Male C57BL/6J mice (7 weeks old) were acquired from Hubei Beiente Biotechnology Co., Ltd. All mice were cultured in a specific pathogen-free environment with a standard light-dark cycle of 25°C for 12 h and had free access to food and water. The DN mouse model was established according to a previous study (Lv et al., 2023). Mice were fed a high-fat diet for 2 months, and then streptozotocin (STZ; 50 mg/kg) was injected intraperitoneally for 7 consecutive days to induce DN mouse model. Seven days after injection, C57BL/6J mice were monitored for blood glucose levels. The control group of mice continued to be fed a standard diet. Then mice were intraperitoneally injected with 25 mg/kg of Xanthohumol, control-plasmid or SNHG10-plasmid, and blood, urine, and renal tissues were collected for the following experiments. The animal experiments were approved by the Ethics Review Committee of Bestcell Model Biological Center (approval number 2024-01-29A).

The HK-2 cells were acquired from ATCC and cultured in DMEM (c11885500BT, Gibco) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin at 37°C in a humidified atmosphere of 5% CO₂. After overnight culture in serum-free medium, the HK-2 cells were pretreated with 50 uM Xanthohumol followed by 30 mM D glucose for 48 h. Cells were divided into following groups: Control (cells without any treatment); HG (cells were treated with 30 mM D glucose for 48 h); HG+Xanthohumol (cells were pretreated with 50 uM Xanthohumol followed by 30 mM D glucose for 48 h); HG+control-plasmid (cells were pretransfected with control-plasmid followed by 30 mM D glucose for 48 h); HG+SNHG10-plasmid (cells were pretransfected with SNHG10-plasmid followed by 30 mM D glucose for 48 h); HG+Xanthohumol+shRNA-SNHG10 (cells were pretreated with Xanthohumol and shRNA-SNHG10 followed by 30 mM D glucose for 48 h). The cells were collected for subsequent experiments.

The control-plasmid and SNHG10-plasmid were conducted by ELK biotechnology and transfected into HK-2 cells using Lipofectamine^® 2000 reagent (11668019, Invitrogen, USA) referring to the manual. After transfection, cells were obtained for following analysis. After 48 h, cells were harvested for the further analysis.

After treatment, Body weights were measured, and blood, serum, urine sample and renal tissues were collected. Urine sample was measured and centrifuged at 2000 g for 10 min to acquire supernatant. Urine albumin concentrations were measured using a mouse urine albumin ELISA kit (Cambridge, United States). Blood glucose level was detected using a blood glucose monitor (Boehringer, Germany). The serum creatinine levels were tested using a QuantiChrom Creatinine Assay Kit according to the instructions. The blood urine nitragen (BUN) level was determined using an automatic biochemistry analyser (Beckman Coulter Inc., United States). The renal to body weight ratio was analyzed.

The 4% paraformaldehyde (80096618, sinoreagent) fixed renal tissues were embedded in paraffin and sectioned at 2∼3 μm of thickness. Then, the sections were dewaxed, hydrated and oxidised by aqueous periodate. Next, the sections were stained with Schiff’s for 30 min and differentiated with 0.5% sodium thiosulfate aqueous solution (20039918, sinoreagent) twice. After washing with tap water, hematoxylin (H9627-25G, Sigma) stained the nuclei for 3 min and 1% hydrochloric acid (10011018, sinoreagent) was used for differentiation. After dehydration and transparency, the slices were sealed using neutral gum for histological analysis.

The 4% paraformaldehyde (80096618, sinoreagent) fixed renal tissues were embedded in paraffin and sectioned at 2∼3 μm of thickness. Then, the sections were dewaxed, hydrated and stained with Masson Staining Kit (G1006, servicebio) according to the scheme for histological analysis. Collagen fibers were dyed blue, muscle fibers and red blood cells were dyed red, indicating the content of collagen fibers in renal tissue and the degree of fibrosis.

