Application of DHA and EPA in antioxidation of cat fibroblasts

Abstract

The invention discloses application of DHA (docosahexaenoic acid) and EPA (eicosapentaenoic acid) to oxidation resistance of cat fibroblasts, and belongs to the field of biological medicines. After the cat fibroblasts are subjected to pretreatment of DHA and EPA, DPPH free radicals can be effectively removed, and the oxidation resistance of the cat fibroblasts is improved.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of biological medicine, and particularly relates to application of DHA and EPA to antioxidation of cat fibroblasts. BACKGROUND

[0002] Cat fibroblasts are fibroblasts isolated from cat skin tissue and are commonly used in scientific research experiments. At present, commercially available frozen cat fibroblasts can be purchased on the market, and the recovery, culture, and subculture of the cells are known.

[0003] However, there is no clear literature on how to improve the antioxidation performance of cat fibroblasts by using DHA and EPA. SUMMARY

[0004] The application provides application of DHA and EPA to antioxidation of cat fibroblasts, which comprises the following steps. The application comprises recovery and culture of cat fibroblasts and co-incubation of cat fibroblasts with DHA and EPA. The recovery and culture of cat fibroblasts comprises the following steps: frozen cat fibroblasts are taken out of a refrigeration device and quickly transferred to a 37℃±2℃ water bath for thawing. After the cells are completely thawed, they are transferred to a centrifuge tube containing a culture medium, centrifuged for a period of time, and then the supernatant is discarded. Fresh culture medium is added to the centrifuge tube, the cells are resuspended by gently blowing, and then the cell suspension is transferred to a culture bottle and placed in a constant-temperature incubator at 37℃±2℃ containing 5%±1% CO2 for static culture. After the cells grow to an appropriate state, subculture is performed. The co-incubation of cat fibroblasts with DHA and EPA comprises the following steps: the cat fibroblasts obtained by the above subculture are inoculated on a cell plate, and cell suspension is added to each well. When the cells grow to at least 80% of the monolayer confluence, the original culture solution is discarded, the cells are washed with PBS buffer for 2-3 times, the culture medium containing DHA and EPA is replaced, and the culture is continued. The feature is that the duration of the continued culture is more than 24 hours.

[0005] Further, the concentration of DHA and EPA is not more than 1000 μmol / L.

[0006] Further, the concentration of DHA and EPA is not more than 500 μmol / L, so as to prevent damage to the cat fibroblasts and improve the relative survival rate.

[0007] Further, when measured by a thermometer with a minimum scale of one decimal place, the water bath temperature reading is 37℃.

[0008] Further, when measured by a thermometer with a minimum scale of one decimal place, the constant-temperature incubator temperature reading is 37℃.

[0009] Further, the centrifuging for a period of time means centrifuging for 5 minutes at a speed of 1000 rpm.

[0010] The application provides application of DHA and EPA to cat fibroblasts, so that researchers who later study the relationship between DHA and EPA and cat fibroblasts can obtain clear reference and followable cell culture and incubation methods, and improve cell survival rate and health level. DETAILED DESCRIPTION

[0011] The technical solutions of the application will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only part of the embodiments of the application, rather than all the embodiments of the application. Based on the embodiments in the application, all other embodiments obtained by those skilled in the art without creative work fall within the protection scope of the application.

[0012] In the description of the application, it should be understood that, if terms such as "upper", "lower", "left", "right" and the like appear, they are only for the convenience of describing the application and simplifying the description, and do not indicate or imply that any structure referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore cannot be understood as a limitation on the application. In particular, the understanding of the term "upper" after a noun in the claims should be understood as the entire inner and outer surface of the structure referred to by the noun conforming to the definition of "upper".

