Application of gamma-linolenic acid and / or methyl caffeate in preparation of medicine for treating diabetic nephropathy

Abstract

The invention provides application of gamma-linolenic acid and / or methyl caffeate in preparation of a medicine for treating diabetic nephropathy, and belongs to the technical field of biological medicine. The gamma-linolenic acid and the methyl caffeate can obviously reduce the ROS content of renal tubular epithelial cells, relieve high glucose-induced cell oxidative stress, inhibit cell apoptosis and improve the cell survival rate, have a protection effect on renal tubular injury of early diabetic nephropathy, can treat diabetic nephropathy, and can be used for treating diabetic nephropathy. The apoptosis of renal tubular epithelial cells of diabetic nephropathy is inhibited.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and in particular relates to an application of gamma-linolenic acid and / or caffeic acid methyl ester in the preparation of a medicine for treating diabetic nephropathy. Background Art

[0002] Diabetic kidney disease (DKD) is a common microvascular complication caused by diabetes mellitus (DM). It usually occurs in patients with type 1 and type 2 diabetes who do not have adequate long-term blood sugar control. According to statistics, 40% of patients with type 2 diabetes and 30% of patients with type 1 diabetes will develop DKD. It is known that the important mechanism of DKD is the increase in ROS and oxidative stress caused by high blood sugar. High sugar can increase the production of intracellular reactive oxygen species (ROS). A large amount of ROS can react with and denature cellular macromolecules including lipids, proteins and nucleic acids, thereby weakening the antioxidant system and aggravating the body's oxidative stress state, leading to pathological changes such as cell apoptosis. Renal tubular damage is an important pathological change in DKD.

[0003] Currently, apart from hypoglycemic drugs and RASi, SGLT2i, GLP-1RA, MRA, etc., there is no optimal solution to effectively control the progression of DKD. Therefore, finding drugs that can effectively protect renal tubular damage in early diabetic nephropathy is crucial for the treatment of diabetic nephropathy. Summary of the invention

[0004] In view of this, the object of the present invention is to provide a use of γ-linolenic acid and / or caffeic acid methyl ester in the preparation of a medicament for treating diabetic nephropathy.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The invention provides application of gamma-linolenic acid and / or caffeic acid methyl ester in preparing medicine for treating diabetic nephropathy.

[0007] The present invention also provides the use of gamma-linolenic acid and / or caffeic acid methyl ester in preparing medicine for protecting renal tubular damage.

[0008] The present invention also provides the use of gamma-linolenic acid and / or caffeic acid methyl ester in preparing a medicine for inhibiting apoptosis of renal tubular epithelial cells.

[0009] The present invention also provides the use of gamma-linolenic acid and / or caffeic acid methyl ester in preparing a medicine for reducing the ROS content in renal tubular epithelial cells.

[0010] Preferably, the concentration of the γ-linolenic acid is 6 to 24 μM; the concentration of the caffeic acid methyl ester is 3 to 12 μM.

[0011] Compared with the prior art, the present invention has the following beneficial effects:

[0012] The gamma-linolenic acid and caffeic acid methyl ester of the present invention can significantly reduce the ROS content of renal tubular epithelial cells, alleviate high sugar-induced cell oxidative stress, inhibit cell apoptosis, and improve cell survival rate, have a protective effect on renal tubular damage in early diabetic nephropathy, can treat diabetic nephropathy, and inhibit apoptosis of renal tubular epithelial cells in diabetic nephropathy. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 It is the best modeling concentration screening diagram;

[0014] Figure 2 This is the screening diagram of the optimal administration concentration of γ-linolenic acid and caffeic acid methyl ester (Note: compared with the control group, ## indicates P < 0.01; compared with the model group, ** indicates P < 0.01, * indicates P < 0.05);

[0015] Figure 3 is the result diagram of HK-2 cell apoptosis in each group of γ-linolenic acid (where A is the control group, B is the model group, C is the low-dose group, D is the medium-dose group, E is the high-dose group, and F is the cell apoptosis rate in each group. Compared with the control group, ## indicates P < 0.01; compared with the model group, ** indicates P < 0.01, and * indicates P < 0.05);