Immunohistochemistry is used to detect renal tissue level of α-SMA, FN, Col-I and E-cadherin. The 4% paraformaldehyde (80096618, sinoreagent) fixed renal tissues were embedded in paraffin and sectioned at 5 μm of thickness. The sections were then incubated in citric acid buffer (10007118, sinoreagent) for 10 min and then heated using a microwave oven for antigen repair. After that, the sections were sealed with 5% BSA (4240GR250, Biofroxx) for 30 min, and then incubated overnight at 4°C using α-SMA (BM0002, Boster, 1:400), Fibronectin (Ab268020, Abcam, 1:500), Collagen Ⅰ (14695-1-AP1, Wuhan Sanying, 1:500) or E-Cadherin (20874-1-AP, Wuhan Sanying, 1:200) antibodies. These sections were then cultured in a secondary antibody for 30 min, stained with hematoxylin (H9627-25G, Sigma) and analyzed using a light microscope (CX31, Olympus) according to the scheme.

After treatment, renal tissues or HK-2 cells were collected. Then, the levels of inflammatory factors (IL-1β, IL-6 and TNF-α) in renal tissues and the HK-2 cells supernatant were detected using IL-1β ELISA Kit (ELK1270/ELK1271, ELK Biotechnology), IL6 ELISA Kit (ELK1156/ELK1157, ELK Biotechnology), and TNF-α ELISA Kit (ELK1190/ELK1387, ELK Biotechnology) following the instructions, respectively. The optical density (OD) value of each well at 450 nm was detected following the manual.

Total RNA was separated from renal tissues or HK-2 cells which used TRIpure Total RNA Extraction Reagent (EP013, ELK, China) according to the synopsis. Total RNA was stored at −80°C. Then cDNA was synthesized using the EntiLink™ 1st Strand cDNA Synthesis Super Mix (EQ031, ELK Biotechnology). Then qRT-PCR was executed using the EnTurbo™ SYBR Green PCR SuperMix (EQ001, ELK Biotechnology). The level of LncRNA SNHG10 was determined using QuantStudio 6 Flex System (Life technologies). Primers sequences are presented in Table 1.

CCK-8 assay was conducted using CCK-8 detection kit (C0038, beyotime). After treatment, HK-2 cells were cultured in a 96-well plate. Then CCK-8 reagent (10 μL) was added to each well, followed by incubation at 37°C for 2 h. To evaluate cell viability, a microplate reader (DR-200Bs, Diatek) was used to measure the absorbance at 450 nm.

Total proteins were extracted by HK-2 cells in RIPA buffer (AS1004, ASPEN) supplemented with 1% PMSF (AS1006, ASPEN) and1% phosphatase inhibitors (AS1008, ASPEN). Lysates were centrifuged for 10 min at 4°C, and the supernatants were acquired. Proteins were resolved by SDS-PAGE kit (AS1012, AS1012) and transferred onto PVDF membranes (IPVH00010, Millipore). The membranes were blocked with 5% skimmed milk (AS1033, ASPEN) for 2 h and then cultivated with primary antibodies against α-SMA (ab124964, Abcam, 1:10000), Fibronectin (15613-1-AP, Proteintech, 1:500), Collagen Ⅰ (AF7001, affbiotech, 1:500) or E-Cadherin (#3195, CST, 1:1000) at 4°C overnight. After washing in TBST for three times, the membranes were cultivated with secondary antibodies (AS1107, ASPEN, 1:10000) for 2 h. The protein signals were visualized by ECL detection system reagents (AS1059, ASPEN) and quantified using ImageJ Software.

After treatment, HK-2 cells were plated into 96-well plates for 48 h. After that, the cells were obtained using centrifugal separation technology at 4°C for 5 min and washed with PBS (GNM20012, genomcell). Then cell apoptosis was measured by annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) apoptosis detection kit (AO2001-02P-G, Tianjin Sungene Biotech Co., Ltd) following the instructions. Finally, apoptotic cells were assessed using Flow cytometer (CytoFLEX, Beckman) and analyzed using Kaluza Analysis software.

TargetScan bioinformatics software was applied to predict the binding sites between SNHG10-plasmid and miR-378b. Then the SNHG10-3′UTR, which contains the miR-378b-plasmid binding site or mutated target site were compounded by genomic PCR and cloned into pGL-6-Luc (Promega, United States) to generate the reporter vector SNHG10-wild-type (SNHG10-WT) or SNHG10-mutated-type (SNHG10-MUT). Then HK-2 cells were transfected with SNHG10 wild-type or mutant portion combined with miR-378b-plasmid by applying Lipofectamine 2000 (11668019, Invitrogen) in accordance with the manual. Then the luciferase activity was determined by Dual-Luciferase Reporter Assay System (11668019, beyotime).

