Establishment of curcumin in cat kidney cell oxidative stress injury model and application of curcumin in treatment of cat kidney oxidative injury

Abstract

The invention provides establishment of a cat kidney cell oxidative stress injury model and application of the cat kidney cell oxidative stress injury model in treatment of cat kidney oxidative injury. In the research, cat kidney cells (F81 cells) are taken as a research model, and a cat kidney cell oxidative stress injury model is successfully constructed. On the basis, the anti-oxidation effect of the curcumin on the F81 cells is screened and verified, and functional genes and related pathways, which influence the F81 cells, of the curcumin are analyzed through transcriptome, so that a certain theoretical basis is provided for application of the curcumin in functional foods for pet cats and related fields of pets for reference; the application verifies that the curcumin has a good treatment effect on kidney-related diseases of pet cats and has an obvious synergistic effect on prevention and treatment of peroxidation stress of kidney injury of the pet cats in combination with CXCL8 neutralizing antibody medication, the effect is obviously improved, and the curcumin is superior to a single medication group.

Description

Technical Field

[0001] The invention belongs to the field of biological medicines, and specifically relates to the establishment of a cat kidney cell oxidative stress injury model and the application of the model in treating cat kidney oxidative damage. Background Art

[0002] With the improvement of national economic living standards, the number of people keeping pets is increasing, and pets are regarded as important family companions. Scientific pet keeping is deeply rooted in people's hearts, and the demand for related industries has also developed rapidly. Kidney disease is one of the important diseases that threaten pets. When exogenous harmful substances reach the kidneys, they will induce the body to produce excess free radicals, which will lead to oxidative stress in the kidneys. When pet cats enter the middle-aged and elderly stage, oxidative damage is more likely to affect the health of kidney function and induce diseases. Such diseases often have a long course of disease, are not easy to be discovered, and ultimately have a more far-reaching impact.

[0003] At present, there are many limitations in the clinical treatment of pet kidney disease, especially pet cat kidney disease. The main treatment methods include drug therapy and surgical intervention (such as angioplasty and bypass surgery). However, these methods are not suitable for all sick pets, and there are safety risks, uncertain efficacy, high recurrence rate, and large side effects. Therefore, it is of positive significance to find effective treatment methods and drugs for pet kidney disease, which can provide new ideas for pet kidney disease treatment and drug development.

[0004] Curcumin is a natural polyphenol with biological activity and a slightly bitter taste. It comes from the rhizome of the genus Curcuma. Studies in recent years have shown that curcumin has different pharmacological effects such as antioxidant, anti-inflammatory, anti-tumor and neuroprotective, and has low toxicity and good clinical application value. In view of the application of curcumin in other human medicine and livestock and poultry industries, it is also used in some pet foods, but whether it has the same effect on pets as it does on animals still needs further experimental confirmation. Summary of the invention

[0005] In view of the above problems, the present invention first provides a method for establishing a cat kidney cell oxidative stress injury model, the method comprising:

[0006] After taking the F81 cells out of liquid nitrogen, quickly place them in a 37°C water bath, then transfer the cells to a centrifuge tube containing 9 mL of complete culture medium, centrifuge at 1000 rpm for 5 min, and discard the supernatant. Add 5 mL of fresh RPMI 1640 complete culture medium containing 10% FBS to resuspend the cells and transfer them to a culture flask, and place them at 37°C with 5% CO 2 The cells were cultured in an incubator at 500 × 10 5 / mL was inoculated into a 96-well cell culture plate, and 100uL of cell solution was added to each well. When the cells grew to 80% of the monolayer, the culture medium was discarded and the cells were washed with PBS and replaced with H 2 O 2 The culture medium was induced for 3 h to construct the feline kidney H 2 O 2 Oxidative damage model.

[0007] Preferably, H 2 O 2 The culture medium contains H 2 O 2 The concentration is selected from 100uM, 150uM and 200uM.