[0016] Figure 4 is the result diagram of HK-2 cell apoptosis in each caffeic acid methyl ester group (where A is the control group, B is the model group, C is the low-dose group, D is the medium-dose group, E is the high-dose group, and F is the cell apoptosis rate in each group. Compared with the control group, ## indicates P < 0.01; compared with the model group, ** indicates P < 0.01, and * indicates P < 0.05);

[0017] Figure 5 is the result of cell cycle determination in each group of γ-linolenic acid (where A is the control group, B is the model group, C is the low-dose group, D is the medium-dose group, E is the high-dose group, and F is the cell survival rate in each group. Compared with the control group, ## indicates P < 0.01; compared with the model group, ** indicates P < 0.01, and * indicates P < 0.05);

[0018] Figure 6is the result of cell cycle determination in each group of caffeic acid methyl ester (where A is the control group, B is the model group, C is the low-dose group, D is the medium-dose group, E is the high-dose group, and F is the cell survival rate of each group. Compared with the control group, ## indicates P < 0.01; compared with the model group, ** indicates P < 0.01, and * indicates P < 0.05);

[0019] Figure 7 is the determination diagram of ROS content in cells of each group of γ-linolenic acid (where A is the control group, B is the model group, C is the low-dose group, D is the medium-dose group, E is the high-dose group, and F is the fluorescence intensity of ROS in cells of each group. Compared with the control group, ## indicates P < 0.01; compared with the model group, ** indicates P < 0.01, and * indicates P < 0.05);

[0020] Figure 8 is a graph showing the determination of ROS content in cells of each caffeic acid methyl ester group (where A is the control group, B is the model group, C is the low-dose group, D is the medium-dose group, E is the high-dose group, and F is the ROS fluorescence intensity of cells in each group. Compared with the control group, ## indicates P < 0.01; compared with the model group, ** indicates P < 0.01, and * indicates P < 0.05). DETAILED DESCRIPTION

[0021] The present invention provides the use of gamma-linolenic acid and / or caffeic acid methyl ester in preparing a medicine for treating diabetic nephropathy

[0022] The present invention also provides the use of gamma-linolenic acid and / or caffeic acid methyl ester in preparing medicine for protecting renal tubular damage.

[0023] The present invention also provides the use of gamma-linolenic acid and / or caffeic acid methyl ester in preparing a medicine for inhibiting apoptosis of renal tubular epithelial cells.

[0024] The present invention also provides the use of γ-linolenic acid and / or caffeic acid methyl ester in the preparation of a drug for reducing the ROS content in renal tubular epithelial cells. In the present invention, the concentration of the γ-linolenic acid is preferably 6 to 24 μM, more preferably 12 μM; the concentration of the caffeic acid methyl ester is preferably 3 to 12 μM, more preferably 6 μM.

[0025] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0026] Materials used in the present invention: Human renal tubular epithelial cells (HK-2) were purchased from Wuhan Pronocell Life Science Technology Co., Ltd.

[0027] γ-linolenic acid and caffeic acid methyl ester were purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0028] DMEM medium, 0.25% trypsin containing EDTA, and fetal bovine serum were all Gibco brands and were purchased from Shanghai Yibaiju Economic and Trade Co., Ltd.;

[0029] PBS, Wuhan Saiweier Biotechnology Co., Ltd.;

[0030] Cell Counting Kit-8 (CCK-8 kit), ANNEXIN V-FITC / PI apoptosis detection kit, and Reactive oxygen species assay kit were purchased from Beijing Solebow Biotechnology Co., Ltd.

[0031] ATP content test kit, Nanjing Jiancheng Bioengineering Institute;

[0032] The brands of 96-well plates, 6-well plates, culture dishes, 2 mL cryovials, 15 mL centrifuge tubes, and 50 mL centrifuge tubes were all selected (LABSELECT) and purchased from Aoqi Kehua Medical Supply Chain Management Service (Tianjin) Co., Ltd.;

[0033] CO2 incubator, clean bench, and centrifuge were purchased from Thermo Fisher Scientific, USA;

[0034] Multifunctional microplate reader, Berton Instrument Co., Ltd., USA;

[0035] Fluorescence inverted microscope, Nikon Corporation, Japan;

[0036] Flow cytometer, Beckman Coulter, USA;

[0037] UV-visible spectrophotometer, Shanghai Yuanxi Instrument Co., Ltd.;

[0038] Ultrasonic cell disruptor, Nanjing Emmanuel Instrument Equipment Co., Ltd.

