Application of atorvastatin in preparation of medicine for preventing and treating renal toxicity caused by vancomycin

Abstract

The invention relates to the field of biological medicine, in particular to application of atorvastatin in preparation of medicine for preventing and treating renal toxicity caused by vancomycin. The protection effect of atorvastatin on the kidney injury caused by vancomycin is provided for the first time, a kidney injury model is established through vancomycin induction, and it is proved through in-vivo and in-vitro experiments that atorvastatin plays the kidney protection effect through anti-inflammation, anti-oxidation, anti-apoptosis and kidney transporter expression regulation; the kidney toxicity induced by vancomycin is effectively improved. The invention provides a basis for using atorvastatin as a novel medicine for treating kidney injury caused by vancomycin.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and to the use of atorvastatin in the preparation of a medicament for preventing and treating vancomycin-induced nephrotoxicity. Background Art

[0002] With the widespread clinical use of antibacterial agents, the infections caused by drug-resistant Gram-positive cocci such as methicillin-resistant Staphylococcus aureus (MRSA) have gradually increased. The treatment of infections caused by them is difficult and the fatality rate is high, which has become a serious problem in clinical practice. Vancomycin (VCM) is a commonly used glycopeptide antibacterial agent in clinical practice, with strong antibacterial activity, especially showing significant therapeutic effects on MRSA infections.However, guidelines in recent years (①He N, Su S, Ye ZK, et al. Evidence-based guideline for therapeutic drug monitoring of vancomycin: 2020 update by the Division of Therapeutic Drug Monitoring, Chinese Pharmacological Society[J]. Clin Infect Dis, 2020.71(Suppl): S363-S371. ②Chinese Expert Consensus on Clinical Application of Vancomycin (2011 Edition)[J]. Chinese Journal of New Drugs and Clinical Remedies, 2011, 30(08): 561-573. ③Rybak MJ, Le J, Lodise TP, Levine DP, Bradley JS, Liu C, Mueller BA, Pai MP, Wong-Beringer A, Rotschafer JC, Rodvold KA, Maples HD, Lomaestro BM. Therapeutic monitoring of vancomycin for serious methicillin-resistant Staphylococcus aureus infections: A revised consensus guideline and review by the American Society of Health-System Pharmacists, the Infectious Diseases Society of America, the Pediatric Infectious Diseases Society, and the Society of Infectious Diseases Pharmacists. Am J Health Syst Pharm. 2020 May 19; 77(11): 835-864.) have pointed out that although increasing the dose and blood drug concentration of vancomycin can improve the efficacy and reduce the generation of drug-resistant strains, the incidence of vancomycin-induced nephrotoxicity (VIN) caused by high-dose or high blood drug concentration of vancomycin also increases significantly, up to 35%. At the same time, because the conditions of many patients using vancomycin are complex, their renal function is severely impaired, or they are combined with other nephrotoxic drugs, the risk of causing nephrotoxicity is further increased.Nevertheless, authoritative domestic and foreign guidelines still recommend vancomycin as the first-choice drug for the clinical treatment of MRSA infections. Therefore, how to effectively reduce the occurrence of VIN has become an urgent clinical problem to be solved. Summary of the Invention

[0003] The object of the present invention is to provide a new application of atorvastatin (ATO).

[0004] To achieve the above object of the invention, the present invention adopts the following technical solutions:

[0005] The present invention provides the application of atorvastatin in the preparation of a drug for preventing and treating vancomycin-induced nephrotoxicity.

[0006] The structural formula of the atorvastatin is as follows:

[0007]

[0008] In the above technical solution, further, the drug takes atorvastatin as the active ingredient.

[0009] In the above technical solution, further, the drug takes atorvastatin as the only active ingredient.

[0010] The present invention also provides the application of a pharmaceutical composition in the preparation of a drug for preventing and treating vancomycin-induced nephrotoxicity, and the pharmaceutical composition takes atorvastatin as the active ingredient.

[0011] In the above technical solution, further, the pharmaceutical composition takes atorvastatin as the only active ingredient.

[0012] In the above technical solution, further, the drug is an oral preparation or an injection preparation.

[0013] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0014] The present invention first proposes the protective effect of atorvastatin on vancomycin-induced kidney injury. A kidney injury model is established by vancomycin induction. In vitro and in vivo experiments prove that atorvastatin exerts a kidney protection effect by anti-inflammatory, antioxidant, anti-apoptotic and regulating the expression of kidney transporters, effectively improving vancomycin-induced nephrotoxicity and reducing kidney injury. The present invention provides a basis for using atorvastatin as a new drug for treating vancomycin-induced kidney injury. Brief Description of the Drawings

[0015] Figure 1Effect of ATO on vancomycin-induced HK-2 cell injury; A. Effect of different concentrations of vancomycin (VCM) on cell viability, B. Dose-response curve of the effect of vancomycin on cell viability, C. Effect of different treatment groups on cell viability; Data are expressed as mean ± SD, n = 3, compared with the CONTROL group, *P < 0.05, **P < 0.01, ***P < 0.001; compared with the vancomycin model group, #P < 0.05, ##P < 0.01, P < 0.001.