[0008] Furthermore, the present application provides a method for screening a substance capable of repairing oxidative stress damage to cat kidney cells, the method comprising:

[0009] 1) Resuscitate F81 cells, transfer the cells to a centrifuge tube containing complete medium, centrifuge, and discard the supernatant;

[0010] 2) Add fresh RPMI 1640 complete medium containing 10% FBS to resuspend the cells and transfer them to a culture flask and place them at 37°C with 5% CO 2 Static culture in an incubator;

[0011] 3) After subculturing, the cells were inoculated into a 96-well cell culture plate and complete medium was added;

[0012] 4) When the cells grow to 80% of the monolayer, discard the culture medium, wash with PBS, and replace with complete culture medium containing the object to be detected, and incubate for 1-5 hours;

[0013] 5) Wash with PBS and replace with H 2 O 2 The cells were cultured and induced for 3 h. The cell viability was detected using the CCK8 kit. The H 2 O 2 The corresponding cell survival rate was affected, and the changes in cell morphology in each group were observed under a microscope.

[0014] 6) Determine whether the potential active substance to be tested can maintain the cell morphology. If it can, the potential active substance to be tested is a substance that can repair the oxidative stress damage of cat kidney cells; if not, it is not a substance that can repair the oxidative stress damage of cat kidney cells.

[0015] Preferably, H 2 O 2 The culture medium contains H 2 O 2 The concentration is selected from 100uM, 150uM and 200uM.

[0016] Furthermore, the present application provides a use of curcumin or a composition thereof in treating diseases related to oxidative damage in cat kidneys;

[0017] Furthermore, the oxidative damage-related disease refers to chronic kidney disease (CKD).

[0018] Furthermore, the composition further contains a neutralizing antibody against CXCL8 and / or TNF-α. Preferably, the amino acid sequence of the light chain variable region of the neutralizing antibody against CXCL8 is:

[0019] QSPSLDQSPSASKDKVTITCMLSQNIKYAAWYQQKPGKAPGLLIHYTSGLGSGIPSRSGSGSGRAYSFSISNCFSEDIAS YYCLQYDGPFPAGKLELA;

[0020] The amino acid sequence of the heavy chain variable region is:

[0021] EVQLVESGLVSPGLKLSCAVASGFTFANYMTWRQAAPKGLWVASISKGASNPYPDSVKGRFTISRDANAKTLYLQMSLAR SEDTATYCARDGTTDYAWGGAMVTVAS.

[0022] Beneficial Effects

[0023] Therefore, cat kidney cells (F81 cells) were used as a research model in this study. These cells are also susceptible to common cat pathogens such as feline calicivirus, parvovirus and herpesvirus, and are important tools for the study of pathogenicity, modeling and new antiviral drugs. The study will attempt to explore whether curcumin also has an antioxidant effect on F81 cells and analyze the functional genes and related pathways of F81 cells through transcriptome analysis, so as to provide a certain theoretical basis for the application of curcumin in functional foods for pet cats and pet-related fields for reference. Studies have confirmed that since reactive oxygen species (ROS) are products of organisms under the action of cell metabolism or environmental factors, they can destroy cell structure and thus affect the normal physiological function of cells. When oxidative stress occurs, the balance between antioxidants and ROS is destroyed. Curcumin can exert its antioxidant effect by inhibiting the production of ROS and scavenging free radicals and peroxides.

[0024] Moreover, through cell experiments and animal experiments, it was verified that curcumin has a good therapeutic effect on kidney-related diseases in pet cats, and the combined use of CXCL8 neutralizing antibodies has a significant synergistic effect on the prevention and treatment of peroxidative stress in pet cat renal injury, and the effect is significantly improved, which is better than the single drug group. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 H 2 O 2 -F81 oxidatively damaged cell morphology;

[0026] Figure 2 H 2 O 2 -Establishment of F81 oxidative damage model;

[0027] Figure 3 Statistics of differential gene expression in F81 cells induced by curcumin;

[0028] Figure 4 GO analysis of differentially expressed genes in F81 cells induced by curcumin;

[0029] Figure 5 KEGG enrichment analysis of differentially expressed genes in F81 cells induced by curcumin;

[0030] Figure 6 Curcumin induces the transcriptional expression of antioxidant and anti-inflammatory related genes in F81 cells.