The data of three independent experiments were exhibited as the mean ± standard deviation (SD). Statistical analyses was evaluated using GraphPadPrism6.0. Differences were estimated with unpaired Student’s t-test or one-way ANOVA among groups. *P < 0.05, and **P < 0.01 indicated statistically significant difference.

To determine the effect of xanthohumol on kidney damage, STZ-induced DN mice were administered with 25 mg/kg xanthohumol. We observed that the body weights were reduced and the ratio of kidney weight/body weight were enhanced obviously in DN mice (Figures 1A,B), and xanthohumol treatment restored these changes. Moreover, STZ treatment led to the remarkable upregulation in the concentrations of blood glucose, NAG activity, urinary albumin, serum creatinine, urine creatinine and BUN (Figures 1C–H), while these influence of STZ were significantly reversed by xanthohumol. ELISA assay manifested that TNF-α, IL-1β, and IL-6 levels (Figures 1I–K) were significantly enhanced in the kidney tissues of STZ-induced DN mice, compared to that in control mice, demonstrating that STZ-induced DN mice had stronger inflammatory responses. However, xanthohumol reduced inflammatory responses in STZ-induced DN mice. These findings demonstrated that xanthohumol alleviated STZ-stimulated kidney injury in DN mice.

To further explore whether xanthohumol relieve renal fibrosis in STZ-induced DN mice, renal fibrosis was assessed by PAS staining and Masson staining and the expression level of fibrotic proteins and epithelial cell markers (FN, a-SMA, Col-I and E-cadherin) were determined by IHC. PAS staining unveiled that glomerular damage and tubulo-interstitial damage were notably increased in STZ-induced DN mice, compared to that in control mice, which was obviously improved by xanthohumol (Figure 2A). Masson staining demonstrated that notably enhanced renal tubulo-interstitial fibrosis in the kidney of DN mice (Figure 2B). Moreover, the expression levels of FN, a-SMA and Col-I were upregulated and E-cadherin was downregulated in DN mice (Figure 2C). However, xanthohumol treatment could remarkably relieve these tubulointerstitial lesions. Together, our findings strongly demonstrated that xanthohumol relieved diabetes-induced renal interstitial fibrosis, thereby blocking DN progression.

Increasing evidences have highlighted that abnormal expression of lncRNAs are associated with renal fibrosis in renal diseases. To detect the functional roles of lncRNA SNHG10 in the progression of DN, we determined the lncRNA SNHG10 expressions in the renal tissues from DN mice. qRT-PCR analysis revealed that lncRNA SNHG10 was downregulated in the renal tissues of DN mice as compared to that in control groups, and this inhibition was reversed by xanthohumol treatment (Figure 3). Our data indicated that abnormal expression of lncRNA SNHG10 may be linked with the pathogenesis of DN.

Moreover, an in vitro DN model was established by simulating renal tubular epithelial cells injury with high glucose, then the HK-2 cells were treated with 50 uM xanthohumol. CCK-8 assay and flow cytometer analysis were applied to determine HK-2 cells viability and apoptosis. As presented in Figures 4A–D-glucose suppressed the HK-2 cells viability and stimulated more apoptotic cells. However, these findings were partly reversed by xanthohumol. We also determined the expression of fibrotic proteins and epithelial cell markers, including FN, a-SMA, Col-I and E-cadherin, were determined by Western blot. Our data suggested that the expression of α-SMA, FN and Col-I were higher in high glucose-induced HK-2 cells than that in Control group. Furthermore, E-cadherin was downregulated in high glucose-induced HK-2 cells, compared with untreated cells. Nevertheless, we observed the opposite results in 50 uM xanthohumol treated cells (Figures 4D,E). To explore the effect of xanthohumol on inflammatory response, we detected TNF-α, IL-1β, and IL-6 levels in culture supernatant of HK-2 cells by ELISA. Our data demonstrated that high glucose notably promoted the secretion of inflammatory factor, and this promotion was inhibited by xanthohumol treatment (Figure 4F). In addition, results from Figure 4G suggested that xanthohumol enhanced the mRNA level of lncRNA SNHG10 in high glucose-induced HK-2 cells, as opposed to Control group. Our data confirmed that xanthohumol ameliorated diabetic nephropathy through suppression of renal fibrosis and inflammatory response by regulating lncRNA SNHG10 levels in the pathogenesis of DN.

