Curcumin/piperine modulates the butyrate levels and alleviates diabetic kidney disease via Nrf2/HO-1 signaling pathway

Abstract

Objectives

This study investigates whether curcumin/piperine (C/P) modulates butyrate levels and mitigates diabetic kidney disease (DKD) via the nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) signaling pathway.

Methods

Eight-week-old mice were treated with C/P for 12 weeks, fecal samples were analyzed for short-chain fatty acids, and inflammatory markers in blood and kidneys were measured. Pathological staining and microscopy assessed the kidney and intestinal tissue morphology. Immunohistochemical detection of the expression of inflammatory markers in the kidneys and intestines. In vitro, the human proximal renal tubular cell line (HK-2) cells were exposed to high glucose (HG) and pretreated with C/P and sodium butyrate (NaB) to observe changes in inflammatory and oxidative stress markers. ML385 helped assess the role of the Nrf2/HO-1 pathway in the effects of C/P and NaB on inflammation and oxidative stress in DKD.

Results

Curcumin/piperine significantly improved renal injury and intestinal structural integrity, upregulated the expression of macrophage antigen CD68 and other inflammatory markers in the kidneys, and increased NaB levels in feces (p < .05). High glucose conditions enhanced inflammation and oxidative stress in HK-2 cells, whereas C/P and NaB pretreatment significantly inhibited these effects, reducing interleukin-6, IL-1β and other markers (p < .05), and increasing Nrf2 and HO-1 expression. Nrf2 inhibitor ML385 reduces the expression of Nrf2 and HO-1, and weakens the beneficial effects of C/P and NaB on HK-2 cells.

Conclusions

Curcumin/piperine enhances renal and intestinal integrity and modulates NaB levels to alleviate DKD through the Nrf2/HO-1 signaling pathway.

1. Introduction

Diabetic kidney disease (DKD), a prevalent and severe microvascular complication in patients with diabetes, exhibits a high incidence rate and has emerged as the leading cause of end-stage renal disease globally [1]. Recent data from the International Diabetes Federation indicate that over 500 million people worldwide are currently affected by diabetes, a number that is steadily rising, thereby imposing a significant societal burden [2,3]. Studies highlight that kidney disease, driven by persistent hyperglycemia, is a major contributor to the increased mortality rates among diabetic patients [4]. Consequently, there is an urgent need to identify new therapeutic targets to alleviate renal dysfunction and retard the progression of DKD.

The pathogenesis of DKD is multifaceted, involving hemodynamic alterations, inflammatory and oxidative stress responses, epithelial–mesenchymal transition, and epigenetic modifications. In particular, inflammation and oxidative stress are deemed pivotal in the onset and progression of DKD [5,6]. Nuclear factor erythroid 2-related factor 2 (Nrf2) is an important factor in transcriptional regulation of over 250 genes, encompassing various functions such as antioxidant responses, mitochondrial bioenergetics, inflammation, and protein homeostasis [7]. Nrf2 is instrumental in mitigating inflammation and oxidative stress, thereby safeguarding cells from DKD-related damage [8,9]. When the body experiences oxidative stress, Nrf2 separates from Kelch like ECH related protein 1 (Keap1) and transfers it from the cytoplasm to the nucleus [10]. This translocation upregulates downstream signaling molecules like heme oxygenase-1 (HO-1) and NAD(P)H: quinone oxidoreductase 1 (NQO1), boosting the expression of antioxidant enzymes and enhancing antioxidant capacity [11]. Extensive research has demonstrated the protective effects of the Nrf2/HO-1 signaling pathway on renal health [12–14].

Curcumin (CUR), a lipophilic polyphenol derived from the traditional Chinese medicine turmeric, can regulate multiple signaling pathways, offering diverse therapeutic possibilities for metabolic diseases [15]. However, its low solubility and rapid metabolism limit gastrointestinal absorption, resulting in low bioavailability [16]. Combining drugs is a more effective strategy to enhance CUR absorption in the intestine. Research indicates that piperine (PIP) can increase CUR’s solubility, extend its plasma residence time, and improve pharmacokinetic properties and cellular absorption [17]. The combined effect of CUR and PIP in the human body may be more efficient than CUR alone.

Patients with DKD often experience dysbiosis of the intestinal flora. Short-chain fatty acids (SCFAs), metabolites of gastrointestinal carbohydrates, positively regulate kidney damage and inflammation [18]. Previous studies have shown that CUR can enhance the expression of intestinal tight junction proteins, alleviate intestinal mucosal barrier damage caused by DKD, regulate the metabolism of intestinal microbiota, promote the proliferation of SCFA-producing bacteria, and increase SCFA levels [19,20]. This article investigates whether curcumin/piperine (C/P) can ameliorate inflammation and oxidative stress in DKD and delay its progression by modulating sodium butyrate (NaB) levels and the Nrf2/HO-1 signaling pathway.

