Application of MC1R in renal injury treatment
By using MC1R gene or protein as a target and using MC1R promoters such as MS05 to activate MC1R, the treatment of acute renal injury is solved, the protection of renal tubular epithelial cells and inflammation regulation is achieved, and the severity and mortality of acute renal injury are reduced.
Patent Information
- Application Number
- CN202510720103.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-05-30
- Publication Date
- 2025-08-29
AI Technical Summary
Targeted treatment for acute renal injury in the prior art is relatively limited, and effective drugs or methods are lacking, resulting in worsening of the patient's condition and high mortality rate.
Using the MC1R gene or protein as a target, the MC1R activity or expression level is increased by screening or using MC1R promoters such as MC1R protein agonist MS05 to alleviate or treat acute renal injury.
MC1R agonist MS05 provides a new therapeutic strategy to reduce the severity of renal injury and mortality by activating MC1R.
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Abstract
Description
Technical Field
[0001] The present invention belongs to the field of biomedicine technology, and particularly relates to the application of MC1R in the treatment of renal injury. Background Art
[0002] Acute kidney injury (AKI) is a clinical syndrome characterized by a rapid deterioration of renal function over a short period of time, characterized by a rapid decrease in glomerular filtration rate (GFR), accumulation of metabolic waste products, and fluid and electrolyte imbalances. Approximately two million people die from AKI annually, with the mortality rate among ICU patients reaching as high as 60%. However, currently, targeted treatments for AKI are relatively limited, primarily focusing on symptomatic supportive care with targeted anti-inflammatory and anti-immune treatments or continuous renal replacement therapy to prevent AKI from becoming chronic or even worsening.
[0003] The melanocortin system comprises adrenocorticotropic hormone (ACT), melanocortin, and melanocortin receptors (MCRs). Melanocortins, through binding to MCRs, play an essential role in physiological processes such as melanogenesis, inflammation and immune regulation, glucocorticoid secretion, hemodynamics, natriuresis, energy metabolism homeostasis, and exocrine secretion. Melanocortin receptor 1 (MC1R), a member of the melanocortin system, is a classic G protein-coupled receptor that exerts its biological effects by binding to melanocortins. However, whether MC1R is involved in acute kidney injury (AKI), its role in AKI, and its specific mechanisms remain unclear. Summary of the Invention
[0004] In view of the problems and shortcomings in the prior art, the present invention aims to provide an application of MC1R in the treatment of renal injury.
[0005] To achieve the purpose of the invention, the technical solution adopted by the present invention is as follows:
[0006] In a first aspect, the present invention provides the use of MC1R gene or MC1R protein in screening candidate drugs for preventing, alleviating and / or treating renal injury.
[0007] According to the above application, preferably, the screening method is: using the MC1R gene or MC1R protein as a target, screening the drugs or reagents to be screened, and selecting drugs or reagents that can increase the activity of the MC1R protein, increase the content of the MC1R protein or increase the expression level of the MC1R gene as candidate drugs for preventing, alleviating or / and treating renal injury.
[0008] According to the above application, preferably, the renal injury is acute renal injury.
[0009] The second aspect of the present invention provides the use of the MC1R gene or MC1R protein in the preparation of a drug for preventing, alleviating or / and treating renal injury.
[0010] According to the above application, preferably, the renal injury is acute renal injury.
[0011] The third aspect of the present invention provides the use of an MC1R promoter in the preparation of a medicament for preventing, alleviating or / and treating renal injury.
[0012] According to the above application, preferably, the MC1R promoter is at least one of a substance that increases MC1R protein content, increases MC1R protein activity, and increases MC1R gene expression.
[0013] According to the above application, preferably, the substance that increases the activity of the MC1R protein is an MC1R protein agonist. More preferably, the MC1R protein agonist is MS05.
[0014] According to the above application, preferably, the substance that increases MC1R gene expression is an expression vector containing a nucleotide sequence encoding MC1R protein or a recombinant virus containing a nucleotide sequence encoding MC1R protein.
[0015] According to the above application, preferably, the renal injury is acute renal injury.
[0016] A fourth aspect of the present invention provides a drug containing the MC1R gene, the MC1R protein or the MC1R promoter.
