immunofluorescence and Electron microscope were executed as described previously.³² Cells were incubated with PI staining (ST512, Beyotime Biotechnology) or Calcein/PI staining (C2015S, Beyotime Biotechnology). Cells were incubated with PI3K (ab32089, 1:100, Abcam) and PAQR3 (sc -515 831, 1:500, Santa Cruz Biotechnology, Inc) at 4°C overnight after blocking with PBS supplemented with 5% BSA for 2 h at room temperature. Cell samples were observed using fluorescence microscope (Zeiss Axio Observer A1, Germany). Ultra-thin sections were observed using a Hitachi H7650 transmission electron microscope (Tokyo, Japan).

Microscale thermophoresis (MST), thermal shift assay (TSA), and cellular thermal shift assay (CETSA)

MST, TSA and CETSA were executed as described previously.³³ 0.10 mg/mL WT PAQR3 protein was used with or without 0.30 mmol/L CA in PBS. 20 μL supernatant was used to Western blotting. CETSA, and the thermal stability were performed using GraphPad Prism software (GraphPad Diego, CA, USA).

Statistical analysis

Data were expressed as mean ± standard deviation (SD) used GraphPad Prism 8. Multiple comparisons performed by one-way ANOVA followed by Tukey’s posttest. p values < 0.05 were considered statistically significant.

Results

CA improved DN in mice model

The current study aimed to examine the impact of CA on STZ-induced DN in mice. The chemical structure of CA is illustrated in Figure 1a. Treatment with CA or ME resulted in reduced blood glucose levels, increased body weight, suppressed water intake, and kidney/body weight ratio, decreased serum creatinine levels, lowered urea nitrogen and urinary albumin levels, and alleviated glomerulus injury in the STZ-induced mouse model of DN (as depicted in Figure 1b–1i). Notably, these findings suggest that CA may have a beneficial effect in ameliorating DN in mice models.

Figure 1. CA improved DN in mice model.

Structural formula of CA (a), blood glucose (b), body weight (c), water intake (d), Kidney/body weight (e), serum creatinine (f), urea nitrogen (g); urinary albumin levels (h), glomerulus injury (HE staining, i). Sham, sham control mice; Model, mice model of DN; Med or High, 15 or 30 mg/kg/d of CA; ME, 195 mg/kg metformin. **P < .01 sham group; ^##P < .01 versus Model group. Data were expressed as mean ± SEM.

Then, CA had a negligible impact on cell viability of CT26, HUVEC and RAW 264.7 cells (Figure S1A-S1C). We did not find any obvious systemic toxicity and pathological signs in the major organs (Figure S1D).

CA reduced kidney injury in amodel of DN

Next, this study further investigated the regulatory effects of CA on kidney injury in a model of DN. Treatment with CA or ME increased E-cadherin mRNA expression and reduced periostin mRNA expression in mice models of DN (Figure 2a). Additionally, MASS and PAS staining demonstrated that CA or ME ameliorated glomerular injury in STZ-induced DN (Figure 2b, c). Furthermore, electron microscopy revealed that CA or ME inhibited the masson-positive area and effacement of the podocyte foot processes in STZ-induced mice models of DN (Figure 2d). Importantly, CA or ME also effectively reduced inflammation levels in STZ-induced mice models of DN and in vitro models (Figure 2e, f). Collectively, CA may have the potential to mitigate kidney injury and inflammation in models of DN.

Figure 2. CA reduced acute kidney injury (AKI) and inflammation in mice model or in vitro model.

E-cadherin and periostin mRNA expression (a), glomerulus injury (MASS staining, b/PAS, c), podocyte ultrastructure changes (electron microscope, d), IL-1β/IL-6/INF-γ/TNF-α in mice model (e) or in vitro model (f). Sham, sham control mice; Model, mice model of DN; Med or High, 15 or 30 mg/kg/d of CA; ME, 195 mg/kg of metformin. Control, control; Model, vitro model; Med or High, 10 and 20 μM of CA; ME, 250 μM of metformin. **P < .01 sham group; ^##P < .01 versus Model group. Data were expressed as mean ± SEM.