2. Materials and methods

2.1. Reagents

Curcumin, NaB, and glucose solutions were purchased from Sigma-Aldrich (Shanghai, China) and PIP was purchased from Shanghai Macklin Biochemical Technology Co., Ltd. (Shanghai, China). Staining kits were purchased from Solarbio (Beijing, China). Cell counting kit-8 (CCK-8) was obtained from APExBIO Technology LLC (Houston, TX), and antibodies such as macrophage antigen CD68 (CD68), neutrophil gelatinase-associated lipocalin (NGAL2), interleukin-1β (IL-1β), Occludin, ZO-1, Nrf2, HO-1, beta actin, horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG, and goat anti-rabbit IgG were purchased from Affinity Biosciences Ltd. (Liyang City, China).

2.2. Animals and models

The 8-week-old control mice (db/m, n = 6) and spontaneous diabetes mice (db/db, n = 18) were sourced from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd. (Beijing, China). All mice were placed in a temperature-controlled room with a 12 h light/dark cycle and were free to access water and standard diet. The db/db mice, except for the db/m group, were randomly divided into three groups: db/db group (db/db, n = 6), CUR group (db/db + CUR, n = 6), and C/P group (db/db + C/P, n = 6). The CUR group received an oral administration of CUR (100 mg/kg·d), while the C/P group received CUR and PIP at a 100:1 ratio daily for 12 weeks. Blood, morning urine, and feces were collected from the mice for subsequent analyses. All procedures were approved by the Animal Health and Use Committee of Shandong University of Traditional Chinese Medicine Affiliated Hospital (AWE-2019-004).

2.3. Biochemical measurements

Centrifuge the collected blood at 3,000 rpm for 10 min to obtain a serum sample. Measure interleukin-6 (IL-6) and tumor necrosis factor (TNF) according to the instructions of the reagent kit (Soleibao, Beijing, China). Use a fully automated biochemical analyzer to detect blood creatinine, blood urea nitrogen, urine creatinine, and urine microalbumin content, and calculate urine microalbumin/creatinine to obtain urine ACR.

2.4. Histopathological staining

After euthanizing the mice under anesthesia, kidney and intestinal tissues were harvested, fixed in 4% paraformaldehyde for 24 h, tissues was embed in paraffin and sliced. Paraffin sections were placed in xylene and underwent conventional ethanol gradient dewaxing. The kidney tissue was stained with periodic acid-Schiff (PAS) following the reagent supplier’s instructions (Soleibao, Beijing, China). Hematoxylin and eosin (H&E) staining was performed on the intestinal tissue. Dewaxed intestinal tissue was stained with hematoxylin, rinsed with tap water after 2 min, followed by a return to blue, and then counterstained with eosin for 1 min. The sections were then dehydrated using ethanol in gradient concentrations. Upon completion of all slicing operations, the sections were sealed with neutral gum.

2.5. Determination of SCFAs in feces

First, 100 mg fecal sample was mixed with 4 mL of ultrapure water and 600 μL of a 50% sulfuric acid solution. Subsequently, 50 μL of an internal standard and 4 mL of ether were added for extraction by shaking. The mixture was centrifuged at 4,000 r/min for 3 min, and the upper layer sample was collected. The extracted samples were quantitatively analyzed for the content of acetic acid, propionic acid, and butyric acid using a gas chromatography-mass spectrometer (GCMS-QP2010 Ultra, Shimadzu Corporation, Kyoto, Japan) equipped with a Wax capillary column (30 m × 0.25 mm × 0.25 μm).

2.6. Transmission electron microscopy

The intestinal tissue was initially fixed with 2.5% glutaraldehyde and subsequently with 2% osmium tetroxide. The tissue was then dehydrated using a graded ethanol series, embedded, and sectioned into ultra-thin slices (70–80 nm). The slices were double-stained with 3% uranyl acetate and lead citrate and observed under transmission electron microscopy to evaluate changes in intestinal structure.

2.7. Immunohistochemistry (IHC)

Paraffin-embedded kidney sections were deparaffinized and dehydrated using xylene and ethanol at varying concentration gradients. Antigen retrieval was conducted in citrate buffer (pH 6.0, Zhongshan Jinqiao, Beijing, China) at 98 °C for 5 min, followed by maintaining the sections at medium heat for 20 min. After natural cooling, the sections were placed in a 3% hydrogen peroxide methanol solution at room temperature in the dark for 10 min. These slices were compared with primary antibodies targeting NGAL2, CD68, IL-1β, ZO-1, Occludin, Nrf2, and HO-1 (1:100, Affinity Biosciences, Liyang City, China), and incubated overnight at 4 °C. The next day, an enhancer was applied and incubated at 37 °C for 20 min, followed by incubation with the HRP-labeled goat anti-rabbit secondary antibody (ZSGB-BIO, Beijing, China) at 37 °C for 20 min. The sections were then colored with DAB solution (ZSGB-BIO, Beijing, China) and washed with phosphate-buffered saline (PBS) three times, each for 2 min. Sections were counterstained with hematoxylin, dehydrated with ethanol, then observed under an optical microscope, and photographed for analysis.

