Use of butyrolactone i in the preparation of a medicament for the treatment of chronic kidney disease

Abstract

The application belongs to the technical field of medicine, and discloses application of butyrolactone I in preparation of a medicine for treating chronic kidney disease, wherein the butyrolactone I has the structure shown in formula 1. The application provides a brand-new use of the known compound butyrolactone I. By evaluating the progress of treating chronic kidney disease of the compound butyrolactone I, it is found that the compound can effectively inhibit the progress of chronic kidney disease of mice, improve kidney function, inhibit oxidative stress damage, reduce secretion of inflammatory factors and the like in a mouse chronic kidney disease model, thereby reducing kidney fibrosis, blocking the progress of acute kidney injury to chronic kidney disease, and the compound can be used as a lead compound for developing a medicine for treating chronic kidney disease.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of medicine, and more particularly relates to an application of butyrolactone I in preparation of a medicine for treating chronic kidney disease, which provides a lead compound for development of a medicine for treating kidney disease. BACKGROUND

[0002] Chronic kidney disease (CKD) is a general term for a heterogeneous disease affecting the structure and function of the kidney, and is specifically manifested as persistent kidney injury (appearance of proteinuria) or decreased kidney function (glomerular filtration rate [GFR] <60 mL / min / 1.73 m 2 The main factors leading to CKD include drugs, poisons, acute kidney injury caused by kidney obstruction (among them, the known inducing factors of kidney obstruction are various, including ureteral calculi, trauma, stenosis and tumors, etc.; for example, unilateral ureteral obstruction (UUO) is a relatively common form of upper urinary tract obstruction, which is usually caused by ureteral calculi, trauma, stenosis and / or tumors), persistent hypertension, diabetes, interstitial nephritis, pyelonephritis, autoimmune diseases, polycystic kidney disease, etc. CKD is one of the important factors leading to high morbidity and mortality of various non-communicable diseases, and poses a serious threat to the health of patients. Patients with end-stage kidney disease can only be cured through kidney transplantation, and the lack of kidney sources and high treatment costs bring heavy burdens to the families and society of patients (reference: Drawz P, Rahman M. Chronic kidney disease [J]. Ann Intern Med.). According to the different causes of chronic kidney disease, drugs for symptomatic treatment are used to reduce the load on the kidney and slow down the development of kidney disease, but as a whole, the currently clinically effective drugs for controlling the progression of CKD are limited, and new drugs need to be developed (reference: Noone D, Licht C. Chronic kidney disease: a new look at pathogenetic mechanisms and treatment options [J]. Pediatr Nephrol).

[0003] Butyrolactone I (BLI for short, also known as olomoucine) is the first butyrolactone compound found in Aspergillus terreus. BLI is a widely studied cyclin-dependent kinase (CDK) inhibitor with anti-tumor and anti-inflammatory effects. There are also reports that BLI is an effective alpha-glucosidase that improves type 2 diabetes by regulating the intestinal flora of db / db mice. Currently, there are only research reports on BLI, and there is no related drug or combination therapy on the market, while on the other hand, the development of CKD treatment drugs has important practical significance. SUMMARY

[0004] In view of the above defects or improvement needs of existing treatment drugs, the purpose of the present application is to provide the use of butyrolactone I in the preparation of a drug for treating chronic kidney disease, providing a new use for the known compound butyrolactone I. By evaluating the progress of the compound butyrolactone I in treating chronic kidney disease, it is found that the compound can effectively inhibit the progression of chronic kidney disease in mice, improve renal function, inhibit oxidative stress damage, reduce inflammatory factor secretion, etc. in a mouse chronic kidney disease model, thereby reducing kidney fibrosis and blocking the progression of acute kidney injury to chronic kidney disease, which can be used as a lead compound for the development of drugs for treating chronic kidney disease. And since butyrolactone I can be obtained from Aspergillus terreus metabolites, the present application is equivalent to finding an effective drug for treating kidney disease from microbial secondary metabolites. In view of the successful experience and important position of natural products in the field of drug development, the present application has important value.

[0005] To achieve the above-mentioned purpose, according to one aspect of the present application, the use of butyrolactone I (Butyrolactone I) in the preparation of a drug for treating chronic kidney disease is provided, characterized in that the butyrolactone I has the following formula 1 structure:

[0006]

[0007]

[0008] As a further preferred embodiment of the present application, the chronic kidney disease is specifically progressive renal function damage caused by renal obstruction, drug-induced renal damage, and / or toxicant-induced renal damage.