Our results have revealed the relationship between DN occurrence and lncRNA SNHG10. To further explain the function of lncRNA SNHG10 in the pathogenesis of DN. STZ-induced DN mice were intraperitoneally injected with control-plasmid or SNHG10-plasmid and we observed that lncRNA SNHG10 was upregulated in the renal tissues from DN mice, compared with Control and control plasmid group (Figure 5A). Our results further confirmed that SNHG10-plasmid relieved STZ-induced renal injury in DN mice, as verified by increased body weights, decreased kidney weight/body weight ratio, blood glucose concentrations, NAG activity, 24-h urinary albumin, serum creatinine, urine creatinine and BUN (Figures 5B–I), compared with control plasmid group. After that, to delineate whether SNHG10-plasmid may reduce the inflammatory response in DN renal tissue, we detected the secretion of inflammatory factors, including TNF-α, IL-1β, and IL-6 levels. As documented in Figures 5J–L, the secretion levels of inflammatory factors in the SNHG10-plasmid group were visibly lower than those in the control plasmid group. Our findings demonstrated that upregulation of SNHG10 alleviated STZ-stimulated kidney injury in DN mice.

Next, PAS and Masson staining unveiled that upregulation of SNHG10 remarkably relieved glomerular damage and renal tubulo-interstitial fibrosis in the kidney of DN mice (Figures 6A,B). Further IHC staining indicated the decreased FN, a-SMA and Col-I expression, as well as enhanced E-cadherin level in the renal tissues of SNHG10-plasmid treated DN mice, as opposed to control plasmid group (Figure 6C). Our data demonstrated that xanthohumol suppressed renal interstitial fibrosis of DN mice by regulating SNHG10.

To further explain the regulation of lncRNA SNHG10 and xanthohumol in the development of DN, we designed three shRNA to silence the expression of SNHG10 in HK-2 cells. RT-qPCR analysis confirmed the knockout efficiency of HK-2 cells, and we observed that shRNA-SNHG10-1 knockout decreased most significantly SNHG10 mRNA levels in HK-2 cells, compared with the other two plasmids (Figure 7A). Thus, shRNA-SNHG10-1 was selected for subsequent cell function experiments. Results from Figure 7B revealed that SNHG10 was upregulated in high glucose-induced HK-2 cells after SNHG10-plasmid or xanthohumol treatment, while this promotion was inhibited by 50 μM xanthohumol+shRNA-SNHG10. Further CCK-8 and FCM assay revealed that SNHG10-plasmid or xanthohumol promoted high glucose-induced HK-2 cells viability and reduced cells apoptosis (Figures 7C–E). However, we observed the opposite data in 50 μM xanthohumol+shRNA-SNHG10 treated cells. In addition, Downregulation of SNHG10 reversed the effects of xanthohumol or SNHG10-plasmid on renal interstitial fibrosis and inflammatory response in high glucose-induced HK-2 cells, as confirmed by enhanced α-SMA, FN, Col-I, TNF-α, IL-1β, and IL-6 levels, as well as suppressed E-cadherin expression (Figures 7F–H). Our findings demonstrated that xanthohumol ameliorates DN through suppressing renal interstitial fibrosis and inflammatory response by regulating SNHG10.

To investigate the possible molecular mechanism of lncRNA SNHG10 in regulating DN procession, TargetScan (http://www.targetscan. org) was applied to identify the latent targets of lncRNA SNHG10. As presented in Figure 8A, miR-378b contained complementary binding sites to the 3′-UTR of lncRNA SNHG10. To reveal the direct binding between lncRNA SNHG10 and miR-378b, SNHG10 (WT) and SNHG10 (MUT) luciferase reporters containing miR-378b NC or miR-378b mimics binding sites were individually constructed. The luciferase reporter assay revealed that miR-378b mimics prominently reduced the luciferase activity of lncRNA SNHG10 (WT) reporter in 293 T cells, while overexpression of miR-378b displayed little effect on luciferase activity in MUT-lncRNA SNHG10 transfected cells (Figure 8B). Our findings suggested that miR-378b directly targets lncRNA SNHG10.