BG is obtained from the whole plant of bitter gourd through enzymatic hydrolysis and ultrafiltration techniques, provided by the Inner Mongolia Bioactive Peptide Engineering Laboratory. Currently, the BG has been granted a national invention patent.

A total of twenty 8-week-old female MRL/lpr lupus mice and ten 6–8-week-old female MRL/MpJ mice were purchased from Changzhou Cavens Experimental Animal Co., Ltd. Upon arrival, all mice were housed under specific pathogen-free (SPF) conditions with a controlled room temperature of 20°C, relative humidity of 40%–60%, a 12-h light/dark cycle, and free access to food and water. The MRL/lpr mice were randomly divided into two experimental groups (n=10 per group): (1) the Lupus Model group, receiving daily intragastric administration of normal saline; (2) the BG Treatment group, receiving daily intragastric administration of BG (BG dosage is 0.06g/kg). The MRL/MpJ mice (n=10) served as the Normal Control group and received normal saline following the same schedule. The intervention lasted for 3 weeks. Characteristics and survival of the experimental mouse groups are shows in Table 1.

For transfection experiments, THP-1 cells were first differentiated into macrophage-like cells using PMA and primed with LPS as described in Section 2.3. miRNA-146a mimics, inhibitors, mimic NC and inhibitor NC, pcDNA-BRD4 and pcDNA-NC were synthesized by Changsha Zebra Biotechnology Co., Ltd. (Changsha, China). The cells were transfected with 25–50 nM miRNA-146a mimic, mimic-NC, miRNA-146a inhibitor and inhibitor NC using LipoHigh liposome efficient transfection reagent (Sangon Biotech).

THP-1-derived macrophage-like cells (THP-1 cells) were seeded into 96-well plates at a volume of 200 μL per well, cultured in a 5% CO₂ incubator at 37°C for 12–24 h, and treated with LPS or BG for 24 h. Then, 10 μL of CCK8 solution was added to each well and cultured for 4 h. The absorbance of each well at 490 nm was measured on a microplate reader to calculate the relative viability of the cells.

The expression of TNF-α, IL-6 and IL-1β in the supernatant of THP-1-derived macrophage-like cells (THP-1 cells) was detected using human TNF-α ELISA kit (Proteintech, KE00154), human IL-6 ELISA kit (Proteintech, KE00139) and human IL-1β-ELISA kit (Proteintech, KE00021). The expression of anti-nuclear antibody (ANA) (Antibodies, ABIN366290), anti-double-stranded DNA antibody (anti-dsDNA) (Antibodies, ABIN366291), and inflammatory factors in mouse serum was detected using mouse ELISA kit (Elabscience, E-MSEL-M0002, E-EL-M0037c, E-EL-M0044c). Each experiment was repeated at least three times.

In the RT-PCR analysis, total RNA was extracted using a kit (Sangon Biotech, B518811) and reverse transcribed using the First Strand cDNA Synthesis Kit (GBTbiotech, P118-100) according to the instructions of manufacturer. The primers used in this study are as follows: GAPDH (forward sequence (F): 5’-GACATCCGATAAAATTGGAACG-3’ and reverse sequence (R): 5’-TTGGACCATTTCTCGATTTGTG-3’), U6 (F: 5’-GACATCCGATAAAATTGGAACG-3’ and R: 5’-TTGGACCATTTCTCGATTTGTG-3’), miR-146a (F: 5’-CCGATGTGTATCCTCAGCTTTG-3’ and R: 5’-TCCCAGCTGAAGAACTGAATTT-3’), BRD4 (F: 5’-CTAGCGTCTCAGAGTGCCTG-3’ and R: 5’-TGCCTCTTGGGCTTGTTAGG-3’). GAPDH and U6 were used as negative control. Finally, the relative expression was calculated using the 2^−ΔΔct method.