[0009] According to another aspect of the present application, the use of butyrolactone I (Butyrolactone I) in the preparation of a drug for treating chronic kidney disease by inhibiting renal cell ferroptosis is provided, characterized in that the butyrolactone I has the following formula 1 structure:

[0010]

[0011] According to another aspect of the present application, the present application provides a use of butyrolactone I in the preparation of a drug for treating chronic kidney disease by inhibiting the JAK-STAT signaling pathway, characterized in that the butyrolactone I has the following formula 1 structure:

[0012]

[0013] Through the above technical scheme conceived by the present application, compared with the prior art, the present application evaluates the activity of the compound butyrolactone I in treating chronic kidney disease, finds that the compound can effectively inhibit the progression of chronic kidney disease in mice, improve kidney function, inhibit oxidative stress damage, reduce inflammatory factor secretion, and other activities in a mouse chronic kidney disease model, thereby reducing kidney fibrosis and blocking the progression of acute kidney injury to chronic kidney disease, and can be used as a lead compound for developing a drug for treating chronic kidney disease. As exemplified in the examples below, the present application evaluates the activity of the compound butyrolactone I in treating chronic kidney disease caused by folic acid or unilateral kidney obstruction, finds that the compound can effectively improve kidney function, inhibit oxidative stress damage, reduce inflammatory factor secretion, and other activities, thereby reducing kidney fibrosis and blocking the progression of acute kidney injury to chronic kidney disease, and can be used as a lead compound for developing a drug for treating kidney injury.

[0014] As exemplified in the examples below, the present application conducts in-depth research on kidney injury caused by folic acid or unilateral kidney obstruction, finds that butyrolactone I has a significant therapeutic effect on chronic kidney disease models caused by kidney injury, and explores the therapeutic effect, and finds that butyrolactone I can particularly inhibit renal cell ferroptosis, and can also inhibit the JAK-STAT signaling pathway.

[0015] The present application is particularly applicable to the treatment of drugs for chronic kidney disease caused by toxic substances, drugs, renal obstruction (inducing factors can be ureteral stones, trauma, stenosis, tumors) and the like, because the kidney disease model caused by folic acid or unilateral renal obstruction established in the present study is two classic models of kidney disease research, has good repeatability, and can summarize most human chronic kidney disease phenotypes (Hammad FT. The long-term renal effects of short periods of unilateral ureteral obstruction. Int J Physiol Pathophysiol Pharmacol. 2022 Apr 15; 14(2): 60-72.; Yan LJ. Folic acid-induced animal model of kidney disease. Animal models and experimental medicine 2021; 4: 329-42; Bao-Y-W, Yuan-Y, Chen-J-H, Lin-W-Q. Kidney disease models: tools to identify mechanisms and potential therapeutic targets. J-Zoological Research. 2018; -39: -72.). Both models confirm the therapeutic effect of butenolide I on kidney damage (that is, the present application confirms that BLI can be applicable to kidney damage caused by drugs, toxic substances and / or progressive kidney function damage caused by kidney damage caused by various causes of renal obstruction such as chronic kidney disease), which lays a foundation for the clinical application of BLI.

[0016] Butenolide I has been discovered for more than 40 years, and research before 2010 focused on cell cycle arrest, cell division regulation. In the past 10 years, there have been reports of anti-inflammatory, intestinal flora regulation, and lipid-lowering, and no other research. The present application first reports the effect of butenolide I on a mouse model of chronic kidney disease, and for the first time, it is found that butenolide I can target JAK1, inhibit the JAK-STAT and its downstream ferroptosis signaling pathway, thereby blocking the progression of renal fibrosis, improving kidney function, and blocking the progression of acute kidney injury to chronic kidney disease. And because the prior art has confirmed that butenolide I can be obtained in large quantities through fermentation strategies, it greatly facilitates the application of butenolide I in the preparation of drugs for treating chronic kidney disease. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 for the protective effect of BLI on folic acid-induced kidney damage in mice; wherein:

[0018] Figure 1 In this context, A corresponds to the mouse kidney index. Among the control group (Con, solvent: NaHCO3 solution), model group (FA-Model, folic acid 250 mg / kg), low-dose group (FA-BLI-L, folic acid 250 mg / kg + 50 mg / kg BLI), high-dose group (FA-BLI-H, folic acid 250 mg / kg + 100 mg / kg BLI), and positive control group (FA-Tel, folic acid 250 mg / kg + telmisartan 5 mg / kg), BLI reduced the mouse kidney index, showing a superior effect compared to the positive control drug telmisartan (abbreviated as Tel in this invention).

[0019] Figure 1 In the figure, B represents HE-stained images of mouse kidney tissue and the corresponding statistical results of kidney injury scores. The results showed that compared with the model group, BLI significantly reduced renal tubular injury, reduced inflammatory cell infiltration, and improved the pathological structure of kidney tissue in mice, with effects comparable to the positive control drug Tel.