[0013] It needs to be additionally pointed out that, if "liquid" or "solution" and the like appear in the application, its meaning is broad, in the application, it should not only be understood as "liquid pure substance" and "solution" that are clear and pure, but also should be understood as including mixture "suspension" formed by suspending small insoluble solid particles in liquid, and mixture "emulsion" formed by dispersing small droplets in another immiscible liquid, and flowable "colloid", and flowable "liquid crystal" and all other substances that basically present as flow phase and can flow freely. In order to facilitate writing and expression, it is abstracted and generalized as "liquid", which is different from the meaning of the ordinary physical term "liquid".

[0014] It needs to be additionally pointed out that, if "tissue" and the like appear in the application, its meaning is broad, in the application, it should not only be understood as cell structure between cells and organs, but also should be flexibly understood as smaller cells, cytoplasm, or larger organs, and even entire organisms and all other certain ranges formed by biological materials according to actual conditions. In order to facilitate writing and expression, it is abstracted and generalized as "tissue", which is different from the meaning of the ordinary biological or medical term "tissue".

[0015] The following describes the specific embodiments, structures, features and effects provided by the present application in accordance with preferred embodiments.

[0016] Example 1 The applicant commissioned the Zhejiang Academy of Agricultural Sciences Institute of Animal Husbandry and Veterinary Medicine to experimentally verify the applicant's ideas. The Zhejiang Academy of Agricultural Sciences Institute of Animal Husbandry and Veterinary Medicine purchased cat fibroblasts from the Kunming Cell Library of the Chinese Academy of Sciences and carried out in vitro verification experiments. With DHA and EPA as the test targets and cat primary fibroblasts as the model, the pre-protective effects of DHA and EPA were systematically evaluated. The cells were interacted with H2O2, and the results showed that the cells in the negative control group remained typical spindle or polygonal, adhered firmly, and the cell arrangement was close, without obvious shedding or fragments. After 100 μM H2O2 alone for 3 h, the positive damage group showed obvious oxidative damage characteristics: cell volume decreased, cell body rounded, intercellular space significantly widened, adhesion rate decreased, a large number of cells shed, cell membrane ruptured, and cell fragments diffused. However, short-term pretreatment with 100 μM or 500 μM DHA or EPA did not effectively alleviate H2O2-mediated cell damage. After pretreatment with DHA or EPA, the cell morphology still showed obvious damage characteristics: cell outline blurred, cell body shrunk, cell number significantly reduced, and high-concentration DHA or EPA group (500 μM) also failed to improve the cell state, with obvious loss of intact cells in the field. CCK-8 quantitative results further confirmed this phenomenon: the cell survival rate of the negative control group was set to 100%, and the cell survival rate of the positive damage group (100 μM H2O2 alone) decreased to about 45%. After pretreatment with 100 μM or 500 μM DHA and EPA, the cell survival rate was about 50%, with no statistically significant difference from the positive damage group. In summary, under the current experimental conditions, whether it was 100 μM or 500 μM DHA or EPA pretreatment, it did not provide effective antioxidant protection to cat fibroblasts, and the cell survival rate was basically the same as that of the untreated oxidative damage group. This result suggests that DHA and EPA may lack direct antioxidant protection in cat fibroblasts, or require higher concentrations, longer pretreatment times, or synergistic effects with other antioxidants to exert effects. However, the detection of MDA indicators in cells found that after 24 h of pretreatment with DHA and EPA, stimulation with H2O2 could reduce the intracellular MDA content by an average of about 37% and 25%, respectively, indicating that DHA and EPA still exerted in vitro antioxidant effects.

[0017] The above experimental results show that the effect of feeding DHA and EPA can not be immediate. The conventional thinking only knows that DHA and EPA have antioxidant properties, and that the antioxidant properties can be improved by using cat fibroblasts. However, the specific principles are not discussed, nor is the use discussed. This is a kind of technical bias. For example, Chinese invention patent CN202311772631.0 discusses that the composition containing DHA and EPA can have antioxidant and anti-inflammatory effects, but the conclusion stops here, without further analyzing the specific principles of antioxidant effects, and without further forming a detailed feeding plan for a specific problem (such as hair condition) of a certain animal (such as a cat).

