Use of macrophage g protein-coupled estrogen receptor 1 in preparation of Anti-renal fibrosis therapeutic drug

Abstract

The use of macrophage G protein-coupled estrogen receptor 1 (GPER1) in the preparation of an anti-renal fibrosis therapeutic drug. By means of mediating GPER1, the progression of UUO-induced renal fibrosis can be delayed, so that the renal protective effect is exerted. The GPER1 agonist G-1 blocks the progression of UUO-induced renal fibrosis by means of inhibiting the polarization of M1-type and M2-type macrophages, attenuating the immune response, and reducing the expression of fibrosis factors.

Description

Application of macrophage G protein-coupled estrogen receptor 1 in the preparation of anti-renal fibrosis therapeutic drugs Technical Field

[0001] The present invention relates to the field of biomedicine technology, in particular to the use of macrophage G protein-coupled estrogen receptor 1 (GPER1) in the preparation of anti-renal fibrosis therapeutic drugs. Background Art

[0002] Chronic kidney disease (CKD) is a common disease that poses a serious threat to human health and life. The prevalence of CKD in my country is 10.8% of the adult population and continues to increase. Renal fibrosis is a common pathway and the primary pathological basis for the progression of CKD to end-stage renal disease (ESRD) from various causes. Currently, there are very limited treatment options that can effectively combat renal fibrosis and delay the progression of CKD to ESRD. Once fibrosis occurs, it is often difficult to reverse. Therefore, identifying new targets that can effectively delay the progression of CKD to end-stage renal disease and developing anti-fibrotic drugs are urgent.

[0003] Estrogen has a renal protective effect, primarily acting on the classical estrogen receptor (including estrogen receptor α and β subtypes). However, the side effects of estradiol limit its clinical application, and the classical estrogen receptor pathway cannot explain the protective effect of estrogen on the kidney. G protein-coupled estrogen receptor 1 (GPER1) is a novel estrogen receptor that is primarily expressed on the cell membrane and has also been found to be expressed on the mitochondrial and endoplasmic reticulum membranes. Unlike the classical estrogen receptor α and β subtypes, which exert "genotype" regulatory effects by regulating the transcription of specific target genes, GPER1 primarily exerts indirect transcriptional regulatory effects through cell signaling pathways. Studies have shown that GPER1 is expressed in multiple organs and participates in a variety of biological processes.

[0004] Macrophages play an important role in the progression of kidney injury, inflammation, and fibrosis. Currently, there is a lack of effective drug treatments and factors that block the effects of macrophages on aggravating renal fibrosis.

[0005] Summary of the Invention

[0006] The purpose of the present invention is to provide an application of macrophage G protein-coupled estrogen receptor 1 (GPER1) in the preparation of anti-renal fibrosis therapeutic drugs.

[0007] Macrophages play a crucial role in the progression of renal fibrosis. Previous studies have revealed that the novel estrogen receptor GPER1 is involved in regulating macrophage activation and related inflammatory pathways. However, the role of GPER1 in inhibiting the progression of renal fibrosis by regulating macrophages remains unclear. Recent studies have shown that GPER1 activation can alleviate tubular damage and proteinuria in salt-sensitive rats fed a high-salt diet. Our previous studies have also shown that GPER1 is involved in immune responses and inflammatory processes in the UUO model, and that antagonizing GPER1 can aggravate renal fibrosis, suggesting that this receptor may be a therapeutic target. Studies have confirmed that GPER1 is involved in regulating macrophage activation and related inflammatory pathways. G1 (a GPER1 agonist) can reduce TLR4 expression in mouse macrophage cell lines and peritoneal macrophages, and reduce LPS-mediated inflammatory responses. These findings suggest that GPER1 activation may alleviate renal fibrosis by inhibiting macrophage inflammation.

[0008] The first aspect of the present invention provides the use of macrophage G protein-coupled estrogen receptor 1 (GPER1) as a target in the preparation of anti-renal fibrosis therapeutic drugs.

[0009] The second aspect of the present invention provides the use of an agent for activating macrophage G protein-coupled estrogen receptor 1 in the preparation of an anti-renal fibrosis therapeutic drug.

[0010] Furthermore, the agent for activating macrophage G protein-coupled estrogen receptor 1 is a macrophage G protein-coupled estrogen receptor 1 agonist.