THP-1-derived macrophage-like cells (THP-1 cells) were lysed with PMSF-containing lysates (Beijing Dingguo Changsheng Company) and protein concentrations were determined using a BCA kit (Beijing Dingguo Changsheng Company). The appropriate SDS PAGE gel was selected according to the molecular size of the protein to be detected, and gel electrophoresis was performed according to the extracted protein concentration. LC3B and SQSTM1 (Sequestosome 1) are two important marker proteins in the detection of autophagy. The changes in LC3B reflect the process of autophagosome formation, while the degradation of SQSTM1 indicates the activity of the autolysosome pathway. By monitoring these two proteins simultaneously, a more comprehensive assessment of the normality and regulatory mechanisms of cellular autophagic function can be achieved. Therefore, WB was performed using specific antibodies against LC3B (proteintech, 18725-1-AP, 1:2500), SQSTM1 (proteintech, 18420-1-AP, 1:10000), BRD4 (proteintech, 28486-1-AP, 1:2000), and β-actin (proteintech, 81115-1-RR, 1:20000). ChemiScope 6100 was used as imaging system.

THP-1-derived macrophage-like cells (THP-1 cells) were permeabilized with 0.1% Triton 100 for 15 min, incubated overnight with primary antibody. After washing with PBS and blocking with 5%BSA were incubated with secondary antibody at 37°C for 1 h. The primary antibody was rabbit anti-LC3B (proteintech, 18725-1-AP), SQSTM1 (proteintech, 66184-1-lg), BRD4 (proteintech, 28486-1-AP), and the secondary antibody was Goat anti-Mouse/Rabbit Poly-HRP (proteintech, PR30009). The kidneys and spleens of mice were subjected to antigen repair after paraffin sections and primary antibodies were added overnight at 4°C, and then secondary antibodies were added to incubate for 50 min at room temperature in the dark. Primary antibodies included F4/80 antibody (Affinity Biosciences, 29414-1-AP, 1:200), CD86 antibody (Affinity Biosciences, #DF6332, 1:500), CD206 antibody (Affinity Biosciences, #DF4149, 1:500) and LC3B antibody (Affinity Biosciences, #AF4650, 1:500). Fluorescent secondary antibodies included FITC-labeled goat anti-rabbit IgG (Servicebio, GB22303, 1:400), FITC-labeled goat anti-mouse IgG (Servicebio, GB22301, 1:400), Cy3-labeled goat anti-rabbit IgG (Servicebio, GB21303, 1:400) and Cy3-labeled goat anti-mouse IgG (Servicebio, GB21301, 1:400). The cell nuclei were stained by 4’,6-diamidino-2 phenylindole (DAPI; Solarbio, C0060).

THP-1-derived macrophage-like cells (THP-1 cells) were seeded into 24-well plates and cultured for 24 h. The cells were collected and fixed with 4% glutaraldehyde (Solarbio, P1126) for 12 h, then fixed with 1% osmium tetroxide (Shanghai Rongbai Biology, RBS0086) for 2 h, dehydrated with gradient ethanol, embedded with epoxy resin, and then made into 50–100nm ultrathin sections. Finally, the sections were stained with 3% uranyl acetate and lead citrate. The autophagosomes of cells were observed under transmission electron microscope (HT7800 (80KV)).

THP-1-derived macrophage-like cells (THP-1 cells) were seeded and treated in 6-well plates. The cells were collected, washed and blocked with FBS for 10-15min. PE anti-mouse CD206 antibody (Biolegend, 141705), APC anti-mouse CD11b antibody (Biolegend, 101211) and FITC anti-mouse CD86 antibody (Biolegend, 105005) were incubated on ice in the dark for 15 min. The proportion of cells was analyzed by flow cytometry (BD FACSCanto) within 1 h.

THP-1-derived macrophage-like cells (THP-1 cells) were divided into the following four groups: BRD4-WT+mimic NC, BRD4-WT+miR-146a, BRD4-MUT+mimic NC, BRD4-MUT+miR-146a. The cells were seeded in 96-well plates and cultured at 37°C and 5% CO₂ to a density of 95%. Subsequently, the BRD4 overexpression vector was transfected with transfection reagents (Gene Pharma, 220721). The detection was performed using a dual luciferase assay kit (Beyotime, RG021S).