[0018] Example 2 Continuing the above experiment, the applicant believes that gene expression regulation may be an important factor affecting cell growth and development and antioxidant enzyme activity, and DHA and EPA pretreatment for 24 hours has affected the gene expression of the cells, and this effect needs more than 24 hours to take effect. The coding gene of SOD, manganese superoxide dismutase, is the main antioxidant enzyme gene of mitochondria. CAT gene encodes catalase, which is distributed in cytoplasm and mitochondria, and can protect cells from oxidative damage by removing hydrogen peroxide produced by mitochondria; GPX gene has multiple types. The applicant requires the Institute of Animal Husbandry and Veterinary Medicine of Zhejiang Academy of Agricultural Sciences to further determine the expression amount of the existing transcriptional change gene by using fluorescence quantitative PCR method. After the frozen cat fibroblasts are taken out from the ultra-low temperature freezer at -80°C, they are quickly transferred to a 37°C water bath for rapid thawing. After the cells are completely thawed, they are transferred to a centrifuge tube containing 9 mL of complete culture medium, centrifuged at 1000 rpm for 5 minutes, and then the supernatant is discarded. Add 5 mL of fresh DMEM / F12 complete culture medium (containing 10% fetal bovine serum) to the centrifuge tube, gently resuspend the cells, and then transfer the cell suspension to a culture bottle and place it in a 37°C, 5% CO2 incubator for incubation. After the cells grow to the appropriate state, they are passaged and then used for subsequent experiments. Cat fibroblasts are inoculated in a 96-well cell plate, and 100 μL of cell suspension is added to each well. When the cells grow to 80% confluence, the original culture medium is discarded, and the cells are gently washed 2-3 times with PBS buffer, and then replaced with culture medium containing different final concentrations of DHA and EPA. Continue to culture for 24 hours. As a result, after incubation with DHA and EPA for 24 hours, the expression of antioxidant enzyme genes SOD, CAT and GPX is up-regulated, and is proportional to the concentration of EPA, so DHA and EPA can promote the expression of antioxidant enzymes and enhance the activity of antioxidant enzymes. At the same time, the transcription of vascular endothelial growth factor VEGFA and insulin-like growth factor IGF-1 in the cells is also up-regulated, which enhances the vascularization around the hair follicle, increases the blood supply to the hair follicle, provides sufficient nutrients for hair growth, promotes the growth cycle of the hair follicle, increases the hair density, and reduces abnormal hair loss. At the same time, EPA and DHA can regulate fibroblast growth factor FGF to regulate the activation of hair follicle stem cells. At the same time, DHA and EPA can also alleviate hair folliculitis by inhibiting inflammatory factors such as IL-4, reduce inflammatory damage, and reduce hair loss caused by skin inflammation.

[0019] The above experimental results reveal that the principle of EPA and DHA improving cat hair is gene expression regulation, and this kind of action principle needs a certain effective time, which suggests that the better way is to give sufficient time and maintain the use of EPA and DHA for a long time to improve the antioxidant performance.

[0020] Example 3 To confirm the effect of feeding fish oil containing DHA and EPA on cats in vivo to verify, the applicant commissioned Zhejiang Academy of Agricultural Sciences Institute of Animal Husbandry and Brand pet feeding research and development center to carry out cat in vivo experiment. It should be noted that the experiment is humane and in line with animal protection ethics, and the content only includes feeding, hair collection (combing or shaving), physical examination, hair test and comprehensive observation, which will not cause any animal pain or casualties. Feeding and hair collection are carried out in Brand pet feeding research and development center cat feeding base (address: Xianghu scenic area, Hangzhou), and hair testing is carried out by Zhejiang Academy of Agricultural Sciences Institute of Animal Husbandry. 15 healthy cats are selected, excluding skin problems, weighing 4.25±1.38 kg, gender random, single cat single cage feeding, sufficient immunization and deworming before the test, feeding time for 7d pre-feeding period and 45d feeding period, change cat litter every day, clean the cage, keep clean, and ensure that the cat has enough rest and entertainment time and space. Divided into three groups, five cats in each group, the blank control group is given ordinary cat food dry food 40g and wet food 40g, the control group 1 is long hair cat, in addition to the above ordinary cat food, 200mg / one / day is added to feed 95% high purity fish oil, fish oil is added to wet food, control group 2 is short hair cat, also in addition to the above ordinary cat food, 200mg / one / day is added to feed 95% high purity fish oil, fish oil is added to wet food, and all cats are given 300ml of drinking water every day.