[0017] According to the above-mentioned drug, preferably, the MC1R promoter is at least one of a substance that increases MC1R protein content, increases MC1R protein activity, and increases MC1R gene expression.
[0018] According to the above-mentioned drug, preferably, the substance that increases the activity of the MC1R protein is an MC1R protein agonist. More preferably, the MC1R protein agonist is MS05.
[0019] According to the above-mentioned drug, preferably, the substance that increases MC1R gene expression is an expression vector containing a nucleotide sequence encoding MC1R protein or a recombinant virus containing a nucleotide sequence encoding MC1R protein.
[0020] According to the above-mentioned medicine, preferably, the renal injury is acute renal injury.
[0021] According to the above-mentioned medicine, preferably, the medicine further contains a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier can be a diluent, excipient, filler, binder, wetting agent, disintegrant, absorption accelerator, adsorption carrier, surfactant or lubricant.
[0022] Compared with the prior art, the present invention has the following positive and beneficial effects:
[0023] The present invention uses mice with normal MC1R function and mice with MC1R dysfunction to explore the role and mechanism of MC1R in acute kidney injury and the role of MC1R agonist MS05 in acute kidney injury through experiments such as folic acid-induced acute kidney injury mouse model and in vitro culture of primary renal tubular epithelial cells. The study found that MC1R-mediated melanocortin signaling can regulate inflammation of renal tubular epithelial cells and polarization of macrophages, thereby affecting the extent of acute kidney injury. The agonist of MC1R protein (MS05) can exert a protective effect in acute kidney injury by activating MC1R. Therefore, MC1R can be used as a drug target in the treatment of acute kidney injury, which can provide a new strategy for the treatment of acute kidney injury. BRIEF DESCRIPTION OF THE DRAWINGS
[0024] Figure 1 Schematic diagram of breeding mice with congenital MC1R loss of function;
[0025] Figure 2 The figures represent the test results of kidney volume and kidney weight-to-body weight ratio of mice with acute kidney injury, where A represents the test result of kidney volume of mice and B represents the test result of kidney weight-to-body weight ratio of mice; WT represents mice with folic acid-induced acute kidney injury with normal MC1R function, and e / e represents mice with folic acid-induced acute kidney injury with MC1R dysfunction;
[0026] Figure 3 The PAS staining and renal injury score results of mouse kidney tissue; A is the PAS staining result of mouse kidney tissue, and B is the renal injury score result calculated based on the PAS staining of mouse kidney tissue; WT represents mice with folic acid-induced acute kidney injury with normal MC1R function, and e / e represents mice with folic acid-induced acute kidney injury with MC1R dysfunction;
[0027] Figure 4 represents the blood creatinine test results of mice with acute kidney injury; WT represents mice with normal MC1R function induced by folic acid, and e / e represents mice with MC1R dysfunction induced by folic acid;
[0028] Figure 5 The results of immunoblotting of kidney tissues of mice with acute kidney injury are shown in Figure 2. A is the band image of immunoblotting, and B is the quantitative analysis result of immunoblotting. WT represents mice with folic acid-induced acute kidney injury with normal MC1R function, and e / e represents mice with folic acid-induced acute kidney injury with MC1R dysfunction.
[0029] Figure 6 The immunofluorescence test results of kidney tissues of mice with acute kidney injury; WT indicates mice with folic acid-induced acute kidney injury with normal MC1R function, and e / e indicates mice with folic acid-induced acute kidney injury with MC1R dysfunction;
[0030] Figure 7 The blood creatinine test results of mice after MS05 administration, where WT represents mice with normal MC1R function induced by folic acid; e / e represents mice with MC1R dysfunction induced by folic acid; WT+MS05 represents mice with normal MC1R function administered with both folic acid and MS05; e / e+MS05 represents mice with MC1R dysfunction administered with both folic acid and MS05.
[0031] Figure 8 The results of PAS staining of mouse kidney tissue after MS05 administration, where WT represents mice with folic acid-induced acute kidney injury with normal MC1R function; e / e represents mice with folic acid-induced acute kidney injury with MC1R dysfunction; MS05 represents MS05 administration; Control represents no MS05 administration;
[0032] Figure 9 The results of immunoblotting of mouse kidney tissue after MS05 administration; A is the band image of immunoblotting, and B is the quantitative analysis result of immunoblotting; WT represents mice with folic acid-induced acute kidney injury with normal MC1R function; e / e represents mice with folic acid-induced acute kidney injury with MC1R dysfunction; WT+MS05 represents mice with normal MC1R function that were administered with both folic acid and MS05; e / e+MS05 represents mice with MC1R dysfunction that were administered with both folic acid and MS05.