2.8. Cell culture and treatment

The human proximal renal tubular cell line (HK-2) was acquired from the Shanghai Fuheng Cell Center (Shanghai, China) and cultured in DMEM/F12 (Thermo Fisher Scientific, Waltham, MA) containing 10% fetal bovine serum (Thermo Fisher Scientific, Waltham, MA) and 1% penicillin/streptomycin (Soleibao, Beijing, China). The cells were maintained at 37 °C in a humidified incubator with 5% CO₂. For cell treatment experiments, HK-2 cells were stimulated with high glucose (HG) and categorized into four groups: a control group (NC, 5.5 mM), a HG group (30 mM), a HG with C/P group, and a HG with NaB group. Cells were initially pretreated with specified concentrations of C/P and NaB for 4 h, followed by a 48-h exposure to HG. In subsequent experiments, ML385 at a dose of 5 μM was used to inhibit cells for 24 h prior to drug pretreatment.

2.9. Cell viability assay

Initially, to assess the effects of various drugs on HK-2 cell activity and determine the appropriate concentrations of CUR and NaB, the CCK-8 kit was utilized. Normal cells were seeded into a 96-well plate, with approximately 5,000 cells per well, with 100 µL of culture medium added, and incubated for 24 h. On the second day, cells were treated with 10 µL of CUR at different concentrations (1, 5, 10, and 15 µM) and NaB (0.1, 0.5, 1, and 2 mM) for 2 h, followed by induction with HG for 48 h, while LG served as the control group. Subsequently, 10 μL of CCK-8 reagent was added to each well, and after 2 h in a humidified incubator, the OD value was detected in a 450 nm spectrophotometric measurement. These data were analyzed to measure cell viability. All experiments were performed in triplicate, with wells containing culture medium but no cells serving as blank controls.

2.10. Quantitative real-time PCR analysis

Total RNA was isolated from HK-2 cells using an RNA extraction kit (Nanjing Vazyme Biotech Co., Ltd., Nanjing, China). The isolated RNA was reverse transcribed using Evo M-MLV reverse transcription reagent mixture (Accurate Biotechnology, Changsha City, China). The synthesized cDNA served as a template for quantitative PCR analysis. The reaction mixture contained 10 µL 2xSYBR Green Pro Taq HS Premium (Accurate Biotechnology, Changsha City, China), 2 µL of cDNA, 0.4 µL of upstream PCR primers, and 0.4 of µL downstream PCR primers, add nuclease-free water to a final volume of 20 µL. Each reaction was conducted in triplicate, and GAPDH was used as an endogenous control for normalizing the data. All primer sequences (Boshang Biological Technology Co., Ltd., Boshang, China) are listed in Table 1.

Table 1.

Primer sequences for quantitative real-time PCR.

Gene	Forward sequence	Reverse sequence
GAPDH	5′-GCACCGTCAAGGCTGAGAAC-3′	5′-TGGTGAAGACGCCAGTGGA-3′
IL-6	5′-CAATGAGGAGACTTGCCTGGT-3′	5′-GCAGGAACTGGATCAGGACT-3′
TNF-α	5′-GAGGCCAAGCCCTGGTATG-3′	5′-CGGGCCGATTGATCTCAGC-3′
Nrf2	5′-ATCCATTCCTGAGTTACAGTGTCTT-3′	5′-TGTCAGTTTGGCTTCTGGACT-3′
HO-1	5′-TGCTGACCCATGACACCAAG-3′	5′-GGGCAGAATCTTGCACTTTGTT-3′

2.11. ROS level assessment

Intracellular reactive oxygen species (ROS) in different treatment groups were measured using 2′,7′-dichlorofluorescein diacetate (DCFH-DA). HK-2 cells were pretreated with C/P (10/2 µM) for 2 h, followed by HG induction for 48 h. Post-HG stimulation, serum-free medium containing 10 μL MDCFH-DA was added to each well and incubated at 37 °C for 20 min. After washing three times with PBS, the fluorescence microscope (Nikon Corporation, Tokyo, Japan) was set to blue light stimulation and directly observed. Images were captured from three randomly selected fields at ×200 magnification. Additionally, cells was treated with ML385 (5 μM) for 24 h before dosing, with subsequent drug treatment procedures remaining unchanged, for comparison with the absence of ML385.