Formalin-fixed paraffin-embedded tissue sections were removed in xylene, rehydrated by graded ethanol, and then placed in a dye vat for hematoxylin-eosin (HE) staining (Pinofide Biotechnology Co., S191003). After staining with HE staining solution I for 3–5 min, the sections were washed with stained cup water to colorless, and then stained with HE staining solution II and III for 3–5 s, and quickly washed with water. Finally, each cylinder was immersed in 85% ethanol, 95% ethanol, HE staining solution IV, anhydrous ethanol I, anhydrous ethanol II, anhydrous ethanol III, n-butanol, xylene I, xylene II for 3–5 min. After staining, the slices were taken out and put into tuyere for drying. Slices were sealed with neutral gum and observed under a microscope.

Proteinuria in mice was measured by Elabscience^® urine protein colorimetric test kit (Elabscience, E-BC-K252-M). The concentration of blood urea nitrogen in mice was detected by Elabscience^® urea (BUN) colorimetric test kit (Elabscience, E-BC-K183-M). The concentration of serum creatinine in mice was detected by creatinine (Cr) assay kit (Nanjing Jiancheng Bioengineering Institute, C011-2-1).

All experiments were performed at least three times. The results are expressed as mean ± SD. SPSS method was used for statistical analysis. The normality of data distribution was assessed using the Shapiro-Wilk test, and homogeneity of variances was assessed using Levene’s test. For comparisons between two groups, an unpaired two-tailed Student’s t-test was used for normally distributed data; otherwise, the Mann-Whitney U test was applied. For comparisons among multiple groups, one-way ANOVA followed by Tukey’s post hoc test was used for parametric data, and the Kruskal-Wallis test with Dunn’s post hoc test was used for non-parametric data. A P value of less than 0.05 was considered statistically significant.

We evaluated the therapeutic effects of BG in lupus-prone MRL/lpr mice. BG treatment significantly reduced proteinuria, serum levels of antinuclear antibodies (ANA), anti-dsDNA antibodies, blood urea nitrogen, and creatinine compared to untreated MRL/lpr controls (Figure 1A–C). It also alleviated the extension of cutaneous lesions (Figure 1D) and lowered serum levels of the pro-inflammatory cytokines TNF-α, IL-6, and IL-1β (Figure 1E). Furthermore, BG treatment prevented spleen enlargement, while no significant change was observed in lymph node size (Figure 1F). Histological analysis of kidney and spleen tissues revealed severe pathology in MRL/lpr mice, including glomerular enlargement, mesangial and endothelial cell proliferation, inflammatory cell infiltration, tubular edema with loss of brush border in kidneys, as well as expanded lymphoid follicles, diffuse white pulp proliferation, increased germinal centers, and red pulp inflammation in spleens. BG treatment markedly reduced these inflammatory and pathological changes in both organs (Figure 1G). No significant difference in survival rate was observed among the groups during the study period. These results demonstrate that BG alleviates key clinical, serological, and histopathological features of lupus in MRL/lpr mice.

The pathogenesis of SLE is usually accompanied by infiltration of immune cells, including macrophages. Using immunofluorescence, we found that F4/80 macrophages infiltrated in the kidney and spleen of MRL/lpr mice. Macrophage infiltration was significantly reduced after BG treatment (Figure 2A). Further detection of macrophage subtypes by immunofluorescence showed an increase in CD86⁺CD206⁺ cells in kidney and spleen from MRL/lpr mice, while BG treatment decreased CD86⁺ cells and increased CD206⁺ cells (Figures 2B, C). To test the effect of BG on macrophage polarization in vitro, we established an LPS-induced macrophage model. First, we determined the effect of LPS and BG at low and high concentrations on THP-1 cell viability using the CCK-8 assay. We found that treatment with LPS and low and high concentrations of BG had no effect on cell viability (Figure 2D). We validated these findings by flow cytometry, we found that the proportion of CD86/CD11b-positive and CD206/CD11b-positive cells were both significantly increased after LPS induction, while the proportion of CD86/CD11b-positive cells decreased, and the proportion of CD206/CD11b-positive cells increased after BG treatment (Figures 2E, F). These results suggests that BG treatment inhibits macrophage M1 polarization and promotes M2 polarization.