[0016] Figure 2 ATO inhibits the production of inflammatory factors in vancomycin-induced HK-2 cells; A. TNF-α, B. IL-6, C. IL-1β; Data are expressed as mean ± SD, n = 3, compared with the CONTROL group, *P < 0.05, **P < 0.01, ***P < 0.001; compared with the vancomycin model group, #P < 0.05, ##P < 0.01, P < 0.001.

[0017] Figure 3 ATO inhibits the production of ROS in vancomycin-induced HK-2 cells; A. ROS fluorescence staining of each group of cells, original magnification ×200, B. Quantitative analysis results of fluorescence intensity of different treatment groups; Data are expressed as mean ± SD, n = 3, compared with the CONTROL group, *P < 0.05, **P < 0.01, ***P < 0.001; compared with the vancomycin model group, #P < 0.05, ##P < 0.01, P < 0.001.

[0018] Figure 4 ATO inhibits apoptosis of vancomycin-induced HK-2 cells; A. Morphological changes and apoptosis of each group of HK-2 cells were observed by Hoechst33342 staining (original magnification ×100) under a fluorescence microscope, including nuclear condensation and fragmentation, B. Quantitative analysis of the percentage of apoptotic cells in the total cell number of different treatment groups, C. Western blot was used to detect the protein expression of Bcl-2 and Bax in HK-2 cells, D. Statistical analysis of Western blot images of Bcl-2 in HK-2 cells, E. Statistical analysis of Western blot images of Bax in HK-2 cells; Data are expressed as mean ± SD, n = 3, compared with the CONTROL group, *P < 0.05, **P < 0.01, ***P < 0.001; compared with the vancomycin model group, #P < 0.05, ##P < 0.01, P < 0.001.

[0019] Figure 5ATO regulates the expression of renal transporters in HK-2 cells; A. Expression of OAT1, OAT3, OCT2 and β-actin proteins in different treatment groups, B. Relative amount of OAT1 protein expression in different treatment groups, C. Relative amount of OAT3 protein expression in different treatment groups, D. Relative amount of OCT2 protein expression in different treatment groups; Data are expressed as mean ± SD, n = 3, compared with the CONTROL group, *P < 0.05, **P < 0.01, ***P < 0.001; compared with the vancomycin model group, #P < 0.05, ##P < 0.01, P < 0.001.

[0020] Figure 6 Protective effect of ATO on vancomycin-induced nephrotoxicity in C57 mice model; A. Renal morphological changes, B. Statistical analysis of kidney wet weight / body weight (mg / g), C. Changes in plasma creatinine, D. Changes in plasma urea nitrogen, E. HE staining histopathological examination, original magnification ×400; Data are expressed as mean ± SD, n = 6, compared with the CONTROL group, *P < 0.05, **P < 0.01, ***P < 0.001; compared with the vancomycin model group, #P < 0.05, ##P < 0.01, P < 0.001.

[0021] Figure 7 ATO inhibits the production of inflammatory factors in C57 mice induced by vancomycin; A-C. qPCR results show that ATO reduces the expression of TNF-α, IL-6, IL-1β inflammatory factors, D-F. ELISA results also show that ATO reduces the expression of TNF-α, IL-6, IL-1β inflammatory factors; Data are expressed as mean ± SD, n = 6, compared with the CONTROL group, *P < 0.05, **P < 0.01, ***P < 0.001; compared with the vancomycin model group, #P < 0.05, ##P < 0.01, P < 0.001.

[0022] Figure 8 ATO has antioxidant effect on C57 mice induced by vancomycin; A. Changes in SOD level, B. Changes in GSH level, C. Changes in CAT level; Data are expressed as mean ± SD, n = 6, compared with the CONTROL group, *P < 0.05, **P < 0.01, ***P < 0.001; compared with the vancomycin model group, #P < 0.05, ##P < 0.01, P < 0.001.

[0023] Figure 9ATO has an anti-apoptotic effect on vancomycin-induced C57 mice; A. TUNEL staining was used to observe apoptosis of mouse renal tubular cells, original magnification ×400, B. Quantitative analysis of TNNEL staining, C. Western blot was used to detect the expression of Bcl-2 and Bax proteins in mouse kidney tissues, D. Statistical analysis of Western blot images of mouse Bcl-2, E. Statistical analysis of Western blot images of mouse Bax; Data are expressed as mean ± SD, n = 3, compared with the CONTROL group, *P < 0.05, **P < 0.01, ***P < 0.001; compared with the vancomycin model group, #P < 0.05, ##P < 0.01, P < 0.001.