2.12. Detection of MDA and GSH

Malondialdehyde (MDA) and glutathione (GSH) levels were determined with commercially available kits (Solarbio, Beijing, China).

2.13. Western blot analysis

HK-2 cells were pretreated with C/P (10 µM/2 µM) and NaB (0.5 mM) for 4 h, followed by HG stimulation for 48 h. According to the quantity, the cells were lysed using an appropriate volume of RIPA lysis buffer (Soleibao, Beijing, China), and protein concentration was assessed with a BCA protein concentration assay kit (Soleibao, Beijing, China). The original protein concentration was according to the standard curve to establish the amount of protein to be loaded. The proteins are dispersed in sodium dodecyl sulfate polyacrylamide gel by electrophoresis, and the proteins on the gel are transferred to the polyvinylidene fluoride (PVDF) membrane in the rotating film clip. The membranes were blocked with 5% skimmed milk powder at room temperature for two hours and then washed three times with TBST for 30 min. Subsequently, the membranes were soaked in anti-Nrf2, anti-HO-1, and anti-beta actin antibodies (Hunan Aikerui Biological Engineering Co., Ltd., Changsha, China) and incubated overnight at 4 °C. The following day, after washing the membrane with TBST for 30 min, incubate with goat anti-rabbit IgG (H + L) secondary antibody HRP conjugate for 90 min. After another TBST wash, protein visualization was performed using a hypersensitive ECL kit (Affinity Biosciences, Liyang City, China).

2.14. Statistical analysis

GraphPad prism 9.5 (La Jolla, CA) was used for statistical analysis of the data. All data were obtained from at least three independent experiments and expressed as the mean ± standard error. Inter group comparisons were analyzed using one-way analysis of variance (ANOVA), followed by post hoc Tukey’s correction test for multiple comparisons. Differences with p < .05 were considered statistically significant.

3. Results

3.1. Curcumin/piperine mitigates renal injury in DKD mice

To investigate the effects of C/P on renal function in db/db mice, relevant biochemical and renal function parameters were measured. Compared to db/m mice, db/db mice exhibited significantly reduced urinary ACR, creatinine, and urea nitrogen levels. After 12 weeks of CUR treatment, these parameters improved significantly, with the combination of PIP showing even more pronounced effects (Figure 1(A–C)). Additionally, CUR pretreatment lowered IL-6 and TNF-α levels, thereby reducing diabetic nephropathy-induced inflammation. However, the combination with PIP did not further alter these inflammatory markers (Figure 1(D,F)). These results indicate that CUR improves renal function and reduces inflammation in db/db mice, and its therapeutic effects are enhanced when combined with PIP.

Figure 1.

Effects of curcumin and curcumin/piperine on biochemistry and renal tissue structure in db/db mice. (A–D, F) Renal function parameters and inflammatory expression in db/db mice treated with curcumin and curcumin/piperine for 12 weeks. (E) Immunohistochemical analysis of CD68, NGAL2, and IL-1β in kidney tissue using PAS stained kidney sections (magnification of ×400). In db/db group, glomerular mesangial hyperplasia and local tissue loss of renal tubules were observed significantly (arrow indication). (G–I) Quantitative analysis of immunohistochemistry. The results are expressed as the mean ± standard deviation of the three experimental groups, *p < .05, **p < .01, ***p < .001, and p < .0001.

To further evaluate the protective effect of CUR, kidney tissues were analyzed using staining and IHC. PAS staining revealed significant mesangial proliferation (black arrow), tubulointerstitial damage, and stiffness or local detachment (red arrow) in the glomeruli of db/db mice. Compared to untreated db/db mice, C/P treatment improved the structural integrity of the kidney tissue (Figure 1(E)). Immunohistochemical staining showed that the expression levels of CD68, NGAL2, and IL-1β in the db/db group were significantly higher than those in the db/m group, and CUR treatment reduced these levels (Figure 1(E)). These results demonstrate that C/P treatment mitigates renal damage in DKD mice.

3.2. Curcumin/piperine enhances the production of short-chain fatty acids in feces and ameliorates intestinal tissue damage and epithelial barrier dysfunction in mice

To evaluate the effect of C/P on SCFAs, the gut microbiota composition in the feces of each group of mice was analyzed, along with the quantitative analysis of SCFA content. Figure 2 shows that the levels of acetic acid, propionic acid, and butyric acid were significantly reduced in the feces of db/db mice. Curcumin treatment increased butyric acid levels; however, the addition of PIP did not show a more significant effect on promoting butyric acid. Nonetheless, compared to the db/db group, butyric acid levels were significantly elevated (Figure 2(A–C)). These results demonstrate that C/P can promote the production of SCFAs.