[0031] Figure 7 Curcumin on H 2 O 2 The effect of damage on F81 cell viability;

[0032] Figure 8 Curcumin promotes the detection of CAT, SOD and GSH-PX enzyme activities in F81 cells. DETAILED DESCRIPTION

[0033] Example 1 Cat Kidney H 2 O 2 Construction of oxidative damage model

[0034] F81 cells (conventional cell line in this field, maintained in this laboratory, Li D, Cui Z, Li G, Zhang L, Zhang Y, Zhao H, Zhang S, Guo Y, Zhao Y, Men F, Zhao S, Shao J, Du D, Huang H, Wang K, Hu G, LiT, Zhao Y. Antiviral effect of copper chloride on feline calicivirus and synergy with ribavirin in vitro. BMC Vet Res. 2020 Jul 6; 16 (1): 231. doi: 10.1186 / s12917-020-02441-0. PMID: 32631322; PMCID: PMC7336648) were taken out of liquid nitrogen and quickly placed in a 37 ° C water bath. The cells were then transferred to a centrifuge tube containing 9 mL of complete medium, centrifuged at 1000 rpm for 5 min, and the supernatant was discarded. Add 5 mL of fresh RPMI 1640 complete medium containing 10% FBS to resuspend the cells and transfer them to a culture flask and place them at 37°C with 5% CO. 2 The cells were cultured in an incubator at 500 × 10 5 / mL were inoculated into 96-well cell culture plates, and 100uL of cell solution was added to each well. When the cells grew to 80% of the monolayer, the culture medium was discarded, and the cells were washed with PBS and replaced with PBS containing different final concentrations of H 2 O 2 The cells were cultured and induced for 3 h. The cell viability was detected using the CCK8 kit. The H 2 O 2 The corresponding cell survival rate was affected, and the changes in cell morphology in each group were observed under a microscope.

[0035] The results showed that different concentrations of H 2 O 2 After 3 h of treatment with F81 cells, the culture medium was used as a negative control to observe the changes in cell morphology, and CCK8 was used to detect cell survival rate. 2 O 2 At 25uM and 50uM, there was no effect on F81 cells. 2 O 2 When the concentration was greater than 100uM, it caused significant damage to F81 cells, with a survival rate of about 50%, and the damage to cells increased with increasing concentration. 2 O 2After the treatment with damaging concentration, the cells showed a tendency to become rounded, the intercellular spaces became larger, the number of cells decreased, and some cells were broken. The number of cells in the high concentration group was very small, and most of them were broken and dissolved ( Figure 1 ). Therefore, 100uM, 150uM and 200uM H 2 O 2 As the experimental concentration causing F81 cell damage, the model was constructed ( Figure 2 ).

[0036] Example 2 Curcumin in cat kidney H 2 O 2 Pathway metabolomics analysis of oxidative damage models

[0037] Based on the model established in Example 1, F81 cells were cultured at 5.0×10 5 / mL were inoculated in 96-well cell culture plates for culture. After the cells grew into a monolayer, a blank control group was set up. F81 cells were pretreated with 10uM and 20uM curcumin for 3h, and then 100 and 200uM H 2 O 2 Damage cells, determine the curcumin in the treatment of cat kidney H 2 O 2 Specific mechanism of oxidative damage model.

[0038] The results showed that curcumin pretreatment helped cells resist H 2 O 2 The higher the concentration of curcumin, the stronger the effect. However, when H 2 O 2 When the concentration reaches 200uM, the cell damage is extremely strong and curcumin cannot help maintain the cell morphology. The cell morphology shows that curcumin can protect F81 cells from H 2 O 2 The higher the concentration of curcumin, the stronger the effect. 2 O 2 When the concentration reaches 200uM, the cells are severely damaged and curcumin cannot help maintain the cell morphology.