[0039] Example 1

[0040] CCK-8 method to screen the best modeling concentration

[0041] Take cells with a cell density of 80% or above to prepare a cell suspension, with 1×10 4 HK-2 cells were randomly divided into 7 groups, and 6 wells in each group were given 0 mg·ml -1 , 50mg·ml -1 , 55mg·ml -1 、60mg·ml -1 、65mg·ml -1 , 70mg·ml -1 、80mg·ml-1 Glucose solutions of different concentrations were used for intervention to screen the optimal concentration for modeling.

[0042] Experimental results: As shown in Table 1 and Figure 1 shown.

[0043] Table 1 Cell survival rate at different glucose concentrations

[0044]

[0045] From Table 1 and Figure 1 It can be seen that the HK-2 cell survival rate decreases with the increase of glucose concentration. The present invention takes the glucose concentration when the cell survival rate is about 50% as the optimal modeling concentration. The optimal modeling concentration of HK-2 cells is 60 mg·mL -1 .

[0046] Example 2

[0047] CCK-8 method to screen the optimal drug concentration

[0048] Take cells with a cell density of 80% or above and add 1×10 4 The cells were inoculated in a 96-well plate, with 6 replicate wells in each group. The cells were divided into a control group, a model group, and a drug group. The control group was cultured with basal medium, and the model group was cultured with 60 mg ml -1 The glucose solution was added to the culture medium for culture; the drug administration group was added with 4μM, 8μM, 12μM, 16μM, 20μM concentrations of γ-linolenic acid and 2μM, 4μM, 6μM, 8μM, 10μM concentrations of caffeic acid methyl ester on the basis of the model group and cultured for 48h. After the intervention, 100μl of 10% CCK-8 reagent was added to each well, and after incubation at 37℃ for 1h, the test was performed under the microplate reader.

[0049] Experimental results: Figure 2 As shown. The CCK-8 results showed that compared with the control group, the cell survival rate in the model group was significantly inhibited, with a significant difference (P < 0.01); the CCK-8 method screened the optimal administration concentrations of γ-linolenic acid and caffeic acid methyl ester to be 12 μM (P < 0.01) and 6 μM (P < 0.01), respectively. This dosage was set as the medium dose, and its 1 / 2 dose and 2 doses were low doses and high doses, respectively, for subsequent experiments.

[0050] Example 3

[0051] Detection of HK-2 cell apoptosis using AV-PI double staining cell apoptosis detection kit combined with flow cytometry

[0052] Take cells with a cell density of 80% or above and add 4 × 105 Cells were inoculated in 6-well plates, and 3 replicates were set for each group. The cells were divided into control group, model group, and drug group. After the HK-2 cells in the drug group adhered to the wall and synchronized, the original culture medium was discarded, and 6, 12, and 24 μM γ-linolenic acid and 3, 6, and 12 μM caffeic acid methyl ester prepared in advance were added for intervention for 24 hours. After the intervention, the cells in the control group, model group, and drug group were digested to prepare cell suspensions and centrifuged. After the centrifugation, the cells were washed twice with PBS, and 400 μL PBS solution was added to each tube to mix well, covered with aluminum foil, and detected by flow cytometry (FACS).

[0053] Experimental results: Figure 3 and Figure 4 As shown. After 24 hours of intervention with different concentrations of γ-linolenic acid and caffeic acid methyl ester, AV-7AAD double staining combined with flow cytometry was used to detect cell apoptosis. Compared with the control group, the apoptosis rate of cells in the model group was significantly increased (P < 0.01); compared with the model group, the apoptosis rate of cells in the γ-linolenic acid and caffeic acid methyl ester groups was decreased (P < 0.01 or P < 0.05).