Figure 2.

The effects of curcumin and curcumin/piperine on intestinal short chain fatty acids and tissue structure in db/db mice. (A–C) The levels of acetic acid, propionic acid, and butyric acid in the feces of db/db mice treated with curcumin and curcumin/piperine for 12 weeks. (D) They are kidney sections stained with H&E (magnification of ×200), transmission electron microscopy of intestinal tissue ultrastructure, and immunohistochemical analysis of occludin and ZO-1 in intestinal tissue (magnification of ×400). In db/db group, the glandular space was widened and inflammatory infiltration was observed significantly (arrow indication). (E, F) Quantitative analysis of immunohistochemistry. The results are expressed as the mean ± standard deviation of the three experimental groups, *p < .05, **p < .01, ***p < .001, and ****p < .0001.

H&E and IHC staining analysis of the mouse colon indicated that CUR treatment significantly improved intestinal mucosal gland damage to varying degrees (Figure 2(D)), widened gaps (indicated by the black arrow), and reduced some inflammatory infiltration (indicated by the red arrow). Additionally, it alleviated the increase in ZO-1 and Occludin expression caused by DKD (Figure 2(D–F)). Electron microscopy of the intestinal tissue revealed swollen mitochondria, disordered and missing microvilli, and loss of tight junction proteins in the db/db group. Curcumin and C/P treatments significantly enhanced intestinal structural integrity, with the combined effect being more pronounced (Figure 2(D)). These results indicate that C/P treatment can promote the production of SCFAs and have a certain protective effect on intestinal tissue integrity and intestinal epithelial barrier.

3.3. Curcumin/piperine and sodium butyrate alleviate HG-induced inflammation and oxidative stress in HK-2 cells

To determine the optimal concentrations of C/P and NaB for the experiment, HK-2 cells were treated with different concentrations of CUR and NaB for 4 h, followed by a cell viability assessment. Results indicated that CUR at 15 μM and NaB at 2 mM significantly reduced cell viability (Figure 3(A,B)). Consequently, CUR at 10 μM and NaB at 1 mM were selected for subsequent experiments, with CUR combined with PIP at 2 μM, maintaining a 5:1 ratio [21]. These results suggest that specific concentrations of CUR and NaB enhance HK-2 cell viability.

Figure 3.

The effects of curcumin/piperine and sodium butyrate on inflammation and oxidative stress in HK-2 cells. (A) Pre-treat HK-2 cells with different concentrations of curcumin (0, 1, 5, 10, and 15 μM) for four hours. (B) Pre-treat HK-2 cells with different concentrations of sodium butyrate (0, 0.1, 0.5, 1, and 2 mM) for four hours. (C) The effects of curcumin, curcumin/piperine, and sodium butyrate on MDA. (D) The effects of curcumin, curcumin/piperine, and sodium butyrate on GSH. (E) Detect intracellular ROS levels using a fluorescence microscope (magnification of ×200), green fluorescence distribution density represents ROS expression intensity. (F) Use Image J software (Bethesda, MD) to analyze the average fluorescence intensity of the images under the microscope (n = 3). The effects of (G–I) curcumin, curcumin/piperine, and sodium butyrate on the mRNA expression of IL-6, IL-1β, and TNF-α induced by HG in HK-2 cells. The results are expressed as the mean ± standard deviation of the three experimental groups, *p < .05, **p < .01, ***p < .001, ****p < .0001, and ^#p < .05 compared to 10 μM, ^##p < .01 compared to 1 mM.

Research has shown that sustained high sugar levels lead to inflammation and oxidative stress in HK-2 cells, which is linked to increased production of ROS [22]. To investigate the effects of C/P and NaB on HG-treated HK-2 cells, DCF fluorescent probes were employed to evaluate intracellular ROS levels. HG exposure resulted in higher DCF fluorescence in HK-2 cells compared to the control group, while ROS levels were significantly reduced in the C/P and NaB groups (Figure 2(E,F)). Additionally, the expression levels of IL-6, IL-1β, and TNF-α in HG-induced HK-2 cells were measured. ELISA and PCR results de­­monstrated that CUR, C/P, and NaB reversed the HG-induced increases in IL-6, IL-1β, and TNF-α levels (Figure 3(G–I)). Evaluating the oxidative/antioxidant system revealed that HG treatment increased MDA levels and decreased GSH levels, but pretreatment with CUR, C/P, and NaB significantly reversed these changes (Figure 3(C,D)). Among all the results, the combination of CUR and PIP exhibited a more pronounced effect than CUR alone. These findings suggest that C/P and NaB can alleviate inflammation and oxidative stress in HK-2 cells.