[0033] Figure 10 The immunofluorescence test results of mouse kidney tissue after MS05 administration, where WT represents mice with folic acid-induced acute kidney injury with normal MC1R function; e / e represents mice with folic acid-induced acute kidney injury with MC1R dysfunction; MS05 represents MS05 administration; Control represents no MS05 administration;
[0034] Figure 11 The immunohistochemical test results of mouse kidney tissue after MS05 administration, where WT indicates mice with folic acid-induced acute kidney injury with normal MC1R function; e / e indicates mice with folic acid-induced acute kidney injury with MC1R dysfunction; MS05 indicates MS05 administration; Control indicates no MS05 administration;
[0035] Figure 12 These are optical microscopic images of renal tubular epithelial cells; WT indicates renal tubular epithelial cells with normal MC1R function, and e / e indicates renal tubular epithelial cells with ineffective MC1R function;
[0036] Figure 13The results of immunofluorescence detection of mucin E-cadherin and DAPI in renal tubular epithelial cells with normal MC1R function and renal tubular epithelial cells with MC1R dysfunction; WT indicates renal tubular epithelial cells with normal MC1R function, and e / e indicates renal tubular epithelial cells with MC1R dysfunction;
[0037] Figure 14 The results of cell viability assay using the MTT method on renal tubular epithelial cells with normal MC1R function and renal tubular epithelial cells with MC1R dysfunction after TNF-α stimulation for 24 h and 48 h. WT represents renal tubular epithelial cells with normal MC1R function treated with TNF-α, and e / e represents renal tubular epithelial cells with MC1R dysfunction treated with TNF-α.
[0038] Figure 15 The results of immunofluorescence detection of p-p65, KIM-1, MCP-1, and E-cadherin in renal tubular epithelial cells with normal MC1R function and renal tubular epithelial cells with MC1R dysfunction after TNF-α stimulation; WT indicates TNF-α-treated renal tubular epithelial cells with normal MC1R function, and e / e indicates TNF-α-treated renal tubular epithelial cells with MC1R dysfunction.
[0039] Figure 16 The immunoblot images show p-p65, KIM-1, MCP-1, and E-cadherin in renal tubular epithelial cells with normal MC1R function and renal tubular epithelial cells with MC1R dysfunction after TNF-α stimulation; WT indicates TNF-α-treated renal tubular epithelial cells with normal MC1R function, and e / e indicates TNF-α-treated renal tubular epithelial cells with MC1R dysfunction.
[0040] Figure 17 The results of immunoblotting quantitative analysis of p-p65, KIM-1, MCP-1, and E-cadherin in TNF-α-stimulated renal tubular epithelial cells with normal MC1R function and MC1R-invalidated renal tubular epithelial cells; WT indicates TNF-α-treated renal tubular epithelial cells with normal MC1R function, and e / e indicates TNF-α-treated renal tubular epithelial cells with MC1R-invalidated renal tubular epithelial cells.
[0041] Figure 18This is a cell viability assay of primary renal tubular epithelial cells from mice with normal MC1R function and mice with MC1R dysfunction after the combined action of TNF-α and MS05; WT represents renal tubular epithelial cells with normal MC1R function treated with TNF-α; WT+MS05 represents renal tubular epithelial cells with normal MC1R function treated with both TNF-α and MS05; e / e represents renal tubular epithelial cells with MC1R dysfunction treated with TNF-α; e / e+MS05 represents renal tubular epithelial cells with MC1R dysfunction treated with both TNF-α and MS05;
[0042] Figure 19 The figures are the results of immunoblotting of primary renal tubular epithelial cells from mice with normal MC1R function and mice with MC1R dysfunction after the combined action of TNF-α and MS05; A is the image of immunoblotting bands, and B is the quantitative analysis result of immunoblotting; WT represents TNF-α-treated renal tubular epithelial cells with normal MC1R function; WT+MS05 represents TNF-α and MS05-treated renal tubular epithelial cells with normal MC1R function; e / e represents TNF-α-treated renal tubular epithelial cells with MC1R dysfunction; e / e+MS05 represents TNF-α and MS05-treated renal tubular epithelial cells with MC1R dysfunction.