[0021] During the test period, it is observed that: the body weight of each test cat changes little during the test period, and the average daily intake is not significantly different. It can be seen that adding high purity fish oil containing DHA and EPA to cat food will not cause obesity problems in cats; adding high purity fish oil containing DHA and EPA to cat food wet food will not cause anorexia in cats.

[0022] During the test period, it is obtained by double-blind sensory evaluation that: the difference in pet cat hair gloss between the test group and the control group is not significant. The difference in pet cat hair softness D30 between the test group 1 long hair group and the control group is extremely significant, and the difference in D45 is significant; the difference in pet cat hair softness between the test group 2 short hair group and the control group is not significant. The difference in pet cat skin elasticity between the test group and the control group is not significant. The difference in softness between the test group 1 and the control group is obvious. It can be seen that high purity fish oil containing DHA and EPA has no obvious improvement on the sensory gloss of pet cat hair; high purity fish oil containing DHA and EPA has strong and obvious improvement on the sensory softness of long hair pet cat hair from the 30th day; high purity fish oil containing DHA and EPA has no obvious improvement on the sensory softness of short hair pet cat hair; high purity fish oil containing DHA and EPA has no obvious improvement on the sensory skin elasticity of pet cat.

[0023] During the test period, it was found that the hair loss weight of the short-haired group in the test group 2 and the control group was not significantly different, the hair loss weight of the long-haired group in the test group 1 was lower than D0 at D5, D30 and D45, and the difference was significant, the skin moisture of the test group and the control group was not significantly different, the skin oil of the test group and the control group was not significantly different, the skin pH of the test group and the control group was not significantly different, the hair density of the test group 1 and the test group 2 was higher than that at the beginning of the test D0, and the difference was significant, and the difference between the control group at the beginning of the test and at the end of the test was not significant. It can be seen that the high-purity fish oil containing DHA and EPA has no obvious effect on the hair loss weight of short-haired pet cats; high-purity fish oil containing DHA and EPA can significantly reduce the hair loss weight of long-haired pet cats (i.e. reduce hair loss); high-purity fish oil containing DHA and EPA has no obvious effect on the skin moisture, oil and pH of pet cats. It needs to be explained additionally that the skin moisture, oil and pH value here are measured by using RBX-916 capacitive skin tester, the measurement position is the exposed skin of the eyebrow, the skin tester probe contacts the skin surface during measurement, the average value of three measurements is taken, and non-human methods such as destroying and taking the skin of the cat are not used.

[0024] During the test period, it was found that the hair loss weight of the short-haired group in the test group 2 and the control group was not significantly different, the hair loss weight of the long-haired group in the test group 1 was lower than D0 at D5, D30 and D45, and the difference was significant, the skin moisture of the test group and the control group was not significantly different, the skin oil of the test group and the control group was not significantly different, the skin pH of the test group and the control group was not significantly different, the hair density of the test group 1 and the test group 2 was higher than that at the beginning of the test D0, and the difference was significant, and the difference between the control group at the beginning of the test and at the end of the test was not significant. It can be seen that the high-purity fish oil containing DHA and EPA has no obvious effect on the hair loss weight of short-haired pet cats; high-purity fish oil containing DHA and EPA can significantly reduce the hair loss weight of long-haired pet cats (i.e. reduce hair loss); high-purity fish oil containing DHA and EPA has no obvious effect on the skin moisture, oil and pH of pet cats. It needs to be explained additionally that the skin moisture, oil and pH value here are measured by using RBX-916 capacitive skin tester, the measurement position is the exposed skin of the eyebrow, the skin tester probe contacts the skin surface during measurement, the average value of three measurements is taken, and non-human methods such as destroying and taking the skin of the cat are not used.