Autophagy is a key physiological pathway for maintaining cellular homeostasis. To investigate whether BG influences autophagy, particularly within the immune context of lupus, we performed a multi-level analysis. First, at the tissue level, Western blot analysis of whole kidney and spleen lysates revealed an upregulation of the autophagy marker LC3B-II and a downregulation of the autophagy substrate SQSTM1/p62 in MRL/lpr mice compared to healthy controls. BG treatment further enhanced these changes (Supplementary Figure 1). These data indicate that BG induces a general activation of autophagic flux in lupus-affected tissues, although the cellular source of these markers cannot be specified from whole-tissue extracts.

To specifically assess autophagy within macrophages, we performed immunofluorescence co-staining for LC3B and the macrophage marker F4/80. In the kidneys and spleens of MRL/lpr mice, we observed an increase in LC3B+F4/80+ cells. Importantly, BG treatment significantly increased the proportion of these double-positive cells (Figures 3A, B), suggesting an enhancement of autophagic activity specifically within tissue-resident macrophages.

This finding was corroborated by transmission electron microscopy (TEM). Ultrastructural analysis of kidney tissues showed an increased number of autophagosomes in MRL/lpr mice, which was further elevated upon BG treatment (Figure 3C). Together, the immunofluorescence and TEM data provide direct visual evidence that BG promotes autophagic activity in vivo, with a clear effect observed within macrophages.

In vitro experiments using LPS-primed human THP-1-derived macrophages yielded consistent results. BG treatment increased the expression of LC3B and decreased SQSTM1/p62 (Supplementary Figures 2-4). Most conclusively, TEM quantification confirmed a significant increase in autophagosome numbers in BG-treated cells compared to LPS controls (Supplementary Figure 5). These in vitro results confirm that BG can directly enhance autophagic flux in human macrophages under inflammatory conditions.

M1 macrophages participate to the inflammatory response in SLE. Thus, we used the LPS-induced THP-1 cell assay to examine the effect of BG on pro-inflammatory cytokine production in vitro. The levels of inflammatory cytokines IL-1β, TNF-α and IL-6 were significantly decreased upon BG treatment (P < 0.05) (Figure 4A). This indicated that BG inhibits the inflammatory response of THP-1 cells induced by LPS. miR-146a is an inhibitor of the classic NF-κB pro-inflammatory pathway. Therefore, qPCR was employed to detect expression of miR-146a in mouse kidney, spleen and LPS-induced THP-1 cells. BG treatment could upregulate the expression of miR-146a in kidney and spleen of MRL/lpr mice and LPS-induced THP-1 cells (Figure 4B, C).

To investigate the role of miR-146a in macrophage polarization and autophagy under inflammatory conditions, we performed gain-of-function experiments in the established LPS-primed, THP-1-derived macrophage model. Briefly, cells were first transfected with miR-146a mimics or a negative control, then differentiated with PMA and subsequently stimulated with LPS as described in the Methods.

Successful transfection was confirmed by qPCR, which showed a significant upregulation of miR-146a expression in the mimic group compared to the control (Supplementary Figure 6). Flow cytometry analysis of surface markers revealed that overexpression of miR-146a significantly decreased the proportion of CD86+CD11b+ cells while increasing the proportion of CD206+CD11b+ cells (Figures 5A, B). This shift in marker expression suggests that miR-146a can attenuate LPS-induced M1-like polarization and promote an M2-like phenotype.

We next examined the effect of miR-146a on autophagy in this model. Western blot analysis showed that miR-146a overexpression further increased the LPS-induced level of LC3B-II and decreased the level of SQSTM1/p62 (Figure 5C; quantified in Supplementary Figure 7). This pattern was corroborated by immunofluorescence (Supplementary Figure 8). Consistently, transmission electron microscopy (TEM) demonstrated a significant increase in the number of autophagosomes in miR-146a-overexpressing macrophages compared to LPS-treated controls (Figure 5D). These combined data indicate that miR-146a enhances autophagic activity in LPS-primed macrophages.