CA reduced high glucose-induced ferroptosis of NRK-52E through the inhibition of mitochondrial damage

This experiment investigated the biological function of CA in DN models. Treatment with CA was found to have several beneficial effects. Firstly, it promoted cell growth and reduced LDH activity levels and apoptosis rates in high glucose-treated NRK-52E cells (Figure 3a–3d). Additionally, CA also increased GSH activity levels and GPX4 protein expression, and inhibited iron concentration in high glucose-treated NRK-52E cells (Figure 3e–3g). Moreover, live imaging demonstrated that CA reduced ROS levels in the kidneys of STZ-induced DN mice models (Figure 3h). Furthermore, CA upregulated GSH activity levels and GPX4 protein expression in the kidney tissue of STZ-induced DN mice models (Figure 3i j). CA increased calcein levels and JC-1 disaggregation, and reduced mitochondrial damage in high glucose-treated NRK-52E cells (Figure 3k–3m). Overall, these findings suggest that CA may attenuate high glucose-induced ferroptosis of NRK-52E cells through the inhibition of mitochondrial damage by inducing the GSH/GPX4 axis, thereby protecting against kidney injury in models of DN.

Figure 3. CA reduced high glucose-induced ferroptosis of NRK-52E through the induction ferroptosis of GSH/GPX4 axle.

Cell viability (a), LDH activity level (b), cell apoptosis (c), proportions of PI positive cells (d), GSH activity (e), lron concentration (f), GPX4 protein expression (g) in vitro model; ROS levels (live imaging, h), GSH activity (i), GPX4 protein expression in kidney tissue (j); JC-1 disaggregation (k), calcein levels (l), CA increased calcein levels and reduced mitochondrial damage in high glucose-treated NRK-52E cells (Figure 3k–3m). in mice model. Sham, sham control mice; Model, mice model of DN; Low, Med or High, 7.5 or 15 or 30 mg/kg/d of CA. Control, control; Model, vitro model; Low, Med or High, 5 or 10 and 20 μM of CA. **P < .01 sham group; ^##P < .01 versus Model group. Data were expressed as mean ± SEM.

Ferroptosis activator reduced the effects of CA on DN

However, it remained uncertain whether ferroptosis played a crucial role in the mechanisms by which CA exerted its effects on DN. To address this, a ferroptosis activator (50 mg/kg of Sorafenib Tosylate) was administered to DN mice models in conjunction with CA treatment. The results showed that the ferroptosis activator reversed the effects of CA on body weight, blood glucose levels, and AKI (Figure 4a–4j). Additionally, treatment with a ferroptosis activator (5 μM of CIL56 or 50 mg/kg of Sorafenib Tosylate) suppressed GPX4 protein expression in mice models or in vitro models treated with CA (Figure 4k,l). Similarly, in an in vitro model, treatment with a ferroptosis activator (5 μM of CIL56) hindered the effects of CA on high glucose-induced ferroptosis of NRK-52E cells (Figure S1). These findings suggest that CA may mitigate high glucose-induced ferroptosis of NRK-52E cells in DN models.

Figure 4. Ferroptosis activator reduced the effects of CA on DN in mice model or in vitro model.

Blood glucose (a), body weight (b), serum creatinine (c), urea nitrogen (d), urinary albumin levels (e), water intake (f), Kidney/body weight (g), E-cadherin and periostin mRNA expression (h and i), glomerulus injury (HE staining, j) in mice model; GPX4 protein expression in vitro model or mice model (k or l). Sham, sham control mice; Model, mice model of DN; Med or High, 15 or 30 mg/kg/d of CA; Ferroptosis, 50 mg/kg of Sorafenib Tosylate. Control, control; Model, vitro model; Med or High, 10 and 20 μM of CA; Ferroptosis, 5 μM of CIL56. **P < .01 sham group; ^##P < .01 versus Model group; ^$P < .01. Data were expressed as mean ± SEM.

CA suppressed PAQR3 expression in model of DN to promote PI3K/AKT axle

Next, we investigated the mechanism by which CA reduces ferroptosis in DN. CA was found to suppress the expression of PAQR3 mRNA/protein and induce the expression of p-PI3K/p-AKT proteins in the kidney tissue of STZ-induced DN mice models or in an in vitro model of DN (Figure 5a,c). Meanwhile, immunohistochemistry and immunofluorescence showed that CA reduced PAQR3 expression level in kidney tissue of STZ-induced mice model of DN or in NRK-52E by high glucose (Figure 5b, e). Bioluminescent imaging showed the CA suppressed PAQR3 expression level in kidney tissue of STZ-induced mice model of DN (Figure 5d). Notably, these findings suggest that CA suppresses PAQR3 expression in DN models, thereby promoting the PI3K/AKT pathway.