[0043] Figure 20 These are the results of immunofluorescence detection of primary renal tubular epithelial cells from mice with normal MC1R function and mice with MC1R dysfunction after the combined action of TNF-α and MS05; WT indicates TNF-α-treated renal tubular epithelial cells with normal MC1R function; MS05 indicates treatment with MS05; e / e indicates TNF-α-treated renal tubular epithelial cells with MC1R dysfunction; control indicates no MS05 treatment. DETAILED DESCRIPTION
[0044] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments. The following detailed description is exemplary and is intended to provide further explanation of the present invention.
[0045] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention belongs. It should be noted that the terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the exemplary embodiments according to the present invention. The experimental methods in the following examples, for which specific conditions are not specified, all adopt conventional techniques in the art or follow the conditions recommended by the manufacturer; the reagents or instruments used, for which the manufacturer is not specified, are all conventional products that can be purchased commercially.
[0046] Example 1: Construction of congenital MC1R loss-of-function mice
[0047] The genotype was Mc1r E / e MC1R heterozygous mice (purchased from The Jackson Laboratory, USA, strain: #000060) were backcrossed for multiple generations to breed offspring (such as Figure 1 The offspring genotype is: Mc1r E / E , Mc1r E / e and Mc1r e / e , among which the genotype is Mc1r e / e The mice are functionally characterized by MC1R dysfunction. Since the MC1R gene regulates the coat color of mice in a recessive manner, the genotype is Mc1r e / e The coat color of MC1R-deficient mice is yellow, and the genotype is Mc1r E / e Heterozygous mice and Mc1r E / E The coat color of the homozygous wild-type mice is black; moreover, these mice have no abnormalities in other physiological functions except for their genotype and hair color. E / e After multiple generations of backcrossing of MC1R heterozygous mice, the offspring mice with yellow fur color are congenital MC1R loss-of-function mice.
[0048] Example 2: Study on the role of MC1R in a mouse model of folic acid-induced acute kidney injury
[0049] A folic acid-induced mouse model of acute kidney injury with normal MC1R function and a folic acid-induced mouse model of acute kidney injury with MC1R dysfunction were established. The folic acid-induced mouse model of acute kidney injury with normal MC1R function was established by intraperitoneal injection of folic acid (250 mg / kg body weight) into mice with normal MC1R function. The folic acid-induced mouse model of acute kidney injury with MC1R dysfunction was established by intraperitoneal injection of folic acid (250 mg / kg body weight) into mice with MC1R dysfunction. The mice were observed for survival, weighed, and blood and urine samples were collected on days 0, 3, 7, and 14 after folic acid administration. Kidney tissue was also collected for subsequent testing. Blood samples were used for serum creatinine testing, and kidney tissue was used for kidney volume measurement, PAS staining, immunoblotting, and immunofluorescence analysis.
[0050] Statistics show that the activity of mice decreased significantly from 1 to 3 days after folic acid administration, and their bodies changed from stretched to curled up and maintained this position for a long time. Their food and water intake decreased, and their urination frequency and urine volume decreased. The mice gradually recovered from 7 to 14 days after folic acid administration.
[0051] The results of the mouse kidney volume and kidney weight-to-body weight ratio were as follows: Figure 2 As shown. Figure 2 It can be seen that the kidney volume of mice was the largest on the 3rd day after folic acid administration, and gradually decreased on the 7th and 14th days after folic acid administration. Moreover, the kidney volume of MC1R-deficient mice was larger than that of MC1R-normal mice at each time point (e.g. Figure 2 The kidney weight-to-body weight ratio was the highest on day 3 after folic acid administration and gradually decreased from day 7 to day 14 after folic acid administration. Moreover, the kidney weight-to-body weight ratio of MC1R-deficient mice was higher than that of MC1R-normal mice at each time point (as shown in Figure 2A). Figure 2 B).