To assess whether miR-146a was involved in the effect of BG on LPS-induced M1-type macrophages, we transfected THP-1 cells with miR-146a inhibitor and induced differentiation with PMA and LPS. The results of qPCR showed that miR-146a inhibitors inhibited the up-regulation of miR-146a expression by BG (P < 0.05) (Supplementary Figure 9). Flow cytometry results revealed that miR-146a inhibitors inhibit the down-regulation of BG on the proportion of CD86/CD11b positive cells and the up-regulation of CD206/CD11b positive cells (Figures 6A, B). The results of WB and immunofluorescence showed that miR-146a inhibitors inhibited the up-regulation of LC3B expression and the down-regulation of SQSTM1 expression by BG (Figure 6C; Supplementary Figure 10, 11). Furthermore, transmission electron microscopy revealed that miR-146a inhibitors could reduce the number of autophagosomes increased by BG (Figure 6D). In addition, we also measured the expression of inflammatory factors and found that miR-146a inhibitors inhibited the down-regulation of BG on IL-1β, TNF-α, and IL-6 levels (P < 0.05) (Figure 6E). Collectively, the above findings suggest that miR-146a is an important regulator of macrophage activity by BG. BG promotes M1 polarization to inhibit M2 polarization and activate autophagy by promoting miR-146a expression. In addition, BG inhibited the inflammatory response of M1-type macrophages by promoting miR-146a expression.

To elucidate the mechanism by which BG regulates macrophage activity, we identified a binding site between miR-146a and BRD4 using miRNA binding site prediction software TargetScan (Figure 7A). Next, we used a luciferase reporter gene assay to verify the targeted binding of miR-146a and BRD4 in THP-1 cells. The results demonstrated that fluorescence activity of the BRD4-WT+miR-146a mimic group was significantly lower than that of the BRD4-WT+miR-146a mimic NC group (P < 0.05). There was no significant difference in the fluorescence activity between the BRD4-MUT+miR-146a mimic NC group and the BRD4-MUT+miR-146a mimic group (Figure 7B). This indicated a targeting relationship between miR-146a and BRD4. Western blot and immunofluorescence results confirmed that overexpression of miR-146a inhibited BRD4 expression (Figure 7C; Supplementary Figure 12). In addition, we detected expression of BRD4 in kidney and spleen of untreated lupus mice and found that BG treatment downregulated this expression (Figures 7D, E).

Our study demonstrated that BRD4 is a target of miR-146a regulation. However, the role of BRD4 in the regulation of BG on macrophages remains to be elucidated. First, we detected expression of BRD4 by immunofluorescence. LPS significantly upregulated BRD4 expression, and BG treatment downregulated BRD4 expression in LPS-induced cells (Supplementary Figure 12). Our results showed that overexpression of BRD4 partially reversed the effect of BG on LPS-induced macrophages. It decreased the proportion of M1 macrophages and increased the proportion of M2 macrophages (Figures 8A, B). Furthermore, BRD4 significantly elevated the concentration of inflammatory factors that were reduced in presence of BG (Figure 8C). Further detection of autophagy-related proteins revealed that LC3B was upregulated and SQSTM1 was downregulated after BG treatment, indicating that BG activated autophagy, whereas overexpression of BRD4 reversed the activation of autophagy by BG (Figures 8D, E; Supplementary Figure 13). Transmission electron microscopy results confirmed that BRD4 inhibited the increased number of autophagosomes caused by BG (Figure 8F).

Splenocytes were obtained from MRL/lpr mice and subjected to functional studies. We found that BG reduced levels of TNF-α, IL-6, and IL-1β (P < 0.05, P < 0.01), levels of Antinuclear antibodies (ANAs) and anti-dsDNA antibodies and increased miR-146a expression (P < 0.01) in a dose-dependent manner (Figures 9A-C). BG treatment downregulated expression of Bromodomain-containing protein 4 (BRD4) and SQSTM1 in splenocytes of MRL/lpr mice, and upregulated expression of LC3B (P < 0.05. P < 0.01) (Figure 9D). In addition, we observed under electron microscope that BG treatment promoted the formation of autophagosomes in spleen cells of MRL/lpr mice (Figure 9E). These results suggest that BG inhibits the inflammatory response of spleen cells and activates autophagy.