[0039] Total RNA of the sample was extracted, and the integrity and total amount of RNA were accurately detected using the Agilent 2100bioanalyzer. rRNA was removed by the kit, and mRNA was enriched and reverse transcribed into cDNA. The adapter was added and PCR amplified to construct the library. After the constructed library was quality-checked to ensure that the library was qualified, the second-generation sequencing was performed. Four fluorescently labeled dNTPs, DNA polymerase, and adapter primers were added to the sequencing flow cell for amplification. The sequencer captured the fluorescent signal and converted the optical signal into a sequencing peak through computer software to obtain the sequence information of the fragment to be tested.

[0040] DESeq2 software (1.20.0) was used to perform differential expression analysis between the treatment group and the control group. The differential expression in the gene expression data was determined using a model based on the negative binomial distribution through the program provided by the software. The obtained P value was adjusted using the method of Benjamini and Hochberg to control the false discovery rate. P ≤ 0.05 was set as the threshold for significant differential expression.

[0041] The edgeR software was used to analyze the differential gene expression between sample groups, and the FPKM value of each transcript in each sample was counted, and this value was used as the expression level of the transcript. The abundance of the transcript reflects the expression level of the gene. The higher the transcript abundance, the higher the gene expression level. The most commonly used differential gene screening criteria are |log2(FoldChange)|≥1&padj≤0.05. The results are shown in the figure ( Figure 3 ), the horizontal axis in the scatter plot and volcano plot represents the expression fold change (log2FoldChange) of the gene in the treatment group and the control group, and the vertical axis represents the significance level of the difference in gene expression between the treatment group and the control group. Compared with the control group, a total of 124 genes in the curcumin treatment group underwent transcriptional changes, of which 92 genes were upregulated and 32 genes were downregulated; each point in the figure represents a specific gene or transcript, and the red points in the figure represent significantly upregulated genes, the blue points represent significantly downregulated genes, and the colored points are non-significantly different genes.

[0042] ClusterProfiler (3.8.1) software was used to perform GO enrichment analysis of differentially expressed genes, correcting for gene length deviation, and P value < 0.05 was considered to be significantly enriched in the GO term through differentially expressed genes. Gene Ontology (GO) was used to perform enrichment analysis of differential gene functions, and differential functional analysis was performed on biological processes (BP), cell composition (CC) and molecular functions (MF) respectively. GO functional enrichment used padj less than 0.05 as the threshold for significant enrichment. Compared with the control group, the DEGs in the curcumin treatment group were mainly involved in transcriptional regulation, catalytic activity, cell cluster formation, cell growth process, signal transduction, immune process, metabolic process, stress response, etc. ( Figure 4 ).

[0043] KEGG is a database resource for understanding the high-level functions or utilities of biological systems from information at the analytical level, especially large data sets generated by genome sequencing and other high-throughput databases. The statistical enrichment of differentially expressed genes in KEGG pathways was analyzed using clusterProfiler (3.8.1) software to determine biological processes, cellular components and molecular functions. The KEGG information base is a knowledge base for systematic analysis of gene functions and linking genomic information and functional information. Using the KEGG database, genes can be classified according to the pathways they participate in or the functions they perform. KEGG pathway enrichment analysis was performed on the differentially expressed genes in the curcumin treatment group, and the most significant 30 KEGG pathways were selected for analysis. The horizontal axis was the ratio of the number of differentially expressed genes annotated to the KEGG pathway to the total number of differentially expressed genes, and the vertical axis was the KEGG pathway. The main upregulated pathways after curcumin treatment include protein digestion and absorption, sensory transduction, renin-tensin system, neutrophil trap formation, glutathione metabolism, and pluripotency signaling pathway regulation. The enriched down-regulated genes were mainly in the RIG-I-like receptor signaling pathway, Toll-like receptor signaling pathway, NOD-like receptor signaling pathway, NF-kB and JAK-STAT and other immune response-related pathways ( Figure 5 ).