[0052] The results of PAS staining and renal injury scoring of mouse kidney tissue (graded from 0 to 4 according to the degree of tubular necrosis, 0: no damage; 1: 10% necrosis; 2: 20% necrosis; 3: 35% necrosis; 4: 50% necrosis) are as follows Figure 3 As shown. Figure 3 It can be seen that mice with acute kidney injury experience tubular dilatation, brush border shedding, epithelial cell shedding, tissue inflammatory infiltration and cellular casts. Among them, tubular damage is most severe on the third day after folic acid administration, and the damage gradually recovers from the 7th to 14th day. Moreover, the kidney damage of MC1R-deficient mice is greater than that of MC1R-normal mice at all time points.
[0053] The results of the mouse blood creatinine test (creatinine determination kit, Nanjing Jiancheng, catalog number C011-2-1) are as follows Figure 4 As shown. Figure 4 It can be seen that the blood creatinine of mice with normal MC1R function and mice with MC1R dysfunction reached the highest on the third day after folic acid administration, and gradually decreased from the 7th to the 14th day. Moreover, the blood creatinine of MC1R dysfunction mice was higher than that of MC1R normal function mice at each time point.
[0054] The results of immunoblotting of mouse kidney tissue were as follows Figure 5 As shown. Figure 5It can be seen that the expression levels of NGAL, KIM-1, and apoptosis-related Cleaved caspase-3 proteins in the mouse kidney tissue were highest on the third day after folic acid administration, and gradually decreased from the seventh to the fourteenth day; the expression levels of inflammation-related proteins p-p65 and MCP-1 in the mouse kidney tissue gradually increased from the third to the seventh day after folic acid administration, and decreased on the fourteenth day; moreover, the expression levels of NGAL, KIM-1, apoptosis-related Cleaved caspase-3 protein, p-p65, and MCP-1 in the kidney tissue of MC1R-deficient mice at each time point were higher than those of mice with normal MC1R function.
[0055] The results of immunofluorescence detection of mouse kidney tissue are as follows Figure 6 As shown. Figure 6 It can be seen that NGAL is mainly expressed at the edges of dilated and damaged tubules, while iNOS and CD206 are mainly expressed in the tubular interstitium; the expression level of NGAL is highest on the third day after folic acid administration and gradually decreases from the 7th to the 14th day; moreover, the expression levels of NGAL and iNOS in MC1R-deficient mice are higher than those in MC1R-normal mice at all time points; the expression level of CD206 in MC1R-deficient mice is lower than that in MC1R-normal mice at all time points.
[0056] These results indicate that renal damage in mice with folic acid-induced acute kidney injury is most severe on day 3, with gradual recovery from day 7 to 14. MC1R dysfunction exacerbates folic acid-induced acute kidney injury in mice.
[0057] Example 3: Study on the protective effect of MC1R agonist MS05 on folic acid-induced acute kidney injury in mice
[0058] Mice with normal MC1R function and mice with MC1R dysfunction were given intraperitoneal injection of folic acid (injection dose of 250 mg / kg body weight) to construct an acute kidney injury mouse model. At the same time, mice with normal MC1R function and mice with MC1R dysfunction were given MS05 (injection dose of 0.7 μmol / kg body weight / d) for continuous subcutaneous injection for 14 days, and blood and urine samples of mice were collected on days 0, 3, 7, and 14 after folic acid administration, and mouse kidney tissue was taken for subsequent testing. Wherein, the blood samples of mice were used for blood creatinine detection, and the kidney tissue of mice was used for kidney tissue PAS staining detection, kidney tissue immunoblotting detection, immunofluorescence and immunohistochemistry detection. In addition, the present invention constructs a folic acid-induced MC1R function normal acute kidney injury mouse model and a folic acid-induced MC1R dysfunction acute kidney injury mouse model as a comparison. The construction method of the folic acid-induced MC1R function normal acute kidney injury mouse model and the folic acid-induced MC1R dysfunction acute kidney injury mouse model is the same as that in Example 2.
[0059] The results of the mouse blood creatinine test (creatinine determination kit, Nanjing Jiancheng, catalog number C011-2-1) are as follows Figure 7 As shown. Figure 7 It can be seen that after continuous injection of MS05, the blood creatinine of mice with normal MC1R function decreased at each time point, among which the decrease was the greatest on the third day after MS05 administration; while the blood creatinine of mice with MC1R dysfunction did not decrease significantly.