[0020] Figure 1 C and Figure 1 The numbers D in the table correspond to the levels of blood urea nitrogen and creatinine in mouse serum, as detected by the kit. The levels of creatinine and urea nitrogen in the serum of mice in the model group were significantly increased, while those in the BLI-treated group were significantly decreased, with effects comparable to Tel, suggesting that BLI can improve renal function in mice.

[0021] Figure 1 E and Figure 1 The F-corresponding kit was used to detect urinary creatinine and microalbumin levels in mice. In the model group, both microalbumin and creatinine levels in the urine were significantly elevated. BLI treatment significantly reduced urinary creatinine and microalbumin levels in mice, with effects comparable to Tel, suggesting that BLI can improve renal function in mice.

[0022] Figure 1 G and Figure 1 The H in the figure corresponds to the detection of KIM-1 and NGAL, biomarkers of kidney injury in mice, respectively. RT-qPCR was used to detect the mRNA levels of KIM-1 and NGAL, biomarkers of kidney injury in mice. The expression levels of KIM-1 and NGAL mRNA were significantly increased in the model group mice. After BLI administration, the expression levels of KIM-1 and NGAL mRNA in the kidney tissue of mice were significantly decreased, with an effect comparable to Tel, suggesting that BLI can significantly reduce kidney injury in mice.

[0023] Figure 1 I in Figure 1 J in Figure 1 K in Figure 1 L and Figure 1The "M" in the table corresponds to the levels of inflammatory cytokines interleukin-1β (IL-1β), interleukin-6 (IL-6), transforming growth factor-β (TGF-β), monocyte chemoattractant protein-1 (MCP-1), and tumor necrosis factor-α (TNF-α) in mouse kidney tissue detected by the kit. The results showed that the levels of inflammatory cytokines IL-1β, IL-6, MCP-1, TNF-α, and TGF-β were significantly upregulated in the kidney tissue of the model group mice. Treatment with BLI significantly reduced the levels of inflammatory cytokines in the kidney tissue of mice, with the low-dose BLI group showing a better effect than Tel, suggesting that BLI can reduce the levels of inflammatory cytokines in kidney tissue.

[0024] Figure 1 N and Figure 1 The "O" in the reagent kit corresponds to the levels of malondialdehyde (MDA) and glutathione (GSH) in mouse kidney tissue homogenates. In the model group, GSH levels were significantly decreased and MDA levels were significantly increased in mouse kidney tissue homogenates. After BLI treatment, GSH levels were significantly increased and MDA levels were significantly decreased in mouse kidney tissues, suggesting that BLI can reduce oxidative stress in kidney tissues, with an effect comparable to that of positive control drugs.

[0025] Figure 1 The "P" in the figure corresponds to the detection of fibrosis-related proteins in mouse kidney tissue. Western blot was used to detect the expression levels of fibrosis-related proteins, namely type I collagen (Col-I), type III collagen (Col-III), type IV collagen (Col-IV), fibronectin (Fn), and α-smooth muscle actin (α-SMA), in mouse kidney tissue. The results showed that the expression of Col-I, Col-III, Col-IV, Fn, and α-SMA proteins in the kidney tissue of the model group mice was significantly upregulated. Compared with the model group, the expression of Col-I, Col-III, Col-IV, Fn, and α-SMA in the kidney tissue of mice after BLI administration was significantly reduced, and the effect was better than that of the positive drug Tel, suggesting that fibrosis in mouse kidney tissue was reduced.

[0026] Figure 2 The protective effect of BLI against renal injury induced by unilateral renal obstruction in mice; among which:

[0027] Figure 2A corresponds to the mouse kidney index. The control group (Con, sham operation group), model group (UUO-Model, unilateral kidney obstruction operation in mice), low-dose group (UUO-BLI-L, unilateral kidney obstruction operation in mice + 50 mg / kg BLI), high-dose group (UUO-BLI-H, unilateral kidney obstruction operation in mice + 100 mg / kg BLI) and positive drug group (UUO-Tel, unilateral kidney obstruction operation in mice + Telmisartan 5 mg / kg), BLI and Tel can reduce the kidney index of mice, and the high-dose group of BLI has significant difference, and the effect of BLI is better than that of the positive drug Tel.

[0028] Figure 2 B is the HE staining picture of mouse kidney tissue and the corresponding kidney injury score statistics. The results show that compared with the model group, BLI can significantly reduce and improve mouse tubular injury, reduce inflammatory cell infiltration, and improve the pathological structure of kidney tissue, and the effect is equivalent to that of the positive drug Tel.