[0044] The total RNA of F81 was extracted using the MEGA kit. The total RNA concentration and purity were detected using an ultra-micro UV spectrophotometer. The qualified RNA samples were reverse transcribed into cDNA and used as templates for real-time fluorescence quantitative PCR to detect the expression of cellular inflammatory mRNA. The PCR reaction system was 20ul, PrimeScript III RT-qPCR Mix (2×) 10μL, ForwardPrimer and Reverse Primer were 0.4μL respectively, the primer concentration was 10uM, the template was 2μL, and DEPC water was used to make up to 20μL. Mix thoroughly and amplify using the Applied Biosystems Q5 Fast Real-Time PCR System. The reaction conditions were: 95℃30s, 95℃10s; 95℃10s, 60℃30s, 40 cycles, and the melting curve was collected. The fluorescence signal was collected. Gapdh was used as an internal reference using 2 -ΔΔCT The relative expression of target gene mRNA was calculated by fluorescence quantitative PCR. The expression of 6 differentially expressed genes in the transcriptome sequencing results, including ANPEP, NQO-1, Nrf-2, Keap1, CXCL8 and TNF-α, was detected. Among them, ANPEP, NQO-1, Keap1 and Nrf-2 were upregulated, and CXCL8 and TNF-α were downregulated. GAPDH gene was used as the internal reference gene, and the relative expression of differentially expressed genes was calculated by 2-ΔΔCt method ( Figure 6), indicating that the results of fluorescence quantitative PCR were consistent with the up- and down-regulation of genes in the transcriptome sequencing results, and that the upregulation of Nrf2 / NQO-1 indicated the activation of the antioxidant pathway, while CXCL8 and TNF-α indicated the inhibition of the immune pathway, i.e., anti-inflammatory effects.

[0045] Example 3 Clinical effect of curcumin and its combination in treating oxidative damage of cat kidney cells

[0046] F81 cells were cultured at 5.0×10 5 / mL were inoculated in a 96-well cell culture plate for culture. After the cells grew into a monolayer, a blank control group was set up. F81 cells were pretreated with curcumin (20uM) for 3h, and then 150uM H 2 O 2 The cells were damaged and induced for 3 h to determine the therapeutic effects of curcumin and its combination drugs in the treatment of oxidative damage in cat kidneys.

[0047] By H 2 O 2 After 3 hours of induction, cells were taken for microscopic examination. The results showed that the negative control group contained only cell culture medium, and the corresponding cell viability was 100%, while 150uM H 2 O 2 After acting on F81 cells alone for 3 hours, the cell viability dropped to about 50%. 2 O 2 Stimulation can maintain cell viability to a certain extent, and cell viability can be 60-70% ( Figure 7 ).

[0048] After the culture is completed, the supernatant is discarded, and the cells are washed with PBS and collected with a cell scraper. After centrifugation, the cells are resuspended with 500ul pre-cooled PBS and ultrasonically broken. Ultrasonic breaking is repeated 3-5 times in an ice water bath at 5000rpm, centrifuged for 10 minutes, and the supernatant is taken. The protein concentration of the supernatant is determined. The reagent addition method is carried out according to the steps in the instruction manual. The main tests include superoxide dismutase (SOD) enzyme activity, catalase (CAT) enzyme activity, and glutathione peroxidase (GSH-PX) enzyme activity.