3.4. Curcumin/piperine and sodium butyrate activate the Nrf2/HO-1 pathway in DKD mice and HK-2 cells

In the subsequent experiments, the relative protein expression of the Nrf2/HO-1 pathway in HK-2 cells treated with C/P and NaB was assessed. IHC staining analysis demonstrated that, compared to db/m mice, the overall levels of Nrf2 and HO-1 proteins in the kidneys of db/db mice were diminished. However, C/P treatment significantly elevated the levels of Nrf2 and HO-1 proteins in the kidneys of db/db mice (Figure 4(A,C,D)). Concurrently, in vitro experiments corroborated these IHC findings. As illustrated in Figure 4, both protein expression and RNA levels of Nrf2 and HO-1 were decreased in HK-2 cells exposed to HG for 48 h, and pretreatment with C/P markedly reversed this reduction (Figure 4(B–H)). Similarly, NaB pretreatment produced the same outcomes (Figure 4(B–H)). These results indicate that C/P and NaB activate the Nrf2/HO-1 pathway in both db/db mice and HK-2 cells.

Figure 4.

Expression levels of Nrf2-HO-1 related genes. (A) Immunohistochemical analysis of Nrf2 and HO-1 in renal tissue (magnification of ×400). (B) Western blot analysis of Nrf2 and HO-1 in HG induced HK-2 cells using curcumin/piperine and sodium butyrate. (C, D) Quantitative analysis of immunohistochemistry. (E, F) Standardized analysis of immunoblot protein expression through grayscale values. The effects of (G, H) curcumin/piperine and sodium butyrate on the mRNA expression of IL-6, IL-1β, and TNF-α. The results are expressed as the mean ± standard deviation of three independent experiments, *p < .05, **p < .01, ***p < .001, and ****p < .0001.

3.5. Curcumin/piperine and sodium butyrate alleviate HG-induced inflammation and oxidative stress in HK-2 cells via the Nrf2/HO-1 pathway

To ascertain whether the protective effects of C/P and NaB on HG-stimulated HK-2 cells in inflammatory response and oxidative stress involve the activation of Nrf2/HO-1 signaling, validation was performed using the Nrf2 inhibitor ML385. Following ML385 administration, Nrf2 and HO-1 expression levels were markedly reduced (Figure 5(B,D,E,H,I)). PCR analyses revealed a significant increase in mRNA expression of IL-6, IL-1β, and TNF-α in ML385-pretreated cells compared to the C/P and B groups (Figure 5(J–K)). Moreover, consistent with expectations, Nrf2 inhibition negated the effects of C/P and NaB, leading to elevated intracellular ROS and MDA levels, while diminishing GSH activity (Figure 5(A,C,F,G)). These results indicate that the protective mechanism of C/P and NaB in HK-2 cells is analogous to that of Nrf2, with a significant connection to HO-1 pathway activation.

Figure 5.

The effect of Nrf2 inhibitor ML385 on inflammation and oxidative stress. (A) Detect the intracellular ROS levels in different groups of cells after ML385 inhibition using a fluorescence microscope (magnification of ×200), green fluorescence distribution density represents ROS expression intensity. (B) Protein blot analysis of Nrf2 and HO-1 in HG induced HK-2 cells induced by curcumin/piperine and sodium butyrate after ML385 inhibition. (C) Use Image J software (Bethesda, MD) to analyze the average fluorescence intensity of the images under the microscope (n = 3). Standardized analysis of immunoblot protein expression using (D, E) grayscale values. (F, G) The effects of curcumin/piperine and sodium butyrate on MDA and GSH after ML385 inhibition. (H–K) The effect of ML385 inhibition on the mRNA expression of Nrf2, HO-1, IL-6, IL-1β, and TNF-α in each group, *p < .05, **p < .01, ***p < .001, and ****p < .0001.

4. Discussion

Curcumin, extracted from the rhizome of Curcuma longa, has been validated by numerous studies for its therapeutic efficacy and safety in treating polycystic ovary syndrome, metabolic syndrome, nonalcoholic fatty liver disease, atherosclerosis, and other conditions [23,24]. Clinical and basic research highlights CUR’s role in reducing edema, mitigating neurotoxicity, and combating ischemia, primarily through its anti-inflammatory and antioxidant properties [25–27]. Nevertheless, CUR’s low bioavailability, poor absorption, high metabolic rate, and limited efficacy in the human body present significant challenges [28]. Consequently, contemporary research increasingly focuses on the synergistic effects of CUR and PIP [29], as PIP reduces the metabolism of various drugs [30], although the precise mechanisms remain unclear. Miyazawa et al. illustrated that CUR and PIP together enhance fat loss and inhibit inflammation in mice on a high-fat diet [31]. Banji et al. found that their concurrent use minimized neuroprotective changes in Purkinje cells [32]. These results underscore the necessity to further investigate the mechanisms of C/P in ameliorating DKD. This study aims to elucidate how C/P mitigates inflammatory response and oxidative stress in DKD both in vivo and in vitro. Initial findings indicate that CUR improves renal function and reduces inflammation in db/db mice, with its efficacy significantly augmented when combined with PIP.