[0060] The results of PAS staining of mouse kidney tissue are as follows Figure 8 As shown. Figure 8 It can be seen that after continuous injection of MS05, the tubular dilatation and brush border shedding of mice with normal MC1R function were alleviated at each time point. The renal tissue damage of mice with normal MC1R function was most significantly alleviated on the third day after MS05 administration; while the renal tissue damage of mice with MC1R dysfunction did not change much after MS05 administration.
[0061] Therefore, based on the results of blood creatinine and renal tissue PAS staining, mice with acute kidney injury (AKI) with normal MC1R function and mice with AKI with MC1R dysfunction on day 3 after MS05 administration were selected for further study.
[0062] The results of immunoblotting of mouse kidney tissue were as follows Figure 9 As shown. Figure 9 It can be seen that after 3 days of continuous subcutaneous injection of MS05, the expression levels of iNOS, p-p65, NGAL, MCP-1, and KIM-1 proteins in mice with normal MC1R function were significantly decreased compared with those in mice without medication, and the expression level of CD206 protein was significantly increased compared with those in mice without medication; while there was no obvious change in mice with MC1R dysfunction after administration of MS05.
[0063] The results of immunofluorescence detection of mouse kidney tissue are as follows Figure 10 As shown. Figure 10 It can be seen that after 3 consecutive days of MS05 injection, the expression of NGAL, MCP-1 and iNOS in mice with normal MC1R function decreased compared with the non-drug group, while the expression of CD206 increased compared with the non-drug group; after MS05 administration to mice with MC1R dysfunction, there was no obvious change in NGAL, iNOS and CD206.
[0064] The results of immunohistochemical examination of mouse kidney tissue are as follows Figure 11 As shown. Figure 11 It can be seen that after 3 consecutive days of MS05 injection, the expression of NGAL and MCP-1 in mice with normal MC1R function decreased compared with the non-drug group, while the changes of NGAL and MCP-1 in mice with MC1R dysfunction after MS05 administration were not obvious.
[0065] The above results indicate that the MC1R agonist MS05 has a protective effect on folate-induced acute kidney injury in WT mice.
[0066] Example 4: Study on the effect of MC1R dysfunction on mouse renal tubular epithelial cell damage
[0067] 1. Morphological observation of renal tubular epithelial cells with normal MC1R function and MC1R dysfunction and immunofluorescence detection of mucin E-cadherin and DAPI in the cells
[0068] Mice with normal MC1R function and mice with MC1R dysfunction were killed and their kidneys were removed. The cortical renal tubular segments were separated and screened and inoculated into culture dishes. The renal tubular epithelial cells crawled out of the renal tubular segments and grew adherently to the wall. After the renal tubular segments were cultured in the culture dishes for 48 hours, the culture medium of the renal tubular epithelial cells with normal MC1R function and the renal tubular epithelial cells with MC1R dysfunction were changed, and then the cells were observed under an optical microscope. At the same time, immunofluorescence detection of mucin E-cadherin and DAPI in the renal tubular epithelial cells with normal MC1R function and the renal tubular epithelial cells with MC1R dysfunction was performed.
[0069] Optical microscopy of renal tubular epithelial cells Figure 12 shown.
[0070] Depend on Figure 12 It can be seen that the primary MC1R normal renal tubular epithelial cells and MC1R dysfunctional renal tubular epithelial cells grown in the renal tubular tissue as the center have spindle-shaped cell morphology, grow adherently, and have the same proliferation rate.
[0071] The immunofluorescence detection results of mucin E-cadherin and DAPI in renal tubular epithelial cells with normal MC1R function and MC1R dysfunction are as follows Figure 13 shown.
[0072] Depend on Figure 13 It can be seen that the present invention extracts primary renal tubular epithelial cells from mice with normal MC1R function and primary renal tubular epithelial cells from mice with MC1R dysfunction with high purity.
[0073] 2. Cell viability assay of renal tubular epithelial cells with normal MC1R function and renal tubular epithelial cells with MC1R dysfunction after TNF-α stimulation
[0074] Mice with normal MC1R function and mice with MC1R dysfunction were killed and their kidneys were removed. Cortical renal tubular segments were isolated and screened and inoculated into culture dishes. When the renal tubular epithelial cells crawled out of the renal tubular segments and grew to 70% confluence, they were stimulated with 20 ng / mL of TNF-α. The cell activity of the renal tubular epithelial cells with normal MC1R function and the renal tubular epithelial cells with MC1R dysfunction after TNF-α stimulation for 24 hours and 48 hours was detected by MTT method. The test results are as follows: Figure 14 As shown in .