[0049] In the experiment, ammonium molybdate was used as an indicator to stop CAT from decomposing H 2 O 2 The reaction can be further combined with the remaining H 2 O 2The reaction produces a light yellow complex, and its activity can be determined by microplate reader. The results show that after treatment with curcumin, the CAT enzyme activity of the test group was significantly improved, which was significantly different from that of the control group. The changes in SOD enzyme activity caused by curcumin treatment of F81 cells can reflect the antioxidant capacity of curcumin. The test results show that the SOD enzyme activity of cells treated with curcumin is greater than that of the control group, reflecting that curcumin can induce and stimulate the activity of SOD, thereby clearing free oxygen radicals in the cell through the action of SOD to enhance the antioxidant capacity. Since glutathione peroxidase can promote the reaction of hydrogen peroxide and reduced glutathione to generate H 2 O and oxidized glutathione, so the activity of glutathione peroxidase can be expressed by the speed of its enzymatic reaction. The experimental results show that the activity of glutathione peroxidase in the curcumin-treated group is significantly enhanced compared with the control group, and the difference is significant ( Figure 8 ), so the results suggest that curcumin contributes to cellular antioxidant effects.

[0050] Twelve domestic cats with chronic kidney disease (CKD), all of whom were patients visiting a pet hospital, were randomly divided into three groups after urine test, blood test and imaging examination. The cats were fed with prescription cat food containing blank control (normal saline), curcumin (10 mg / kg body weight), and curcumin + antibody combination (10 mg / kg body weight curcumin + 100 μL (50 μg / mL) CXCL8 neutralizing antibody (tracheal instillation)) for 2 months. Routine blood test, routine urine test and direct microscopic examination of urine sediment were performed before and after feeding (Table 1).

[0051] Among them, the antibody used in the curcumin + antibody combination group is the CXCL8 antibody developed by the applicant in the early stage, and the amino acid sequence of the light chain variable region is:

[0052] QSPSLDQSPSASKDKVTITCMLSQNIKYAAWYQQKPGKAPGLLIHYTSGLGSGIPSRSGSGSGRAYSFSISNCFSEDIAS YYCLQYDGPFPAGKLELA;

[0053] The amino acid sequence of the heavy chain variable region is:

[0054] EVQLVESGLVSPGLKLSCAVASGFTFANYMTWRQAAPKGLWVASISKGASNPYPDSVKGRFTISRDANAKTLYLQMSLAR SEDTATYCARDGTTDYAWGGAMVTVAS.

[0055] Table 1 Changes in biochemical levels of sick cats before and after the experiment

[0056]

[0057] From the results, it can be seen that urea nitrogen is the main end product of protein metabolism in animals and is mainly excreted by glomerular filtration. Its content reflects the ability of animal nitrogen metabolism and the functional characteristics of animal kidneys. There was no significant difference in serum total protein (TP) in each group of domestic cats before and after the experiment (P>0.05). There was no significant change in the serum urea nitrogen (BUN) content of the control group of sick domestic cats before and after the experiment (P>0.05), but the serum urea nitrogen content of the treatment group decreased significantly before and after the experiment (P<0.05), and the effect of the antibody combined treatment group was significantly better than that of the single curcumin group. The above results also indicate that the supplementation of CXCL13 neutralizing antibodies can alleviate inflammatory cell infiltration, increased lung inflammation, oxidative damage to lung tissue, and pathological damage to lung tissue in chronic kidney disease (CKD). This beneficial effect may be achieved by reducing immune cell infiltration and inhibiting inflammatory response. The results of urine specific gravity also verified the above conclusions. The effect of the drug treatment group was significantly better than that of the blank control, and the effect of combined medication was the best. The relevant indicators were already at normal levels (between 1.035 and 1.06). The results of urine sediment microscopy also showed that in the early stage of the disease, the urine sediment microscopy results of the sick cats showed magnesium ammonium phosphate crystals, with an average of 2.4. After 2 months of feeding, the urine sediment microscopy results of the cats in the curcumin treatment group showed a small amount of magnesium ammonium phosphate crystals, and the urethral stones were improved. No obvious crystals were found in the urine of the curcumin + antibody combination group. The urine output of all cats increased and the symptoms improved.