[0011] Furthermore, the macrophage G protein-coupled estrogen receptor 1 agonist is G-1.

[0012] Furthermore, in the application, the agent that activates macrophage G protein-coupled estrogen receptor 1 inhibits the phenotypic transformation of M0 macrophages to pro-inflammatory (M1 type) and pro-fibrotic (M2 type) macrophages by inhibiting the activation of the MAPK signaling pathway.

[0013] Furthermore, in the application, the reagent that activates macrophage G protein-coupled estrogen receptor 1 reduces the inflammatory response, reduces the expression of inflammatory factors in renal tubular epithelial cells, reduces the secretion of pro-fibrotic factors, and delays renal fibrosis.

[0014] The advantages of the present invention are:

[0015] 1. The present invention found that by mediating GPER1, the immune response can be weakened and the expression of fibrotic factors can be reduced, thereby delaying the progression of UUO-induced renal fibrosis and thus exerting a renal protective effect.

[0016] 2. The present invention found that activating macrophage GPER1 can inhibit the MAPK signaling pathway and inhibit the polarization of M1 and M2 macrophages, thereby reducing inflammatory damage to renal tubular epithelial cells and inhibiting the transformation of renal fibroblasts to myofibroblasts.

[0017] 3. This invention explores the role and mechanism of GPER1 in activated macrophages in delaying the progression of renal fibrosis induced by unilateral ureteral obstruction (UUO) model, providing an important basis for the development of new targets and new drugs for anti-renal fibrosis progression. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1. GPER1 colocalizes with CD68-positive cells in CKD patients and UUO model mice; (A) Representative images of GPER1 and CD68 immunostaining in CKD patient kidney tissue. [GPER1 (red), CD68 (green), and DAPI (blue)]. (B) Representative images of GPER1 (red) and CD68 (green) immunostaining in female castrated mouse kidney tissue from the UUO model. CD68 is a marker for macrophages. Arrows mark the colocalization of GPER1 and CD68. Scale bar: 50 μm.

[0019] Figure 2. The GPER1 agonist G-1 blocks fibrosis progression in the unilateral ureteral obstruction model in female castrated mice; (A) Schematic diagram of the experimental design. (B) H&E, Masson's, and PAS staining of each group in the UUO model in female castrated mice. Scale bar: 50 μm. (C) Quantification of collagen area stained with Masson's staining (n = 5 per group). (D) Relative expression of Fn, Col1a1, and Col3a1 mRNA in each group (n = 8 in the Vehicle group, n = 9 in the G1 group). (E) Protein expression of Fn and α-SMA in each group (n = 3). Data are expressed as mean ± SEM. Statistical analysis was performed using two-way ANOVA with Tukey's test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

[0020] Figure 3. The GPER1 agonist G-1 inhibits macrophage infiltration in a unilateral ureteral obstruction model in female castrated mice; (A) Relative expression of Cd68 and Lyz2 mRNA in each group (Vehicle group, n = 8; G1 group, n = 9). (B) Immunohistochemical staining of total macrophages (F4 / 80), M1 macrophages (CD86), and M2 macrophages (CD206) in each group. Scale bar: 50 μm. (C) Quantitative analysis of immunohistochemical staining (n = 6 per group). Data are expressed as mean ± SEM. Two-way ANOVA with Tukey's test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

[0021] Figure 4. GPER1 agonist G-1 inhibits macrophage infiltration in a unilateral ureteral obstruction model in female castrated mice. (A) Relative mRNA expression of M1 macrophage-related proinflammatory genes Cd86, Nlrp3, Il-1β, Tnf-α, and Nos2 in each group (Vehicle group, n = 8; G1 group, n = 9). (B) Relative mRNA expression of M2 macrophage-related genes Mrc1, Il10, Arg-1, and Ccl17 in each group (Vehicle group, n = 8; G1 group, n = 9). (C) Dot plots of flow cytometry analysis of macrophages in each group. (D) Quantitative analysis of macrophages in each group (Vehicle group, n = 6; G1 group, n = 5). Data are expressed as mean ± SEM. Two-way ANOVA with Tukey's test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

[0022] Figure 5. GPER1 agonist G-1 inhibits M0 primary macrophage polarization; (A) Relative mRNA expression of related genes Ccl2, Cxcl10, Cxcl2, Ccl3, Cxcl1, Ccl5, Cxcl4, Il6, Cd86, Retn1a, Mrc1, Arg-1, and Il10 in BMDMs (n = 3). (B) Heat map of differentially expressed genes in M0 macrophages between the two groups. (C and D) Gene Ontology analysis of differentially expressed genes in M0 macrophages between the two groups.