[0025] In summary, feeding pet cats with high-purity fish oil containing DHA and EPA, whether long-haired or short-haired, will obtain obvious hair improvement, among which long-haired cats improve more obviously, can immediately reduce hair loss within a few days, and significantly improve the softness of touch from the beginning of continuous feeding for 30 days. But short-haired cats will also improve, although this improvement is not obvious to the naked eye, in fact, it can be known from the observation under the electron microscope that it has obtained healthier hair. Therefore, the applicant believes that in order to improve the hair of pet cats, high-purity (95%) fish oil containing DHA and EPA should be fed to pet cats, 40-60 mg per kilogram of body weight per day, and continuously provided for more than 30 days.

[0026] The in vivo test results further confirm the conclusion of the above in vitro test results, that is, the antioxidant effect of DHA and EPA needs to act on cat fibroblasts for more than 24 hours to achieve.

[0027] The above-described embodiments are merely preferred embodiments of the present application, and it should be noted that, for those skilled in the art, some improvements and replacements can be made without departing from the technical principles of the present application, and these improvements and replacements should also be considered as the protection scope of the present application.

Claims

1. An application of DHA and EPA for antioxidant activity in feline fibroblasts, comprising the resuscitation and culture of feline fibroblasts, and co-incubation of feline fibroblasts with DHA and EPA; wherein, The resuscitation and culture of feline fibroblasts includes: rapidly removing frozen feline fibroblasts from the refrigeration equipment and transferring them to a 37℃±2℃ water bath for thawing; once the cells are completely thawed, transferring them to centrifuge tubes containing culture medium, centrifuging for a period of time, discarding the supernatant, adding fresh culture medium to the centrifuge tubes, gently resuspending the cells by pipetting, and then transferring the cell suspension to a culture flask and placing it in a constant temperature incubator at 37℃±2℃ with 5%±1% CO2 for static culture; after the cells have grown to a suitable state, performing passage operations; co-incubating feline fibroblasts with DHA and EPA includes: seeding the feline fibroblasts obtained from the above passage operation into cell plates, adding cell suspension to each well; when the cells grow to at least 80% monolayer confluence, discarding the original culture medium, washing 2-3 times with PBS buffer, replacing with culture medium containing DHA and EPA, and continuing culture; characterized in that the continued culture time is more than 24 hours.

2. The application of DHA and EPA for antioxidant activity in cat fibroblasts according to claim 1, characterized in that, The concentrations of DHA and EPA should not exceed 1000 μmol / L.

3. The application of DHA and EPA for antioxidant activity in cat fibroblasts according to claim 1, characterized in that, The concentrations of DHA and EPA should not exceed 500 μmol / L, thereby preventing damage to feline fibroblasts and improving their relative survival rate.

4. The application of DHA and EPA for antioxidant activity in cat fibroblasts according to claim 1, characterized in that, When measured with a thermometer having one decimal place as the smallest scale division, the water bath temperature reading is exactly 37°C.

5. The application of DHA and EPA for antioxidant activity in cat fibroblasts according to claim 1, characterized in that, When measured with a thermometer having one decimal place as the smallest scale division, the temperature reading of the constant temperature incubator is exactly 37°C.

6. The application of DHA and EPA for antioxidant activity in cat fibroblasts according to claim 1, characterized in that, The centrifugation period refers to centrifuging at 1000 rpm for 5 minutes.