Figure 5. CA suppressed PAQR3 expression in model of DN to promote PI3K/AKT axle.

PAQR3 mRNA expression and PAQR3/PI3K/AKT protein expression in mice model (a) or in vitro model (c), PAQR3 expression in kidney tissue (Immunohistochemistry, b/Bioluminescent imaging, d), PAQR3 expression in vitro model (immunofluorescence, e). Sham, sham control mice; Model, mice model of DN; Low, Med or High, 7.5 or 15 or 30 mg/kg/d of CA. Control, control; Model, vitro model; Low, Med or High, 5 or 10 and 20 μM of CA. **P < .01 sham group; ^##P < .01 versus Model group. Data were expressed as mean ± SEM.

PAQR3 activator reduced the effects of CA on DN in vitro model

To investigate the functional involvement of PAQR3 in the effects of CA in DN models, we conducted experiments in which we upregulated PAQR3 expression levels through PAQR3 plasmid transfection and treated NRK-52E cells induced with glucose with CA. Our results showed that PAQR3 upregulation inhibited the activation of the PI3K/AKT/GPX4 axis induced by CA treatment, as evidenced by the suppression of protein expression levels (as shown in Figure 6a,b). Additionally, PAQR3 upregulation reduced GSH levels and cell growth, while increasing iron concentration and LDH activity in NRK-52E cells induced with glucose by CA treatment (Figure 6c–6f). Moreover, PAQR3 upregulation increased apoptosis and inflammation levels, and promoted the proportion of PI positive cells in NRK-52E cells induced with glucose by CA treatment (Figure 6g–6l). Collectively, our findings suggest that PAQR3 plays an activator role in the effects of CA on DN.

Figure 6. PAQR3 activator reduced the effects of CA on DN in vitro model.

PAQR3/PI3K/AKT/GPX4 protein expression in vitro model (a and b), GSH activity (c), lron concentration (d), Cell viability (e), LDH activity level (f), cell apoptosis (g), proportions of PI positive cells (h), IL-1β/IL-6/INF-γ/TNF-α (i, j, k, l) in vitro model. Control, control; Model, vitro model; Med or High, 10 and 20 μM of CA; PAQR3, PAQR3 up-regulation. **P < .01 sham group; ^##P < .01 versus Model group; ^$P < .01. Data were expressed as mean ± SEM.

CA directly targeted the PAQR3 protein to advance ubiquitination of PAQR3 protein

This experiment aimed to elucidate the mechanism of CA targeting the PAQR3 protein. As shown in Figure 7a, analysis of the drug-protein linkage revealed that CA binds to PAQR3 protein. We mutated the PAQR3 protein at positions 13-GLU, 14-GLU, 29-ARG, 30-LEU, 35-GLN, 211-SER, 244-TYR, and 254-TYR (Figure 7b). Upon binding with CA, the thermophoretic motion of PAQR3 was affected, and the melting temperature of PDPK1 was elevated from ~ 55 to ~ 60°C (Figure 7c,d). This enhancement in thermal stability was observed in the wild-type PAQR3, while CA did not affect the thermal stability of the mutant PAQR3 at positions 13-GLU, 14-GLU, 29-ARG, 30-LEU, 35-GLN, 211-SER, 244-TYR, and 254-TYR (Figure 7e, f). In in vitro models of ALI, CA was found to accelerate PDPK1 ubiquitination (Figure 7g). These results suggest that CA reduces PAQR3 ubiquitination to induce PAQR3 activity, which may contribute to the effects of CA in the DN model.

Figure 7. CA directly targeted the PAQR3 protein to advance ubiquitination of PAQR3 protein.

3D image revealed that CA bond to the binding pocket and formed with PAQR3 (a); PAQR3 protein expression (b); Microscale thermophoresis (MST) of PAQR3 incubated with CA (c); TSA results in the presence or absence of CA (d); the thermal stability of WT PDPK1 and Mut PAQR3 plasmid after treatment with CA using CETSA (e and f); PAQR3 Ubiquitination (g).