[0075] Depend on Figure 14 It can be seen that the activity of renal tubular epithelial cells gradually decreased after TNF-α stimulation, and the activity of renal tubular epithelial cells with MC1R dysfunction decreased more than that of renal tubular epithelial cells with normal MC1R function at each time point.
[0076] Immunofluorescence detection of p-p65, KIM-1, MCP-1 and E-cadherin in MC1R normal renal tubular epithelial cells and MC1R dysfunction renal tubular epithelial cells after TNF-α stimulation for 0h, 12h, 24h and 48h. Figure 15 shown.
[0077] Depend on Figure 15 It can be seen that p-p65 is expressed in the cell nucleus, KIM-1, MCP-1 and E-cadherin are expressed in the cytoplasm. After TNF-α stimulation, the expression of damage indicators p-p65, KIM-1 and MCP-1 gradually increased, and the expression of E-cadherin gradually decreased. At each time point, the damage of renal tubular epithelial cells in MC1R-deficient mice was more severe than that in mice with normal MC1R function.
[0078] Immunoblotting was performed to detect p-p65, KIM-1, MCP-1, and E-cadherin in renal tubular epithelial cells with normal MC1R function and renal tubular epithelial cells with MC1R dysfunction after TNF-α stimulation for 0 h, 12 h, 24 h, and 48 h. Figure 16 and Figure 17 shown.
[0079] Depend on Figure 16 and Figure 17 It can be seen that after TNF-α stimulation, the expression of renal tubular epithelial cell damage indicators p-p65, KIM-1, and MCP-1 proteins gradually increased, and the expression of E-cadherin and p-GSK3β proteins gradually decreased. At each time point, the renal tubular epithelial cell damage in MC1R-deficient mice was more severe than that in mice with normal MC1R function.
[0080] The above results indicate that MC1R dysfunction can lead to aggravated damage to renal tubular epithelial cells during inflammatory stimulation.
[0081] Example 5: Study on the protective effect of MC1R agonist MS05 on mouse renal tubular epithelial cells
[0082] Primary renal tubular epithelial cells from mice with normal MC1R function and primary renal tubular epithelial cells from mice with MC1R dysfunction were treated with TNF-α (20 ng / mL) and MS05 (10 -7 M) were used for experiments after 48 h of co-action.
[0083] The MTT method was used to detect the cell viability of primary renal tubular epithelial cells from mice with normal MC1R function and mice with MC1R dysfunction after TNF-α and MS05 co-treatment for 48 hours; at the same time, primary renal tubular epithelial cells from mice with normal MC1R function and mice with MC1R dysfunction after TNF-α (20 ng / mL) treatment alone for 48 hours were used as a comparison. Figure 18 As shown in .
[0084] Depend on Figure 18 It can be seen that after 48 hours of co-action of TNF-α and MS05, MS05 increased the activity of renal tubular epithelial cells with normal MC1R function, while the activity of renal tubular epithelial cells with MC1R dysfunction did not change much.
[0085] Western blotting was used to detect the expression levels of p-p65, KIM-1, MCP-1 and E-cadherin in primary renal tubular epithelial cells of MC1R normal mice and primary renal tubular epithelial cells of MC1R dysfunction mice after 48 hours of combined treatment with TNF-α and MS05; at the same time, primary renal tubular epithelial cells of MC1R normal mice and primary renal tubular epithelial cells of MC1R dysfunction mice after 48 hours of treatment with TNF-α (20 ng / mL) alone were used as a comparison. Figure 19 shown.
[0086] Depend on Figure 19 It can be seen that after the administration of MS05, the protein expression of damage indicators p-p65, KIM-1, and MCP-1 in the renal tubular epithelial cells with normal MC1R function was significantly decreased compared with the non-drug group, and the protein expression of E-cadherin was significantly increased compared with the non-drug group; after the administration of MS05, the protein expression of damage indicators p-p65, KIM-1, MCP-1, and E-cadherin in the renal tubular epithelial cells with ineffective MC1R did not change significantly compared with the non-drug group.