[0058] In summary, curcumin has a good therapeutic effect on kidney-related diseases in pet cats, and the combined use of CXCL8 neutralizing antibodies has a significant synergistic effect on the prevention and treatment of peroxidative stress in pet cat renal injury, and the effect is significantly improved, which is better than the single drug group.

[0059] The above description of the embodiments is to facilitate the understanding and use of the present invention by those skilled in the art. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative work. Therefore, the present invention is not limited to the above embodiments. Improvements and modifications made by those skilled in the art based on the principles of the present invention without departing from the scope of the present invention should be within the protection scope of the present invention.

Claims

1. A method for establishing a cat kidney cell oxidative stress injury model, characterized in that: The method comprises: taking F81 cells out of liquid nitrogen and quickly placing them in a 37°C water bath, then transferring the cells to a centrifuge tube containing 9 mL of complete culture medium, centrifuging at 1000 rpm for 5 min, and discarding the supernatant; adding 5 mL of fresh RPMI 1640 complete culture medium containing 10% FBS to resuspend the cells and transfer them to a culture bottle, placing the cells in a 37°C incubator containing 5% CO2 for static culture; subsequently subculturing and changing the medium for use of the cells; culturing the cells at 5.0×10 5 / mL was inoculated into a 96-well cell culture plate, 100uL of cell solution was added to each well, and when the cells grew to 80% of the monolayer, the culture medium was discarded, and the medium containing H2O2 was replaced after washing with PBS and induced for 3h to construct a cat kidney H2O2 oxidative damage model; Wherein, the concentration of H2O2 in the H2O2-containing culture medium is selected from 100uM, 150uM and 200uM.

2. A method for screening a substance capable of repairing oxidative stress damage in cat kidney cells, characterized in that: The method is: 1) Resuscitate F81 cells, transfer the cells to a centrifuge tube containing complete medium, centrifuge, and discard the supernatant; 2) Add fresh RPMI 1640 complete medium containing 10% FBS to resuspend the cells and transfer them to a culture flask, and place them in an incubator at 37°C with 5% CO2 for static culture; 3) After subculturing, the cells were inoculated into a 96-well cell culture plate and complete culture medium was added; 4) When the cells grow to 80% of the monolayer, discard the culture medium, wash with PBS, and replace with complete culture medium containing the object to be detected, and incubate for 1-5 hours; 5) After washing with PBS, replace the medium containing H2O2 and induce for 3 hours. Use CCK8 kit to detect cell viability, calculate the effect of different concentrations of H2O2 on cell survival rate, and use a microscope to observe the changes in cell morphology in each group. 6) Determine whether the potential active substance to be tested can maintain the cell morphology. If so, the potential active substance to be tested is a substance that can repair oxidative stress damage to cat kidney cells; If not, it is not a substance that can repair oxidative stress damage to cat kidney cells.

3. The method of claim 2, wherein the concentration of H2O2 in the H2O2-containing culture medium is selected from 100uM, 150uM and 200uM.

4. Use of curcumin or a composition thereof in the preparation of a medicament for treating diseases related to oxidative damage of cat kidney, characterized in that: The oxidative damage-related disease refers to chronic kidney disease (CKD).

5. The use according to claim 4, wherein the composition further comprises a neutralizing antibody against CXCL8 and / or TNF-α.

6. The use according to claim 5, wherein: The amino acid sequence of the light chain variable region of the CXCL8 neutralizing antibody is: QSPSLDQSPSASKDKVTITCMLSQNIKYAAWYQQKPGKAPGLLIHYTSGLGSGIPSRSGSGSGRAYSFSISNCFSEDIAS YYCLQYDGPFPAGKLELA; The amino acid sequence of the heavy chain variable region is: EVQLVESGLVSPGLKLSCAVASGFTFANYMTWRQAAPKGLWVASISKGASNPYPDSVKGRFTISRDANAKTLYLQMSLAR SEDTATYCARDGTTDYAWGGAMVTVAS.

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