Maintaining the normal structure of intestinal tissue and the integrity of the intestinal epithelial barrier is essential for protecting the body from stress stimuli associated with inflammation and oxidative imbalances [33]. The gut–kidney axis highlights the importance of intestinal tissue in DKD. Prior research has demonstrated that traditional Chinese medicines like CUR effectively alleviate intestinal ecological imbalances in DKD, inhibiting inflammation and oxidative stress [34]. In our study, H&E staining of intestinal tissue in db/db mice revealed pathological changes in the intestinal mucosa. The high-glucose environment caused atrophy of intestinal mucosal glands, widened gaps, inflammatory cell infiltration, and loss or localized defects in crypt structures, significantly damaging the intestinal epithelial barrier. The expression of tight junction proteins Occludin and ZO-1 was markedly reduced; however, CUR treatment mitigated these damages and increased Occludin and ZO-1 expression. Although the therapeutic effect of the C/P combination was not significantly superior to CUR alone, it still showed substantial efficacy compared to untreated db/db mice. These results indicate that C/P offers a protective effect on the intestinal structure and barrier integrity of db/db mice.

SCFAs are synthesized by the intestinal flora by fermenting indigestible carbohydrates, providing essential nutrients to the intestinal epithelium [35], enhancing its barrier function [36], and delaying the progression of type 2 diabetes, chronic kidney disease, inflammatory bowel disease, among other conditions through various mechanisms [37–39]. Acetate, propionate, and butyrate, produced in the highest concentrations, are particularly significant [40]. Numerous studies indicate that CUR increases gut microbiota diversity, modulates SCFA levels, and boosts the production of butyrate-producing species [41,42]. In this study, SCFA levels in the feces of mice from each group were measured, revealing decreased levels of acetic, propionic, and butyric acids in the db/db group. Curcumin treatment reversed these reductions, with the C/P combination showing even more pronounced effects. Butyric acid not only maintains normal intestinal function but also has demonstrated renal protective effects and a strong association with DKD in increasing studies [43,44]. Future research will involve further exploration using NaB.

Inflammation and oxidative stress are pivotal factors in the progression of DKD, with numerous researchers continuously seeking effective strategies to inhibit these processes and thereby mitigate DKD’s advancement [45,46]. Neutrophil gelatinase-associated lipocalin is recognized as an early biomarker indicative of systemic infection and is primarily used to detect acute kidney injury. However, studies indicate that NGAL expression is also elevated in chronic kidney disease, serving as a potential predictor of renal function deterioration [47,48]. CD68, IL-6, and IL-1β are essential pro-inflammatory mediators in DKD’s pathogenesis, with TNF-α predominantly released by macrophages and lymphocytes to regulate systemic inflammation. In our in vivo experiments, IL-6 and TNF-α levels in the blood, along with NGAL2, CD68, and IL-1β expression in the kidney tissue of db/db mice, were significantly elevated compared to db/m mice, whereas CUR treatment downregulated this expression. In vitro experiments revealed that HG significantly upregulated IL-6, IL-1β, and TNF-α in HK-2 cells, whereas pretreatment with CUR and NaB reduced these inflammatory markers. Additionally, the C/P combination exhibited more pronounced effects than CUR alone. Notably, C/P increased IL-6 expression, suggesting high sensitivity of IL-6, necessitating further validation of these drug effects on cells. Oxidative stress is a manifestation of the imbalance between the oxidative and antioxidant systems in the body, is closely linked to DKD, particularly the excessive production of ROS, leading to DNA damage, hormonal dysregulation, glucose metabolism impairment, and compromised kidney function [49,50]. Our research demonstrates that in HG-induced HK-2 cells, ROS and MDA levels (markers of oxidative stress) significantly increase, while endogenous antioxidant GSH levels significantly decrease. Pretreatment with CUR and NaB reversed these alterations, with C/P proving more effective than CUR alone. These findings substantiate that C/P and NaB interventions can alleviate renal inflammation and restore oxidative system balance.