[0023] Figure 6. GPER1 activation inhibits polarization of M0 macrophages to M1 macrophages and reduces the inflammatory response in co-cultured PTECs. (A-C) mRNA expression of macrophage markers in each group (n = 3). (D) Gene ontology analysis of differentially expressed genes in transcriptome data from M1 macrophages induced by LPS / INF stimulation and G-1 treatment. (E) Expression of TNF-α, IL-6, and G-CSF in the supernatants of macrophages from each group (n = 3). (F) Western blot analysis of proteins related to the MAPK pathway. (G) Schematic diagram of the transwell co-culture assay. (H) mRNA expression of PTECs-related inflammatory factors Ccl2, Cxcl10, Cxcl2, Ccl5, Tnf-α, Il-1β, Nlrp3, and Lcn2 (n = 3). (I) Gene ontology analysis of transcriptome data from renal tubular epithelial cells co-cultured with G-1-treated M1 macrophages. Values ​​are expressed as mean ± SEM. Two-way ANOVA with Tukey's test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0024] Figure 7. GPER1 activation inhibits polarization of M0 macrophages to M2 macrophages and downregulates the expression of pro-fibrotic factors in fibroblasts. (A) mRNA expression of M2 macrophage markers in each group (n = 3). (B) Gene Ontology analysis of differentially expressed genes in transcriptome sequencing of IL-4-stimulated M2 macrophages. (C) Western blot analysis of proteins related to the MAPK pathway. (D) Schematic diagram of the Trans-well co-culture assay. (E) mRNA expression of fibroblast-associated pro-fibrotic factors Tgf-β1, Fn, Col1a1, Col3a1, and Vim (n = 3). Values ​​are expressed as mean ± SEM. Two-way ANOVA with Tukey's test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

[0025] Figure 8. Schematic diagram of the mechanism by which GPER1 delays renal fibrosis induced by unilateral ureteral obstruction (UUO) model. DETAILED DESCRIPTION

[0026] The specific implementation methods provided by the present invention are described in detail below with reference to the examples.

[0027] Example:

[0028] 1. Experimental methods:

[0029] 1. Immunofluorescence staining was used to co-localize the expression of GPER1 in macrophages in the renal tissues of CKD patients and UUO model mice.

[0030] 2. In vivo experiments: (1) Wild-type female castrated mice were used to construct the UUO model and were treated with GPER1 agonist (G-1). According to the principle of randomized control, they were divided into sham operation group, sham operation + G-1 group, UUO group, and UUO + G-1 group; (2) Wild-type male mice were used to construct the UUO model and were treated with GPER1 agonist. The grouping was the same as (1); (3) CRISPR / Cas9 gene editing technology was used to construct Gper1 - / - Mouse model, divided into wild-type sham operation group, Gper1 - / - Sham operation group, wild type UUO group, Gper1 - / - UUO group. All the above groups were killed 7 days after UUO modeling, and renal tissues were collected. Histopathological staining (H&E, PAS, and MASSON) was performed to compare the degree of renal fibrosis. qPCR and Western blot were used to detect the changes in the mRNA and protein expression levels of fibrosis factors. Immunohistochemistry, qPCR, and flow cytometry were used to detect the expression of M1 and M2 macrophages and changes in inflammatory factors in renal tissues.

[0031] 3. In vitro experiments:

[0032] (1) Primary bone marrow macrophages (BMDMs) from wild-type male mice were extracted and cultured for 7 days to differentiate into M0 macrophages. G-1 was then added to intervene. Transcriptome sequencing and qPCR were used to observe the changes in the polarization of M0 to M1 and M2 macrophages and the changes in the MAPK signaling pathway. Gper1 was used to detect the changes in the MAPK signaling pathway. - / - Validated in mice.

[0033] (2) M0 BMDMs were stimulated with LPS / INF to induce differentiation into M1 type, G-1 was added for intervention, and co-cultured with primary renal tubular epithelial cells (PTECs). Changes in M1 type polarization markers, expression and secretion of inflammatory factors, and MAPK signaling pathways were detected to evaluate PTECs damage.