[0087] The expression levels of p-p65, KIM-1, MCP-1 and E-cadherin in primary renal tubular epithelial cells of MC1R normal mice and primary renal tubular epithelial cells of MC1R dysfunction mice after 48 hours of combined treatment with TNF-α and MS05 were detected by cell immunofluorescence. At the same time, primary renal tubular epithelial cells of MC1R normal mice and primary renal tubular epithelial cells of MC1R dysfunction mice after 48 hours of treatment with TNF-α (20 ng / mL) alone were used as a comparison. The test results are shown in Figure 2. Figure 20 shown.
[0088] Depend on Figure 20It can be seen that after the administration of MS05, the expression of damage indicators p-p65, KIM-1, and MCP-1 in the renal tubular epithelial cells of mice with normal MC1R function was significantly decreased compared with the non-drug group, and the expression of E-cadherin was significantly increased compared with the non-drug group; the expression of damage indicators p-p65, KIM-1, MCP-1 and E-cadherin in the renal tubular epithelial cells of mice with MC1R dysfunction did not change significantly compared with the non-drug group.
[0089] The above results indicate that the MC1R agonist MS05 has a protective effect on renal tubular epithelial cells of WT mice.
[0090] This study, using mice with normal and non-functional MC1R, explored the role and mechanism of MC1R in acute kidney injury, as well as the effect of the MC1R agonist MS05, using a folic acid-induced acute kidney injury mouse model and in vitro cultured primary renal tubular epithelial cells. The study demonstrated that MC1R-mediated melanocortin signaling can regulate inflammation in renal tubular epithelial cells and polarization of macrophages through the MC1R / NF-κB pathway, thereby affecting the severity of acute kidney injury. MS05 may exert a protective effect in acute kidney injury by activating MC1R. This study suggests a novel concept for the treatment of acute kidney injury by targeting the specific MC1R in renal tubular epithelial cells.
[0091] Finally, it should be noted that the above embodiments are merely preferred embodiments of the present invention and are not intended to limit the present invention in any other manner. Any person skilled in the art may utilize the above technical content as a guide to make changes or modifications. These are equivalent embodiments of equivalent modifications. However, any simple modifications, equivalent changes, and modifications to the above embodiments that do not depart from the technical concept of the present invention and are based on the technical essence of the present invention remain within the scope of protection of the claims of the present invention.
Claims
1. Application of MC1R gene or MC1R protein in screening candidate drugs for preventing, alleviating and / or treating renal injury.
2. The use according to claim 1, characterized in that The screening method is: using the MC1R gene or MC1R protein as a target, screening the drugs or reagents to be screened, and selecting drugs or reagents that can increase the activity of the MC1R protein, increase the content of the MC1R protein, or increase the expression level of the MC1R gene as candidate drugs for preventing, alleviating, or / and treating renal injury.
3. Use of the MC1R gene or MC1R protein in the preparation of drugs for preventing, alleviating and / or treating renal injury.
4. Use of MC1R promoters in the preparation of drugs for preventing, alleviating or / and treating renal injury.
5. The use according to claim 4, characterized in that The MC1R promoter is at least one of a substance that increases the MC1R protein content, increases the MC1R protein activity, and increases the MC1R gene expression.
6. The use according to claim 5, characterized in that The substance that increases the activity of MC1R protein is an MC1R protein agonist; the substance that increases the expression of MC1R gene is an expression vector containing a nucleotide sequence encoding MC1R protein or a recombinant virus containing a nucleotide sequence encoding MC1R protein.
7. The use according to claim 6, characterized in that The MC1R protein agonist is MS05.
8. A drug, characterized in that The medicine contains MC1R gene, MC1R protein or MC1R promoter.
9. The drug according to claim 8, characterized in that The MC1R promoter is at least one of a substance that increases the MC1R protein content, increases the MC1R protein activity, and increases the MC1R gene expression.
10. The drug according to claim 9, characterized in that The substance that increases the activity of MC1R protein is an MC1R protein agonist; the substance that increases the expression of MC1R gene is an expression vector containing a nucleotide sequence encoding MC1R protein or a recombinant virus containing a nucleotide sequence encoding MC1R protein.