[0024] Figure 10 ATO can regulate the expression of renal transporters in C57 mice; A. Expression of OAT1, OAT3, OCT2 and β-actin proteins in different treatment groups, B. Relative quantity of OAT1 protein expression in different treatment groups, C. Relative quantity of OAT3 protein expression in different treatment groups, D. Relative quantity of OCT2 protein expression in different treatment groups; Data are expressed as mean ± SD, n = 3, compared with the CONTROL group, *P < 0.05, **P < 0.01, ***P < 0.001; compared with the vancomycin model group, #P < 0.05, ##P < 0.01, P < 0.001. Detailed implementation manners

[0025] The present invention will be further described below in conjunction with specific embodiments, but the present invention is not limited in any way.

[0026] Example 1

[0027] A mouse model of vancomycin-induced kidney injury was established, and the alleviating effect and mechanism of ATO on vancomycin-induced nephrotoxicity were investigated through in vivo and in vitro experiments.

[0028] I. Experimental methods

[0029] 1. In vitro experiments: The CCK8 assay was used to screen the dosage of vancomycin for modeling and the safe and protective dosages of ATO. This experiment was divided into 5 groups (n = 3): blank control group, ATO single administration group (10 μM), vancomycin modeling group (4 mM), low-dose ATO protection group (vancomycin 4 mM + ATO 2 μM), and high-dose ATO protection group (vancomycin 4 mM + ATO 10 μM). Subsequently, inflammatory factors (TNF-α, IL-6, and IL-1β) were detected by qPCR; the intracellular oxidative stress level was detected using a ROS kit; apoptosis levels were analyzed by Hoechst 33342 staining and Western blot (Bax, Bcl-2); and the expression of transporter-related proteins (OAT1, OAT3, OCT2) was detected by Western blot.

[0030] 2. In vivo experiments:

[0031] Thirty male C57BL / 6 mice, 6 in each group, were randomly divided into the following 5 groups according to the principle of uniform body weight: ① blank control group, ② ATO single administration group (10 mg / kg / d), ③ vancomycin modeling group (intraperitoneal injection of vancomycin 600 mg / kg / d), ④ low-dose ATO protection group (intraperitoneal injection of vancomycin 600 mg / kg / d + ATO 5 mg / kg / d), and ⑤ high-dose ATO protection group (intraperitoneal injection of vancomycin 600 mg / kg / d + ATO 10 mg / kg / d).

[0032] ATO was administered by oral gavage once a day for 10 consecutive days. Starting from the 4th day, after oral gavage of ATO, vancomycin was intraperitoneally injected 0.5 h later. On the 11th day, the mice were anesthetized and sacrificed with isoflurane. The whole blood of the mice was placed in heparinized EP tubes and centrifuged at 5000 rpm for 10 min. Subsequently, the supernatant was taken and placed in new EP tubes and frozen at -80 °C for later detection; the appearance of the kidneys was photographed and kidney tissues were taken for subsequent experiments.

[0033] Corresponding kits were used to detect the plasma creatinine and urea nitrogen contents of the mice to evaluate renal function; HE staining was performed to evaluate kidney morphology; ELISA kits and qPCR were used to detect inflammatory factors (TNF-α, IL-6, and IL-1β); SOD, GSH, and CAT kits were used to detect oxidative stress; apoptosis levels were analyzed by TUNEL staining and Western blot (Bax, Bcl-2); and the expression of transporter-related proteins (OAT1, OAT3, OCT2) was detected by Western blot.

[0034] II. Experimental results

[0035] 1. Results of in vitro experiments:

[0036] As Figure 1 shown, vancomycin can significantly produce cytotoxicity above 4 mM concentration, causing a large number of HK-2 cells to die and the cell survival rate to decrease significantly ( Figure 1 A), and the IC50 value of vancomycin is 4.078 mM ( Figure 1 B). Therefore, we selected 4 mM vancomycin as the modeling dose for cytotoxicity. ATO has low toxic side effects, and 10 μM ATO is non-toxic and does not cause cell death. Moreover, ATO has a protective effect on the cytotoxicity caused by vancomycin (4 mM), can increase the survival rate of HK-2 cells, and this protective effect is concentration-dependent ( Figure 1 C). Therefore, in subsequent experiments, we selected two concentrations of low and high concentrations (2 μM and 10 μM) for the protective group dose of ATO.

[0037] As Figure 2 shown, the qPCR results showed that compared with the CONTROL group, the mRNA levels of TNF-α ( Figure 2 A), IL-6 ( Figure 2 B), and IL-1β ( Figure 2 C) in the VCM group were significantly increased, while they were significantly down-regulated in the ATO protection group, indicating the anti-inflammatory effect of ATO.