[0034] (3) M0 was stimulated with IL-4 to induce differentiation into M2 type, G-1 was added for intervention, and co-cultured with primary mouse kidney fibroblasts to detect changes in M2 polarization markers, expression and secretion of profibrotic factors, and MAPK signaling pathways, and to detect the expression of profibrotic factors in fibroblasts.

[0035] 2. Experimental results:

[0036] 1. In the kidney tissues of CKD patients and mice with unilateral ureteral obstruction, GPER1 was expressed in CD68-positive macrophages (Figure 1A-B).

[0037] 2. In vivo experiments: In wild-type female castrated mice, G-1 treatment reduced tubulointerstitial damage and extracellular matrix deposition, and decreased the mRNA and protein expression levels of pro-fibrotic factors (Col1a1, Col3a1, and Fn), suggesting that GPER1 activation can alleviate renal fibrosis (Figures 2A-E). Compared with the UUO group, the UUO+G-1 group showed decreased mRNA levels of macrophage-encoding genes Cd68 and Lyz2, and immunohistochemical staining showed a significant decrease in total macrophage (F4 / 80) infiltration (Figures 3A-C). Further analysis of the activated macrophage subsets M1 and M2 revealed that RT-PCR results indicated a decrease in the mRNA levels of M1 polarization markers (Cd86, Nlrp3, Il-1β, Tnf-α, Nos2) and M2 polarization markers (Arg-1, Mrc1, Il-10, Ccl17) (Figures 4A-B). Similarly, immunohistochemical staining demonstrated a significant decrease in both CD86-positive (M1) and CD206-positive (M2) cells after G-1 treatment (Figures 3B-C). Flow cytometry also confirmed a significant decrease in the total number of macrophages (F4 / 80+), M1 (F4 / 80, CD86+), and M2 (F4 / 80, CD206+) in the kidneys of G-1-treated UUO mice (Figures 4C-D). - / - Verification results in mice also showed that GPER1 activation can reduce macrophage infiltration and inhibit renal fibrosis.

[0038] 3. In vitro experiments:

[0039] (1) In the G-1 group (G-1 stimulated M0), the mRNA levels of M1 and M2 macrophage-related biomarker genes (Ccl2, Cxcl10, Cxcl2, Cxcl1, Ccl5, Cxcl4, Il6, Cd86, Retnla, Mrc1, Arg-1, and Il10) were significantly decreased, suggesting that G-1 inhibited the polarization of M0 to M1 or M2 phenotypes (Figure 5A). Gene transcriptome sequencing and Gene Ontology data analysis of differentially expressed genes showed that compared with the control group, the inflammatory response and immune response in the G-1 group were downregulated, and the activation of the MAPK signaling pathway was inhibited (Figure BD). These results indicate that GPER1 activation inhibits the phenotypic transformation of macrophages into inflammatory and profibrotic macrophages.

[0040] (2) The mRNA levels of M1 polarization markers (Ccl2, Il-6, Ccl3, Ccl7, Cxcl10, and Nos2) in the LPS / INF group (LPS / INF stimulated M0 type) were significantly increased compared with the DMSO group; while the levels of the above inflammatory factors in the LPS / INF+G-1 group were significantly decreased compared with the LPS / INF group (Figure 6A-C). Transcriptome data analysis showed that compared with the LPS / INF group, the LPS / INF+G-1 group inhibited the proliferation and differentiation of T lymphocytes (Figure 6D). Protein level verification showed that after G-1 intervention, the phosphorylation levels of MAPK / JNK, MAPK / ERK, and MAPK / P38 were all downregulated, suggesting that G-1 regulates the differentiation of M0 macrophages into M1 macrophage phenotype by inhibiting the MAPK signaling pathway, thereby reducing the secretion of inflammatory factors (Figure 6F). Further studies revealed that compared with the DMSO group, the LPS / IFN group showed significantly increased levels of TNFa, IL6, and G-CSF in the supernatant of M1 macrophages, and G-1 treatment downregulated these inflammatory factors (Figure 6E). To further investigate the cross-talk between macrophages and PTECs, PTECs were co-cultured with G-1-pretreated M1 macrophages using a transwell system (Figure 6G). The results showed that the mRNA expression of PTEC-associated inflammatory factors (Ccl2, Cxcl10, Cxcl2, Ccl5, Tnf-a, and Il-1β) was significantly downregulated, suggesting that GPER1 activation ameliorated the inflammatory activity of M1 macrophages, thereby exerting a protective effect against co-cultured PTECs (Figure 6H). Transcriptome sequencing analysis revealed that compared with the LPS / INF group, the LPS / INF+G-1 group showed downregulation of inflammatory and immune responses in renal tubular epithelial cells (Figure 6I). These results indicate that GPER1 activation inhibits the activation of the MAPK signaling pathway, inhibits the polarization of M0 to M1, reduces the inflammatory response, and reduces the expression of inflammatory factors in renal tubular epithelial cells, thereby delaying the progression of renal fibrosis.