[0054] Example 4

[0055] Measurement of HK-2 cell cycle by flow cytometry

[0056] Take HK-2 cell suspension with a cell density of 80% or above and plate 4×10 5 The cells were inoculated on a 6-well plate and divided into a control group, a model group, and a drug-treated group, with 3 wells in each group as a repeated control. In the drug-treated group, 6, 12, and 24 μM of γ-linolenic acid and 3, 6, and 12 μM of caffeic acid methyl ester were added to the model group for intervention for 24 h. After the intervention, the cells in the control group, model group, and drug-treated group were digested to prepare a cell suspension, which was washed twice with PBS solution, and 2 mL of 75% ethanol solution was added to each well and placed at 4 ° C overnight. After centrifugation, the control group, model group, and drug-treated group were aspirated with a pipette to remove ethanol, and each well was washed twice with PBS solution. 5 μL of 7-aminoactinomycin D reagent was added to each well, incubated in the dark for 15 min, and the cell cycle was examined by flow cytometry.

[0057] Experimental results: Figure 5 and Figure 6As shown. HK-2 cells were treated with γ-linolenic acid and caffeic acid methyl ester for 24 hours and the cell cycle changes were detected by flow cytometry. Compared with the control group, the proportion of G0 / G1 phase in the model group increased (P<0.01), and the proportion of G2 / M phase + S phase decreased (P<0.01); compared with the model group, the proportion of G0 / G1 phase in the γ-linolenic acid and caffeic acid methyl ester administration groups decreased (P<0.01 or P<0.05), and the proportion of G2 / M phase + S phase increased (P<0.05). It shows that γ-linolenic acid and caffeic acid methyl ester can alleviate the inhibitory effect of high sugar on the proliferation of HK-2 cells.

[0058] Example 5

[0059] Reactive oxygen detection kit to detect ROS content

[0060] Take cells with a cell density of 80% or above and add 4 × 10 5 The cells were evenly inoculated into 6-well plates with a volume of 100 μL, and randomly divided into a control group, a model group, and a drug-treated group. The control group was cultured with HK-2 special complete medium; the model group was cultured with HK-2 special medium plus 60 mg mL -1 The cells were cultured with glucose solution; the drug-treated group was intervened for 24 hours by adding 6, 12, and 24 μM γ-linolenic acid and 3, 6, and 12 μM caffeic acid methyl ester on the basis of the model group. Diluted DCFH-DA was added to the cells of the control group and the model group; after the cells of the drug-treated group were cultured for 24 hours, the original culture medium was removed with a pipette, DCFH-DA was added, and the cells were cultured in a CO2 cell culture incubator for 20 minutes. The cells were taken out and rinsed with basal culture medium. The cells were detected by flow cytometry.

[0061] Experimental results: Figure 7 and Figure 8 As shown. By fluorescence observation: compared with the control group, the ROS content in the HK-2 cells of the model group was significantly increased (P < 0.01); compared with the model group, the ROS content in the γ-linolenic acid group and the caffeic acid methyl ester group was reduced, among which the γ-linolenic acid medium-dose group (12μM), the caffeic acid methyl ester medium-dose group (6μM) and the caffeic acid methyl ester high-dose group (12μM) had the most significant effect in reducing the ROS content (P < 0.01), indicating that γ-linolenic acid and caffeic acid methyl ester can significantly reduce the ROS content in HK-2 cells.

[0062] It can be seen from the above examples that the γ-linolenic acid and caffeic acid methyl ester of the present invention can significantly reduce the ROS content of renal tubular epithelial cells, reduce high sugar-induced cell oxidative stress, inhibit cell apoptosis, and improve cell survival rate, have a protective effect on renal tubular damage in early diabetic nephropathy, can treat diabetic nephropathy, and inhibit apoptosis of renal tubular epithelial cells in diabetic nephropathy.

[0063] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Use of γ-linolenic acid and / or caffeic acid methyl ester in the preparation of drugs for treating diabetic nephropathy.

2. Use of γ-linolenic acid and / or caffeic acid methyl ester in the preparation of drugs for protecting renal tubular damage.

3. Use of γ-linolenic acid and / or caffeic acid methyl ester in the preparation of drugs for inhibiting apoptosis of renal tubular epithelial cells.

4. Use of γ-linolenic acid and / or caffeic acid methyl ester in the preparation of drugs for reducing the ROS content in renal tubular epithelial cells.

5. The use according to any one of claims 1 to 4, characterized in that: The concentration of the gamma-linolenic acid is 6-24 μM; the concentration of the caffeic acid methyl ester is 3-12 μM.

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