PI3K inhibitor the effects of CA on DN in a model

Based on the above results, it is likely that the effects of CA on DN in a DN model are mediated through the PI3K signaling pathway. Treatment with PI3K inhibitor LY294002 (at 25 mg/kg or 50 μM) suppressed the expression levels of p-PI3K/p-AKT/GPX4 proteins in the kidney tissue of STZ-induced DN mice models that were treated with CA or in vitro models treated with CA (Figure 8a,b and S2A). Furthermore, treatment with PI3K inhibitor reversed the detrimental effects of CA on AKI and inflammation in STZ-induced DN mice models (Figure 8c–8n). Similarly, treatment with PI3K inhibitor (at 50 μM of LY294002) impeded the positive effects of CA on GSH, iron concentration, cell growth, and increased LDH activity levels in NRK-52E cells induced by glucose (as illustrated in Figure S2B-S2G). Collectively, these findings suggest that CA activates the PI3K/AKT/GPX4 axis in both mice models and in vitro models of DN.

Figure 8. PI3K inhibitor the effects of CA on DN in mice model.

PI3K/AKT/GPX4 protein expression (a and b), glomerulus injury (HE staining, c), Blood glucose (d), Kidney/body weight (e), water intake (f), serum creatinine (g), urea nitrogen (h), urinary albumin levels (i), GSH/TNF-α/IL-1β/IL-6/INF-γ(j, k, l, m, n). Sham, sham control mice; Model, mice model of DN; Med or High, 15 or 30 mg/kg/d of CA; Inhibitor, PI3K inhibitor. **p < .01 sham group; ##P < .01 versus Model group; $P < .01. Data were expressed as mean ± SEM.

CA reduced the interaction between PAQR3 and P110α pathway to induce PI3K/AKT signaling pathway

we conducted experiments using a PI3K inhibitor (LY294002) in d-glucose-induced NRK-52E cells to elucidate the role of PAQR3 in the effects of CA on PI3K/AKT signaling pathway. As shown in Figure 9a, LY294002 reversed the effects of CA on p-PI3K/p-AKT proteins, indicating that CA functionally interacted with PI3K to induce the insulin pathway. Additionally, CA treatment reduced the distribution of PAQR3 and p110α in d-glucose-induced NRK-52E cells (Figure 9b). Moreover, co-immunoprecipitation (co-IP) verified that CA stimulation decreased the interaction between P110α protein and PAQR3 protein in NRK-52E cells, while treatment with CA diminished the interaction between P110α protein and PAQR3 protein (Figure 9c). Furthermore, immunofluorescence also showed that CA treatment reduced the co-localization of PAQR3 protein and p110αprotein in NRK-52E cells (Figure 9d). Meanwhile, co-IP results indicated that CA also increased the interaction between p110α protein and p85α protein in d-glucose-induced NRK-52E cells, implying that CA treatment promoted the formation of a PI3K dimer (Figure 9e). Furthermore, PAQR3 up-regulation reversed the effects of CA on the inhibition of PAQR3/P110α distribution in d-glucose-induced NRK-52E cells (Figure 9f). Additionally, PAQR3 up-regulation abolished the formation of the PI3K dimer (P110α/P85α) induced by CA treatment (Figure 9g). As expected, PAQR3 up-regulation also reversed the CA-induced activation of the PI3K/AKT axis (Figure 9h). Therefore, CA promotes the PI3K/AKT signaling pathway by inhibiting the interaction between PAQR3 protein and the P110α protein in DN.

Figure 9. CA reduced the interaction between PAQR3 and P110α pathway to induce PI3K/AKT signaling pathway.

LY294002 reversed the effects of CA on p-PI3K and p-AKT protein expression (a), the expression of PAQR3 and p110α protein expressions (b), the combination of PAQR3 and p110α (c), PAQR3 and P110α expression (Immunofluorescence staining, d), the combination of p110α and p85α (e), the expression of PAQR3 and p110α protein (f), the interaction between P110α and P85α protein expressions (g), PAQR3 up-regulation reversed the indcution of p-PI3K and p-AKT (h). **p < .01. Data were expressed as mean ± SEM.