[0029] Figure 2 C and Figure 2 D corresponds to the kit detection of mouse serum urea nitrogen and creatinine levels. The creatinine and urea nitrogen content in the serum of the model group mice is significantly increased, and the BLI treatment can reduce the creatinine level in the serum of mice, and the effect is better than that of the positive drug Tel, which indicates that BLI can improve the kidney function of mice.

[0030] Figure 2 E and Figure 2 F respectively corresponds to the kit detection of mouse urine creatinine and microalbumin levels. The creatinine and microalbumin in the urine of the model group mice are significantly increased, and the BLI treatment can reduce the creatinine content in the urine of mice, and the effect is slightly better than that of the positive drug, but there is no significant difference between the positive drug Tel.

[0031] Figure 2 G and Figure 2 H respectively corresponds to the detection of mouse kidney injury markers. RT-qPCR detects the mRNA levels of mouse kidney injury markers KIM-1 and NGAL. The expression levels of KIM-1 and NGAL mRNA in the model group mice are significantly increased, and the expression levels of KIM-1 and NGAL mRNA in the kidney tissue of mice are significantly reduced after the administration of BLI and Tel. The effect of BLI in down-regulating KIM1 is better than that of the positive drug, which indicates that BLI can significantly reduce the kidney injury of mice.

[0032] Figure 2 I, Figure 2 J, Figure 2 K, Figure 2 L and Figure 2The numbers M in the table correspond to the levels of inflammatory cytokines interleukin-1β (IL-1β), interleukin-6 (IL-6), transforming growth factor-β (TGF-β), monocyte chemoattractant protein-1 (MCP-1), and tumor necrosis factor-α (TNF-α) in mouse kidney tissue detected by the kit. The results showed that the levels of inflammatory cytokines IL-1β, IL-6, MCP-1, TNF-α, and TGF-β were significantly upregulated in the kidney tissue of the model group mice. Treatment with BLI significantly reduced the levels of inflammatory cytokines in the kidney tissue of mice, with the high-dose BLI group showing a significantly better effect than the positive control drug Tel, suggesting that BLI can reduce the levels of inflammatory cytokines in kidney tissue.

[0033] Figure 2 N and Figure 2 The "O" in the reagent kit corresponds to the levels of malondialdehyde (MDA) and glutathione (GSH) in mouse kidney tissue homogenates. In the model group, GSH levels were significantly decreased and MDA levels were significantly increased in mouse kidney tissue homogenates. After BLI treatment, GSH levels were significantly increased and MDA levels were significantly decreased in mouse kidney tissues, suggesting that BLI can reduce oxidative stress in kidney tissues, with an effect comparable to that of positive control drugs.

[0034] Figure 3 The "P" in the figure corresponds to the detection of fibrosis-related proteins in mouse kidney tissue. Western blot was used to detect the expression levels of fibrosis-related proteins, namely, type I collagen (Col-I), type III collagen (Col-III), type IV collagen (Col-IV), fibronectin (Fn), and α-smooth muscle actin (α-SMA), in mouse kidney tissue. The results showed that the expression of Col-I, Col-III, Col-IV, Fn, and α-SMA proteins in the kidney tissue of the model group mice was significantly upregulated. Compared with the model group, the expression of Col-I, Col-III, Col-IV, Fn, and α-SMA proteins in the kidney tissue of mice after BLI administration was significantly reduced, suggesting that fibrosis in mouse kidney tissue was reduced, and the effect of BLI was comparable to that of the positive control drug Tel.

[0035] Figure 3 BLI inhibits renal cell ferroptosis; among which:

[0036] Figure 3 In the study of folic acid-induced chronic kidney disease model, RNA-seq was used to detect changes in mRNA expression in the kidney tissues of mice in each experimental group. The results showed that, compared with the model group, BLI significantly regulated the mRNA expression of genes related to three pathways closely related to ferroptosis: fatty acid metabolism, glutamate metabolism, and ferroptosis.

[0037] Figure 3B corresponds to RT-qPCR detection of ferroptosis-related gene expression. The results show that BLI can significantly reverse the mRNA expression change trend induced by folate.