Previous text has established that C/P and NaB can mitigate inflammation and oxidative stress in DKD. This section delves into their mechanisms of action. An increase in Nrf2 activity is well known to inhibit inflammation, prevent the decline of antioxidant defenses in DKD, reduce ROS production by enhancing antioxidant mechanisms, and ameliorate renal tubular injury [8,51]. HO-1, a primary antioxidant response element downstream of Nrf2, has its anti-inflammatory and antioxidant effects in DKD development confirmed by numerous studies [52–54]. Current research indicates that both CUR and NaB can target the Nrf2 signaling pathway, safeguarding cells from inflammation and oxidative stress, thereby maintaining homeostasis [55–57]. However, the specific role of butyrate in DKD through the Nrf2/HO-1 pathway remains unclear. In light of these insights, the effects of C/P and NaB on the Nrf2/HO-1 signaling pathway in DKD were investigated. The study revealed a significant reduction in Nrf2 and HO-1 expression in the kidneys of db/db mice, which was reversed by CUR treatment. This finding was corroborated by in vitro experiments, where pretreatment with NaB mitigated the HG-induced reduction in Nrf2 and HO-1. The application of the Nrf2 inhibitor ML385 diminished and reversed the anti-inflammatory and oxidative alleviating effects of C/P and NaB on DKD, demonstrating their roles in DKD through activating the Nrf2/HO-1 signaling pathway.

In summary, CUR regulates gut microbiota balance, increases butyrate content, and effectively alleviates DKD by the activation of the Nrf2/HO-1 signaling pathway. The combination of CUR with PIP further enhances this therapeutic effect. In previous studies, Tu et al. demonstrated that the combination of CUR and PIP enhances the lipid-lowering effects of CUR by increasing the activity of cholesterol 7α-hydroxylase and apolipoprotein A1, thereby treating high-fat diet-induced hyperlipidemia in rats [58]. In another study, supplementation with CUR and PIP significantly increased body fat loss and suppressed inflammatory responses in high-fat diet-induced mice compared to CUR alone [31]. Our results also confirm that the combined use of CUR and PIP is more effective than CUR alone. However, PIP, as an alkaloid, has inherent gastrointestinal irritant properties [59]. Future research should focus on long-term toxicological studies to determine the appropriate dosage for combined use in humans, thereby minimizing potential risks associated with the compounds themselves. Existing clinical studies have shown that novel delivery systems, such as nanotechnology and in situ gel formation, can significantly improve the delivery efficiency of CUR and PIP [59]. Nevertheless, identifying a safe and effective delivery mechanism remains a major challenge for clinical application. Addressing these challenges could further expand the clinical synergy of CUR and PIP, unlocking greater translational potential and providing new possibilities for clinical treatment.

Traditional approaches to the management of DKD primarily involve pharmacological interventions and lifestyle modifications. Pharmacological treatments include glucose-lowering and antihypertensive agents, as well as newer therapeutic options such as sodium-glucose cotransporter-2 (SGLT2) inhibitors and glucagon-like peptide-1 (GLP-1) receptor agonists. Lifestyle modifications encompass dietary control and regular physical activity. In recent years, stem cell therapy and exosome-based treatments have emerged as novel therapeutic strategies for DKD [60]. Despite these advancements, the treatment of DKD remains a significant challenge. Compared to other therapeutic approaches for DKD, CUR may offer distinct advantages in early intervention and comprehensive renal protection, presenting a promising potential for improving DKD management. Currently, research on NaB’s impact on DKD is relatively scarce. Our study suggests that NaB may be a promising therapeutic target for DKD, providing new avenues for treatment research. However, our research has limitations. The relationship between NaB’s effects on inflammation reaction and oxidative stress in mice and the Nrf2/HO-1 signaling pathway was not validated in vivo. Future studies should further investigate the connection between NaB and DKD.

5. Conclusions

In conclusion, this study demonstrates that C/P modulates NaB levels, alleviating inflammatory response and oxidative stress by activating the Nrf2/HO-1 pathway. This interaction underscores their therapeutic potential in DKD, providing a new theoretical foundation for the clinical treatment of C/P in treating this condition.

Funding Statement

This study was supported by the Youth Project of Shandong Provincial Natural Science Foundation (No. ZR2020QH063).

Author contributions

Zhu Y.N. designed and completed the research, and wrote the manuscript. Sun L.N. and Guo Z.A. provided project funding and financial support, and ultimately supervised and reviewed the manuscript. Xin P.Q., Huang F., and Song L. participated in data organization and project research. Man Y.L., Ren M.M., and Ma Z.W. participated in data organization and analysis. Wang Y. provided the necessary technical support for the research. All authors reviewed the manuscript.

Ethical approval

All procedures were approved by the Animal Health and Use Committee of Shandong University of Traditional Chinese Medicine Affiliated Hospital (AWE-2019-004).

Disclosure statement

No potential conflict of interest was reported by the author(s).