Discussion

Diabetes can cause acute life-threatening complications such as ketoacidosis, non-ketotic hyperosmolar coma, and chronic complications such as coronary heart disease, retinopathy, nephropathy, neuropathy, dermatopathy, infection, and osteoporosis, seriously damaging the health of patients. At the same time, the incidence rate of diabetes is high and the number of patients is large, so the prevention and treatment of diabetes is very important.³⁴ On the one hand, people who have suffered from diabetes need to do a good job in chronic disease management, control blood sugar and reduce complications; On the other hand, timely screening should be carried out to find high-risk groups of diabetes. The risk of high-risk groups suffering from diabetes is higher than that of healthy people. Early guidance and intervention and good health management are beneficial to the prevention of diabetes. Currently, there are 24 million patients in China suffering from chronic kidney disease caused by diabetes, with diabetes nephropathy being the primary cause of progression to end-stage renal disease.³⁴ From 1990 to 2016, the mortality rate of all age diabetes nephropathy increased by 33%, which seriously affected the quality of life of patients.³⁵ The pathogenesis of diabetes nephropathy is not clear yet.⁴ How to diagnose diabetes nephropathy early is the key link to prevent diabetes nephropathy from further aggravating.³⁶ Interestingly, our experiment revealed that CA or ME administration led to reductions in blood glucose levels, and prevented DN in a streptozotocin-induced mouse model of DN. Schlernitzauer et al. indicated that CA is one having potential anti-diabetic properties.³⁷ Therefore, these findings demonstrate for the first time that CA can ameliorate DN in a mouse model, suggesting a protective role for CA in DN.

In clinical research, diabetes nephropathy is the main factor leading to death and renal failure of diabetes patients; In a broad sense, diabetes nephropathy covers a variety of diseases, including vascular diseases and infectious diseases, among which vascular diseases include large vessels and microvascular diseases⁷. Among patients with microvascular diseases, glomerulosclerosis is the main representative, which can be divided into diffuse, nodular, and exudative diseases according to the specific pathological conditions of patients. Research has confirmed that patients with diabetes nephropathy are prone to renal failure in the late stage, even serious cardiovascular adverse events.⁷ Therefore, in clinical treatment, emphasis is placed on the early diagnosis results of patients with diabetes nephropathy, which has a good guiding role in patient condition control and treatment. With the improvement of living standards, the prevalence of diabetes nephropathy has also increased.^7.8 In the present study, CA demonstrated a reduction in kidney injury and inflammation in both mouse and in vitro models. Lee et al. suggest that CA has an anti-inflammatory effect in model of allergy-related inflammatory disorders.³⁸ Ding et al. showed that CA reduced renal tubular injury in model of chronic kidney disease.³⁹ Hence, it can be inferred that CA improves kidney injury by suppressing inflammation in the model of diabetic nephropathy, although the specific mechanism remains unclear. CA might been a potential drug for DN, however, the efficacy of CA is not as good as that of ME. In the next experiment, we will improve its activity and enhance its targeting effect.

Iron death, as a new type of programmed cell death, involves complex mechanisms. It has been proven that iron metabolism disorders, abnormalities in the SystemXc -/GSH/GPX4 pathway, oxidative stress caused by Nrf2 pathway inhibition, membrane lipid peroxidation, targeted induction of tumor suppressor p53, and abnormalities in the NADPH/FSP1/CoQ10 pathway are closely related to complications such as DM and DCM, DN, NAFLD, DPN, DR, etc, The above diseases can be treated from the perspective of intervention in iron death.⁴⁰ Compared to Western medicine, traditional Chinese medicine has the advantage of multiple pathways and targets, which can greatly improve the therapeutic effect, reduce the harm of toxic side effects to the body, and bring good news to the vast number of patients.⁴¹ Ferroptosis is directly characterized by mitochondrial shrinkage and increased mitochondrial membrane density.^40.41 This experiment demonstrated that CA reduced high glucose-induced ferroptosis in NRK-52E cells. Mechanistically, the ferroptosis activator attenuated the effects of CA on DN. Wang et al. suggested that CA inhibited ferroptosis in mice of LPS-induced endometritis.⁴² These results demonstrate that CA may alleviate ferroptosis in DN models by inducing the GSH/GPX4 axis.

Mitochondria, as an important energy supplying organelle, perform important metabolic functions in cells and provide driving force for them. The types of cell death also include necrotic apoptosis, pyroptosis, and ferroptosis As for the mechanism of Ferroptosis, it is generally believed that it is related to the iron overload in the body and the disorder of oxidation/antioxidant system.⁴³ The biochemical manifestations are mainly antioxidant depletion, ROS accumulation, and the production of toxic lipid peroxidation product MDA.⁴³ Studies have shown that GPX4 is the key antioxidant enzyme used for lipid peroxide reduction in cells, and GSH is its substrate.^44.44.45 Additionally, we showed that CA suppressed PAQR3 expression in model of DN to promote PI3K/AKT/Nrf2/GPX4 axle. Importantly, PI3K inhibitor attenuated the effects of CA on DN. The present study is the first to report such a finding, CA directly targeted the PAQR3 protein to advance PAQR3 protein ubiquitination. Importantly, CA decreased the binding between PAQR3 and P110α protein, leading to an increase in P110α protein level or activity, which in turn enhanced the interaction between PAQR3 and P85 protein, resulting in the formation of PI3K dimer.