[0038] As Figure 3 shown, by detecting ROS with a fluorescence microscope, compared with the CONTROL group, the ROS content in the VCM group was significantly increased, while it was significantly down-regulated in the ATO protection group, indicating the antioxidant effect of ATO.

[0039] As Figure 4 shown, the morphological changes and apoptosis of HK-2 cells were observed by Hoechst 33342 staining under a fluorescence microscope. Compared with the CONTROL group, partial nuclear shrinkage, nuclear condensation, and nuclear fragmentation occurred in the HK-2 cells in the VCM group, suggesting apoptosis, while the ATO protection group could significantly reduce apoptosis ( Figure 4 A, 4B). In addition, the Western blot results showed that vancomycin down-regulated the expression of Bcl-2 protein and up-regulated the expression of Bax protein, while ATO could reverse it ( Figure 4 C-4E). The above results consistently indicated the anti-apoptotic effect of ATO.

[0040] As Figure 5 shown, the WB results showed that the expressions of renal transporters OAT1, OAT3, and OCT2 in the VCM group were all significantly decreased, while ATO could up-regulate the expression of transporters, indicating that ATO could regulate the expression of renal transporters in HK-2 cells, promote the excretion of toxins, and relieve renal toxicity.

[0041] 2. In vivo experimental results:

[0042] As Figure 6 shown, compared with the CONTROL group, the kidneys of mice in the VIN model group were significantly enlarged and yellowish-white ( Figure 6 A), and the kidney weight / body weight ratio was significantly increased ( Figure 6 B), and the plasma creatinine ( Figure 6 C) and blood urea nitrogen ( Figure 6 D) levels were significantly increased. After administration of ATO, compared with the VIN model group, the kidneys became smaller in appearance, the color tended to be normal, the kidney weight / body weight ratio gradually decreased, and the plasma creatinine and blood urea nitrogen levels were significantly reduced. In the VIN model group, HE staining showed severe kidney injury, manifested as extensive dilation, desquamation, vacuolization, necrosis, atrophy, interstitial inflammatory cell infiltration and edema of the renal tubules. Compared with the model group, the renal tissue injury in the ATO protection group was alleviated in a dose-dependent manner ( Figure 6 E).

[0043] As Figure 7 shown, the results of qPCR ( Figure 7 A-7C) and ELISA ( Figure 7 D- Figure 7 F) showed that compared with the CONTROL group, the levels of inflammatory factors TNF-α, IL-6, and IL-1β in the VCM group were significantly increased, while they were significantly down-regulated in the ATO protection group, indicating the anti-inflammatory effect of ATO.

[0044] As Figure 8 shown, compared with the CONTROL group, the levels of SOD ( Figure 8 A), GSH ( Figure 8 B), and CAT ( Figure 8 C) in the renal tissue of the VCM group were significantly reduced, while after administration of ATO, they increased in a dose-dependent manner, indicating the antioxidant effect of ATO.

[0045] As Figure 9 shown, the results of the TUNEL kit detection showed that the apoptosis ratio in the ATO protection group was significantly lower than that in the VCM group ( Figure 9 A, 9B), and the WB results showed that ATO could significantly increase the protein expression of BCL-2 and inhibit the protein expression of Bax ( Figure 9 C-9E), indicating the anti-apoptotic effect of ATO.

[0046] As Figure 10 shown, the WB results showed that the expressions of renal transporters OAT1, OAT3, and OCT2 in the VCM group were all significantly reduced, while ATO could up-regulate the expression of the transporters, indicating that ATO could regulate the expression of renal transporters in C57 mice, promote the excretion of toxins, and relieve renal toxicity.

[0047] In summary, the in vitro and in vivo experimental results both indicate that atorvastatin can exert a renal protective effect by means of anti - inflammation, anti - oxidation, anti - apoptosis and regulating the expression of renal transporters, effectively improving vancomycin - induced nephrotoxicity.

Claims

1. The use of atorvastatin in the preparation of drugs for preventing and treating vancomycin-induced nephrotoxicity.

2. The use according to claim 1, characterized in that: The medicine uses atorvastatin as an active ingredient.

3. The use according to claim 1, characterized in that: The drug contains atorvastatin as the only active ingredient.

4. Use of a pharmaceutical composition in the preparation of a drug for preventing and treating vancomycin-induced nephrotoxicity, wherein the pharmaceutical composition contains atorvastatin as an active ingredient.

5. The use according to claim 4, characterized in that: The pharmaceutical composition has atorvastatin as the only active ingredient.

6. The use according to claim 1 or 3, characterized in that: The medicine is an oral preparation or an injection preparation.

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