[0038] Figure 3 C corresponds to the staining of kidney tissue samples from each mouse in the FA group (including: Con, FA-Model, FA-BLI-L, FA-BLI-H, FA-Tel) and the UUO group (including: Con, UUO-Model, UUO-BLI-L, UUO-BLI-H, UUO-Tel) to observe the levels of lipid peroxidation product 4HNE, iron ion, ferritin heavy chain 1 (FTH1), and transferrin receptor protein 1 (TfR1) in the mouse kidney tissue. The results show that compared with the FA and UUO model groups, BLI administration can significantly reduce the level of lipid peroxidation product 4HNE in both models, with a better effect in the FA model than in the positive drug Tel. Enhanced Prussian blue staining shows that BLI can significantly reduce the accumulation of iron ions in the kidney tissue in both models, with a better effect in the UUO model than in the positive drug Tel. FTH1 and TfR1 immunohistochemical staining results show that BLI can significantly reduce the levels of ferritin and transferrin in the kidney tissue, and the effect of BLI is better than that of the positive drug Tel.

[0039] Figure 4 D corresponds to the collection of mouse kidney tissue samples to extract tissue proteins, and western blot detection of the expression levels of marker proteins in the ferroptosis process, such as glutathione peroxidase 4 (GPX4), solute carrier family 7 member 11 (SLC7A11), ferredoxin 1 (FSP1), FTH1, ferritin light chain (FTL), and TfR1. The results show that in the FA and UUO models, BLI can significantly up-regulate the levels of GPX4, SLC7A11, and FSP1, while down-regulate FTH1, FTL, and TfR1, with a better effect than the positive drug Tel.

[0040] Figure 4 BLI targets JAK1 to inhibit ferroptosis; wherein:

[0041] Figure 4 A corresponds to transcription factor enrichment analysis of kidney RNA-seq data. The results show that the STAT family of transcription factors changes significantly under the action of BLI.

[0042] Figure 4 B corresponds to detection of changes in STAT1 and STAT3 protein expression in mouse kidney tissue. The results show that BLI can significantly inhibit the phosphorylation of transcription factors STAT1 and STAT3, with a better effect than Tel.

[0043] Figure 4 C corresponds to the ability of BLI to bind to STAT1, STAT3, JAK1, JAK2, JAK3 using molecular docking calculation. The results show that the binding energy of BLI to JAK1 is relatively the lowest.

[0044] Figure 4 D corresponds to detecting the change of JAK1 protein phosphorylation level in mouse kidney tissue. The results show that BLI can significantly inhibit JAK1 phosphorylation in FA and UUO models, and the effect is better than Tel.

[0045] Figure 4 E corresponds to the cell heat transfer experiment (CETSA) and drug affinity reaction target stability experiment (DARTS) to verify the binding ability of BLI to JAK1. The results show that BLI can enhance the thermal stability and denaturation stability of JAK1, suggesting that BLI and JAK1 may exist direct binding.

[0046] Figure 1 F corresponds to observing the effect of BLI on the expression of key signal proteins and genes of ferroptosis at the cellular level. The results show that BLI can significantly reverse the protein and gene expression caused by ferroptosis inducer Era, and the effect is equivalent to JAK1 inhibitor Jak-In8, and the effect of BLI is abolished by JAK1 agonist RO8191. DETAILED DESCRIPTION

[0047] In order to make the objectives, technical solutions and advantages of the present application clearer, the present application will be further described in detail below with reference to the accompanying drawings and examples. It should be understood that the specific examples described herein are only used to explain the present application and do not limit the present application. In addition, the technical features involved in each embodiment of the present application described below can be combined with each other as long as they do not conflict with each other.

[0048] The butyrolactone I used in the following examples was extracted from the rice fermentation product of A. terreus by chromatographic separation according to the prior art, and the purity was determined by HPLC to be > 99.0%. Related prior art, for example, see: Rao, K. V., Sadhukhan, A. K., Veerender, M., Mohan, E. V. S., Dhanvantri, S. D., Sitaramkumar, S., Babu, M. J., Vyas, K., Reddy, O. G., Butyrolactones from Aspergillus terreus. Chem. Pharm. Bull., 2000, 48, 559-562.

[0049] The kits used for detecting the contents of creatinine and urea nitrogen in serum, creatinine and trace albumin in urine, the contents of inflammatory factors interleukin-1β (IL-1β), interleukin-6 (IL-6), monocyte chemotactic protein (MCP-1), tumor necrosis factor-α (TNF-α), transforming growth factor-β (TGF-β), glutathione (GSH), and malondialdehyde (MDA) were all purchased from the market.

[0050] The following are specific examples:

[0051] Example 1

[0052] Butyrolactone I (BLI) treatment of FA-induced kidney injury.