[0041] (3) In the IL4 group (IL4 stimulated M0), the mRNA levels of M2 polarization markers (Mrc1, Tgfβ1, Retnla, Il10) were significantly increased compared with the DMSO group; while the levels of the above markers in the IL4+G-1 group were significantly decreased compared with the IL4 group (Figure 7A). Transcriptome differential expression data analysis suggested that compared with the IL4 group, the IL4+G-1 group inhibited the activation of the MAPK signaling pathway (Figure 7B). Protein level verification showed that after G-1 intervention, the phosphorylation levels of MAPK / JNK, MAPK / ERK and MAPK / P38 were all downregulated, suggesting that G-1 inhibited the differentiation into the pro-fibrotic macrophage phenotype by inhibiting the MAPK signaling pathway, thereby alleviating renal fibrosis (Figure 7C). Mouse fibroblasts were co-cultured with G-1-pretreated M2 macrophages. The results showed that the mRNA expression of mouse fibroblast-associated profibrotic factors (Tgf-β1, Fn, Col1a1, Col3a1, and Vim) was significantly suppressed, indicating that activated GPER1 can inhibit the secretion of profibrotic factors by fibroblasts co-cultured with macrophages (Figure 7D-E). These results indicate that GPER1 activation inhibits the polarization of M0 to M2 by inhibiting the activation of the MAPK signaling pathway, reducing the secretion of profibrotic factors and delaying renal fibrosis.

[0042] 3. Conclusion

[0043] Selective activation of GPER1 inhibits the MAPK pathway, suppressing the phenotypic transformation of M0 macrophages to pro-inflammatory (M1) and pro-fibrotic (M2) macrophages, alleviating the inflammatory response and delaying renal fibrosis (Figure 8).

[0044] The preferred embodiments of the present invention have been specifically described above, but the present invention is not limited to the described embodiments. Those skilled in the art may make various equivalent modifications or substitutions without departing from the spirit of the present invention. These equivalent modifications or substitutions are all included in the scope defined by the claims of this application.

Claims

1. Application of macrophage G protein-coupled estrogen receptor 1 as a target in the preparation of anti-renal fibrosis therapeutic drugs.

2. Application of reagents for activating macrophage G protein-coupled estrogen receptor 1 in the preparation of anti-renal fibrosis therapeutic drugs.

3. Use of the reagent for activating macrophage G protein-coupled estrogen receptor 1 according to claim 2 in the preparation of an anti-renal fibrosis therapeutic drug, characterized in that: The reagent for activating macrophage G protein-coupled estrogen receptor 1 is a macrophage G protein-coupled estrogen receptor 1 agonist.

4. Use of the reagent for activating macrophage G protein-coupled estrogen receptor 1 according to claim 3 in the preparation of an anti-renal fibrosis therapeutic drug, characterized in that: The macrophage G protein-coupled estrogen receptor 1 agonist is G-1.

5. Use of the reagent for activating macrophage G protein-coupled estrogen receptor 1 according to claim 2 in the preparation of an anti-renal fibrosis therapeutic drug, characterized in that: The reagent for activating macrophage G protein-coupled estrogen receptor 1 inhibits the phenotypic transformation of M0 macrophages to pro-inflammatory M1 macrophages and pro-fibrotic M2 macrophages by inhibiting the activation of MAPK signaling pathway.

6. Use of the reagent for activating macrophage G protein-coupled estrogen receptor 1 according to claim 2 in the preparation of an anti-renal fibrosis therapeutic drug, characterized in that: The reagent for activating macrophage G protein-coupled estrogen receptor 1 reduces inflammatory response, reduces the expression of inflammatory factors in renal tubular epithelial cells, reduces the secretion of profibrotic factors, and delays renal fibrosis.

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