PI3K/AKT can regulate oxidative stress, cell apoptosis, inflammatory factors in the body, and regulate the development of diabetes nephropathy.⁴⁶ PI3K/AKT is the target for clinical treatment of diabetes nephropathy, which can inhibit the inflammatory reaction of renal tissue, alleviate proteinuria symptoms, and improve kidney damage.⁴⁷ Phosphorylated Akt can promote Keap1/Nrf2 nuclear transfer and antioxidant response element binding, promote downstream antioxidant system expression regulation of GPX4, thereby clearing oxygen-free radicals and inhibiting intestinal mucosal cell ferroptosis; At the same time, the clearance of oxygen-free radicals helps to restore the normal energy metabolism function of mitochondria, promote ATP production and increase ATPase activity.⁴⁸ Consequently, this facilitated the rapid formation of p-PI3K to induce PI3K/AKT signaling pathway. Ding et al. that CA alleviated acute lung injury through the promotion of GSH activity.⁴⁹ In this study, CA promoted PI3K/AKT/GPX4 axle through the inhibition of PAQR3 in model of DN. In this study, we have discovered that PAQR3 plays a crucial role as a target for CA. The next experiment uses mass spectrometry CETSA to achieve high-throughput detection of more targets. Meanwhile, the half-life of PAQR3 with or without CA is unclear. Our next experiment will mainly investigate the impact of CA on the half-life of PAQR3. Indeed, we did not use the PAQR3 CKO mice in this study to investigate the antidiabetic nephrotic activity of CA on PAQR3, which is a deficiency of this study. Our next step will be to supplement relevant research and improve the relevant conclusions.

Conclusions

Our study revealed a new target of CA in inhibiting high glucose-induced ferroptosis in renal tubular epithelial cells. CA advanced PAQR3 ubiquitination to ameliorate mitochondrial damage-dependent Ferroptosis in DN through the activation of PI3K/AKT signaling pathways by the relieving of the interaction between PAQR3 protein and P110α protein (Figure 10). Importantly, CA is a possible therapeutic option to relieve glucose-induced ferroptosis in diabetes for DN or other diabetes.

Figure 10. Chicoric acid advanced PAQR3 ubiquitination to ameliorate Ferroptosis in diabetes nephropathy through the relieving of the interaction between PAQR3 and P110α pathway.

Authors’ contribution

Zhichen Pu and Yong Liu conceived the study, designed the study and prepared the manuscript. Yong Liu and Jiajun Zhou conducted the experiments and data analysis, involved in preparation of the figures and manuscript. 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.

Abbreviations

DN	=	Diabetic nephropathy
CA	=	Cichoric acid
P AQR3	=	Progestin and adipo Q acceptor 3,
GPX4	=	glutathione peroxidase 4
PI3K	=	Phosphatidylinositol-4,5-bisphosphate 3-kinase
Akt	=	protein kinase B
Nrf2	=	Nuclear factor E2-related factor 2
HFD	=	high-fat diet
STZ	=	streptozotocin
Qpcr	=	Quantitative polymerase chain reaction
CCK8	=	Cell counting kit‑8
MST	=	Microscale thermophoresis
TSA	=	Thermal shift assay
CETSA	=	cellular thermal shift assay
co-IP	=	co-immunoprecipitation

Acknowledgments

This work was supported by National Natural Science Foundation of China (811731333); Key Natural Science Projects of the Department of Education of Anhui province (2022AH051239, 2023AH051767); Talent Introduction Program of Yijishan Hospital of Wannan Medical College (YR202005); Science and Technology Innovation Team of Yijishan Hospital of Wannan Medical College (YPF2019016, YR20230105), and Key projects of Wannan Medical (WK2021ZF11, KY23740650). Thanks to the central laboratory for its support to this research.

Disclosure statement

We wish to confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome.

Additional information

Funding

The work was supported by the National Natural Science Foundation of China [811731333].