[0053] In this experiment, 50 SPF male 8-week-old C57BL / 6 mice were raised in the SPF experimental animal center of Tongji Medical College, Huazhong University of Science and Technology, and were given standard diet and drinking water, and the room temperature was controlled at 23±2℃. The experimental plan involving mice in the experiment was approved by the Animal Experiment Ethics Committee of Tongji Medical College, Huazhong University of Science and Technology. After 7 days of adaptation, the mice were randomly divided into control group (Con), model group (FA-Model, folic acid 250mg / kg), low-dose group (FA-BLI-L, folic acid 250mg / kg+50mg / kg BLI), high-dose group (FA-BLI-H, folic acid 250mg / kg+100mg / kg BLI) and positive drug group (FA-Tel, folic acid 250mg / kg+telmisartan 5mg / kg), 10 mice in each group. After the grouping was completed, the corresponding dose of test drug (i.e., BLI or telmisartan) was given to each group once a day, and the administration was continued for 14 days. One hour after the first administration, 250mg / kg of folic acid solution was injected intraperitoneally to construct a kidney injury model. The samples were taken after 14 days of administration. The results of kidney index showed that BLI could reduce the kidney index of mice (see Fig. 1A in the specification). The results of kidney H&E staining showed that BLI could reduce the tubular injury and inflammatory cell infiltration of kidney tissue of mice, and improve the pathological changes of kidney (see Fig. 1B in the specification). The results of kidney function detection suggested that the contents of serum creatinine, urea nitrogen, urine creatinine and trace albumin in the model group were significantly increased, and were significantly decreased after BLI administration (see Fig. 1C, D, E, F in the specification). The expression levels of kidney injury markers KIM-1 and NGAL mRNA in mice were detected, and the expression of KIM-1 and NGAL mRNA in the kidney tissue of mice in the model group was significantly up-regulated, and the expression of KIM-1 and NGAL mRNA in the tissue samples of mice was significantly down-regulated after BLI treatment (see Fig. 1G and Fig. 1H in the specification). Figure 1 Figure 1 Figure 1 Figure 1 Figure 1 ​​​​H) in FIG. 1. The levels of inflammatory factors in the kidney tissue homogenate of the mice were detected, and the contents of inflammatory factors IL-1β, IL-6, TGF-β, MCP-1, and TNF-α in the model group were significantly increased. After the mice were given BLI, the levels of inflammatory factors in the kidney tissue of the mice were significantly decreased, indicating that BLI can reduce the levels of inflammatory factors in the kidney tissue (see FIG. 1 Figure 1 I, J, K, L, and M in FIG. 1. The levels of GSH and MDA in the kidney tissue homogenate of the mice were detected, and the content of GSH in the kidney tissue homogenate of the mice in the model group was significantly decreased, and the content of MDA was significantly increased. After the mice were given BLI, the content of GSH in the mice was significantly increased, and the content of MDA was decreased, indicating that BLI can improve the oxidative stress in the kidney of the mice (see FIG. 1 Figure 1 N in FIG. 1, and Figure 1 O in FIG. 1. The expression of fibrosis-related proteins Col-I, Col-III, Col-IV, Fn, and a-SMA in the kidney tissue samples of the mice was detected, and the expression of Col-I, Col-III, Col-IV, Fn, and a-SMA proteins in the kidney tissue of the mice in the model group was significantly increased. After the mice were given BLI, the expression of fibrosis-related proteins in the kidney tissue of the mice was significantly decreased (see FIG. 1 Figure 2 P in FIG. 1, indicating that BLI can reduce the fibrosis in the kidney of the mice.

[0054] Example 2

[0055] Effect of butyrolactone I (BLI) on kidney injury caused by unilateral renal obstruction.

[0056] In this experiment, 50 SPF male 8-week-old C57BL / 6 mice were raised in the SPF experimental animal center of Tongji Medical College of Huazhong University of Science and Technology, and were given standard diet and drinking water. The room temperature was controlled at 23±2°C. The experimental scheme involving mice in the experiment was approved by the Animal Experiment Ethics Committee of Tongji Medical College of Huazhong University of Science and Technology. After 7 days of adaptation, the mice were randomly divided into a control group (Con, sham operation group), a model group (UUO-Model, unilateral renal obstruction operation in mice), a low-dose group (UUO-BLI-L, unilateral renal obstruction operation in mice+50mg / kg BLI), a high-dose group (UUO-BLI-H, unilateral renal obstruction operation in mice+100mg / kg BLI), and a positive drug group (UUO-Tel, unilateral renal obstruction operation in mice+Telmisartan 5mg / kg), 10 mice in each group. After the operation, the mice recovered for 12 hours, and then the drugs were administered. Each group was given the corresponding dose of the test drug once a day, and the administration was continued for 14 days. After 14 days of administration, the samples were taken. The results of kidney index showed that BLI can reduce the kidney index of the mice (see FIG. 1 Figure 2 A) in FIG. 1. The results of kidney H&E staining showed that BLI can reduce the tubular injury and inflammatory cell infiltration in the kidney tissue of the mice, and improve the pathological changes of the kidney (see FIG. 1 Figure 2(B in the text). Renal function test results showed that serum creatinine, urinary creatinine, and microalbumin were significantly elevated in the model group, and all levels decreased significantly after BLI treatment (see [link]). Figure 2 (C, D, E, F in the text). The expression levels of KIM-1 and NGAL mRNA, markers of kidney injury in mice, were detected. KIM-1 and NGAL mRNA expression was significantly upregulated in the kidney tissue of the model group mice, and significantly downregulated in tissue samples after BLI treatment (see [reference needed]). Figure 2 G and Figure 2 (H in the text). The levels of inflammatory factors in mouse kidney tissue homogenates were detected. In the model group, the levels of inflammatory factors IL-1β, IL-6, TGF-β, MCP-1, and TNF-α were significantly upregulated. Treatment with BLI significantly downregulated the levels of inflammatory factors in mouse kidney tissue. BLI treatment can reduce the levels of inflammatory factors in kidney tissue (see [link to article]). Figure 2 The levels of GSH and MDA in mouse kidney tissue homogenate were detected. In the model group, GSH was significantly decreased and MDA was significantly increased in the tissue homogenate. After BLI treatment, GSH was significantly increased and MDA was decreased, suggesting that BLI can improve oxidative stress in mouse kidneys (see [reference needed]). Figure 2 N and Figure 3 The expression of fibrosis-related proteins Col-I, Col-III, Col-IV, Fn, and α-SMA in mouse kidney tissue samples was detected. The expression of Col-I, Col-III, Col-IV, Fn, and α-SMA proteins was significantly upregulated in the kidney tissue of the model group mice, while the expression of fibrosis-related proteins was significantly reduced in mouse kidney tissue after BLI treatment (see O). Figure 3 The presence of P in the data suggests that BLI can reduce renal fibrosis in mice.

[0057] Example 3

[0058] BLI inhibits ferroptosis in renal tubular epithelial cells.

[0059] This experiment used mouse kidney tissue samples from Example 1 for RNA-seq sequencing, and KEGG signaling pathway enrichment analysis was performed on the mRNAs showing significant changes. The results showed that BLI significantly altered the mRNA expression of genes related to three signaling pathways: fatty acid metabolism, glutamate metabolism, and ferroptosis (see...). Figure 3 (A) RT-qPCR was used to detect the expression of a series of ferroptosis-related genes in mouse kidney tissue samples from Example 1. The results showed that BLI significantly reversed the trend of folic acid-induced changes in ferroptosis-related gene mRNA expression (see [reference]). Figure 3(B in Example 1). Kidney tissue samples from mice in Examples 1 and 2 were used to detect the expression levels of lipid peroxidation product 4HNE, iron ion content, and FTH1 and TfR1 protein expression levels in mouse kidney tissue using 4HNE fluorescence staining, DAB-enhanced Prussian blue staining, and FTH1 and TfR1 immunohistochemical staining. The results showed that in FA and UUO model mice, the levels of 4HNE and iron ions, as well as the expression of FTH1 and TfR1 proteins, were significantly upregulated in mouse kidney tissue. BLI administration significantly reduced the level of lipid peroxidation product 4HNE and the accumulation of iron ions in kidney tissue in both models, while also reducing FTH1 and FTL protein levels. Furthermore, the effect of BLI was superior to that of the positive control drug Tel (see [link to example 1]). Figure 4 (C) Tissue proteins were extracted from mouse kidney tissue samples collected in Examples 1 and 2. Western blot was used to detect the expression levels of marker proteins GPX4, SLC7A11, FSP1, FTH1, FTL, and TfR1 during ferroptosis. The results showed that in both FA and UUO models, BLI significantly upregulated the levels of GPX4, SLC7A11, and FSP1, while downregulating FTH1, FTL, and TfR1, demonstrating a superior effect compared to the positive control drug Tel (see C). Figure 4 (D in the middle).

[0060] Example 4

[0061] BLI inhibits ferroptosis by targeting JAK1.

[0062] This experiment used the RNA-seq data from Example 3 for transcription factor enrichment analysis. The results showed that the STAT family of transcription factors underwent significant changes after BLI treatment (see...). Figure 4 (A) Proteins from mouse kidney tissues in Examples 1 and 2 were analyzed to detect the phosphorylation levels of STAT1 and STAT3 proteins. The results showed that the phosphorylation levels of STAT1 and STAT3 proteins were significantly increased in the FA and UUO model groups. After BLI treatment, the phosphorylation levels of STAT1 and STAT3 proteins were significantly downregulated in both models, demonstrating a superior effect compared to the positive control drug Tel (see A). Figure 4 (B in the text). Molecular docking calculations were performed using AutoDockTools software to determine the binding affinity of BLI to STAT1, STAT3, JAK1, JAK2, and JAK3. The results showed that BLI had the lowest binding affinity to JAK1, suggesting a possible direct interaction between JAK1 and BLI (see [link to calculation]). Figure 4C; the structures of STAT1, STAT3, JAK1, JAK2, JAK3 proteins for docking are from PDB database https: / / www.rcsb.org / , their codes are shown in the figure). The JAK1 protein phosphorylation level changes in the mouse kidney tissue samples in Examples 1 and 2 were detected, and the JAK1 phosphorylation was significantly up-regulated in the FA and UUO model mice, and the JAK1 protein phosphorylation level in the mouse kidney tissue was significantly down-regulated after BLI treatment, which was better than Tel (see Figure 4 D) in the figure. The human kidney tubular epithelial cell line HK2 and the rat kidney tubular epithelial cell line NRK-52E were cultured, and the cells in the logarithmic growth phase were taken for CETSA experiment. After being treated with BLI or DMSO for 90 min, the cells were collected and divided into 7 equal parts, which were placed at 37℃, 42℃, 45℃, 48℃, 51℃, 54℃, and 57℃ for 3 min, respectively, and then the samples were collected and the proteins were extracted. Subsequently, western blot was used to detect JAK1 protein in the protein samples, and the results showed that BLI could significantly enhance the thermal stability of JAK1 protein in HK2 and NRK-52E cells. The DARTS experiment took the lysates of HK2 and NRK-52E cells and divided them into two equal parts, and then added BLI and DMSO respectively, and incubated at room temperature for 15 min. After that, the samples were divided into 4 equal parts, and treated with proteases of different proportions for 10 min. After termination of the enzyme reaction, western blot was used to detect JAK1 protein in the protein samples, and the results showed that BLI could significantly enhance the protease stability of JAK1 protein in HK2 and NRK-52E cells (see ​ E) in the figure. The HK2 cell ferroptosis model was induced by ferroptosis inducer Era, and the effect of BLI on HK2 ferroptosis was observed. The results showed that BLI could significantly inhibit the phosphorylation of JAK1, STAT1 and STAT3 induced by Era, inhibit the expression of HAMP, and up-regulate the levels of FPN and SLC7A11, which was comparable to the effect of JAK1 inhibitor Jak-In8x, and the effect of BLI was abolished by JAK1 agonist RO8191. The results of RT-qPCR detection of HAMP, FPN and SLC7A11 mRNA expression were consistent with the results of protein level detection (see ​ F) in the figure. Results and analysis:

[0063] Butyrolactone I has a good therapeutic effect on the progression of chronic kidney disease caused by folic acid and unilateral kidney obstruction, and can effectively improve the renal tubular injury, oxidative stress, inflammatory infiltration and fibrosis-related protein expression. The mechanism is that the compound can target JAK1, inhibit the JAK-STAT signaling pathway, and reduce the occurrence of ferroptosis in kidney tissue, which can be used as a lead compound for the development of drugs for the treatment of chronic kidney disease.

[0064] From the above examples, it is not difficult to see that butyrolactone I can effectively inhibit the progression of chronic kidney disease in mice, improve kidney function, inhibit oxidative stress damage, reduce inflammatory factor secretion, etc. in the mouse chronic kidney disease model, thereby reducing kidney fibrosis, blocking the progression of acute kidney injury to chronic kidney disease, and can be used as a lead compound for the development of drugs for the treatment of chronic kidney disease. Of course, based on the present application, in addition to using butyrolactone I alone, butyrolactone I can also be used in combination with other drugs for the treatment of chronic kidney disease (such as Tel).

[0065] Those skilled in the art will readily understand that the above description is only of the preferred embodiments of the present application and is not intended to limit the present application, and any modifications, equivalent replacements and improvements made within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. Use of butyrolactone I in the manufacture of a medicament for the treatment of chronic kidney disease, characterized in that, The butyrolactone I has the following formula 1 structure: Formula 1 The chronic kidney disease is specifically progressive kidney function damage caused by kidney damage due to kidney obstruction, drug-induced kidney damage and / or toxicant-induced kidney damage.

2. The use according to claim 1, characterized in that, The butyrolactone I (Butyrolactone I) plays a role in treating chronic kidney disease by inhibiting renal cell ferroptosis.

3. The use according to claim 1, characterized in that, The butyrolactone I (Butyrolactone I) plays a role in treating chronic kidney disease by inhibiting the JAK-STAT signaling pathway.

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