Application of chlorogenic acid in preparation of product for preventing and treating GCHV viral inflammation

Abstract

The invention belongs to the technical field of fish disease prevention and treatment, and particularly relates to application of chlorogenic acid in preparation of a product for preventing and treating GCHV viral inflammation. The regulation and control effects of chlorogenic acids with different concentrations (0.1%, 0.5% and 1%) on inflammatory factors (TNF-alpha, IL-1beta and IL-16) infected by the GCRV II virus are systematically analyzed, and the condition that the expression of the inflammatory factors (TNF-alpha, IL-1beta and IL-16) is obviously inhibited from the third day to the seventh day after infection by 0.5% chlorogenic acid is found; 0.01), the effect of which is not statistically different from that of the blank group (CK) (pgt; 0.05) of the method. If the high-concentration group has proinflammatory effect inversion (IL-1beta superscript 15.4%, p = 0.031) on the ninth day, dose-dependent toxicity is prompted. Results show that 0.5% chlorogenic acid can effectively inhibit viral inflammatory response, and long-term use of high dosage of chlorogenic acid may cause immune disorder.

Description

Technical Field

[0001] The present invention belongs to the technical field of fish disease prevention and control, and particularly relates to the application of chlorogenic acid in the preparation of products for preventing and treating GCHV viral inflammation. Background Art

[0002] Grass carp hemorrhage virus (GCHV), which the International Committee on Taxonomy of Viruses calls reovirus of grass carp (GCRV), is a fish virus belonging to the Reoviridae family and the genus Aquareovirus. It has a diameter of 70 - 80 nm, is an icosahedral spherical particle, and contains 11 segments of double-stranded RNA. Different strains exist in different regions. Currently, 10 isolates have been reported. This virus mainly causes hemorrhagic disease in grass carp, the main variety in freshwater aquaculture, at the fingerling stage, with a mortality rate as high as over 90%, causing huge losses to the aquaculture industry.

[0003] Ma Ning, Huang Yanhong, Tang Geshi, etc. Progress in the application of epigallocatechin gallate in aquaculture [J]. Feed Research, 2024(003):047. It is disclosed that EGCG has been proven to be able to inhibit a variety of DNA viruses and RNA viruses, and its mechanisms include inhibiting the synthesis of viral nucleic acids, destroying virus particles, interfering with the attachment of viruses to host cells, and affecting viral protein metabolism

[19] . In aquaculture, EGCG can inhibit the activities of viruses such as grass carp reovirus (GCRV), cyprinid herpesvirus Ⅱ (CyHV-2), largemouth bass virus (LMBV), white spot syndrome virus (WSSV), and Singapore grouper iridovirus (SGIV). Summary of the Invention

[0004] In order to provide new products with good prevention and treatment effects on grass carp hemorrhage virus, the present invention provides the following technical solutions:

[0005] The present invention discloses the application of chlorogenic acid in the preparation of products for preventing and treating GCHV viral inflammation, and the chlorogenic acid is used as an active ingredient in the products.

[0006] Preferably, the product is a medicine.

[0007] Preferably, the mass fraction of chlorogenic acid in the product is 0.01 - 0.05%.

[0008] Preferably, the mass fraction of chlorogenic acid in the product is 0.05%.

[0009] Preferably, the GCHV viral inflammation is caused by at least one of the inflammatory factors TNF-α and IL-16.

[0010] The present invention also discloses an application of chlorogenic acid in the preparation of a product for preventing and treating GCHV virus, and the chlorogenic acid is used as an active ingredient in the product.

[0011] Preferably, the product is a medicine.

[0012] Preferably, the mass fraction of chlorogenic acid in the product is 0.01-0.05%.

[0013] Preferably, the mass fraction of chlorogenic acid in the product is 0.05%.

[0014] Advantages of the present invention:

[0015] By systematically analyzing the regulatory effects of chlorogenic acid at different concentrations (0.1%, 0.5%, 1%) on inflammatory factors (TNF-α, IL-1β, IL-16) after GCRVⅡ virus infection, it is found that 0.5% chlorogenic acid (0.5L group) significantly inhibits the expression of inflammatory factors from the 3rd to 7th day after infection (TNF-α↓38.2%, IL-16↓47.1%, p<0.01), and its effect has no statistical difference compared with the blank group (CK) (p>0.05). The high-concentration group (1L) shows a reversal of the pro-inflammatory effect on the 9th day (IL-1β↑15.4%, p = 0.031), suggesting dose-dependent toxicity. The results show that 0.5% chlorogenic acid can effectively inhibit the viral inflammatory response, while long-term use at high doses may cause immune imbalance. Description of the drawings

[0016] Figure 1 Comparison of grass carp muscle and intestine in different groups on the seventh day of infection;

[0017] Figure 2 It is a heat map of inflammatory factor concentrations. Heat map description: Color scale: Red represents high concentration (>300 pg / mL), and blue represents low concentration (<200 pg / mL). Detailed implementation manners

[0018] To make the objectives, technical solutions and advantages of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0019] Example 1 Materials and Methods

[0020] 1. Experimental Materials and Preliminary Preparation

[0021] Grass carp were selected as experimental subjects, with a body length range of 12 - 15 cm and an average weight of about 50 g. The grass carp were placed in an aquaculture tank with a volume of 30 L in the fish house for breeding. Under the stable condition that the temperature was maintained at (25 ± 2) °C, the grass carp were acclimated for 7 days to adapt them to the experimental environment, and then the experiment was officially carried out.

[0022] 2. Experimental Groups The following groups were set up in this experiment:

[0023] Normal Control Group The grass carp in this group were not given any special treatment and were fed with ordinary feed. The feeding method was regular and quantitative, with a fixed feeding amount of 1 g each time and 2 times a day to maintain the normal growth and physiological state of the grass carp.

[0024] GCRVⅡ Model Group An LC 400 concentration of GCRV II virus solution was slowly and evenly poured into the water tank where the grass carp were cultured, so that the grass carp were soaked and infected for 15 min to establish a grass carp hemorrhagic disease virus infection model. After infection, they were also fed with ordinary feed, and the feeding amount and frequency were the same as those in the normal control group, that is, 1 g each time and 2 times a day.

[0025] Chlorogenic Acid Treatment Group First, an LC 400 concentration of GCRV II virus solution was poured into the water tank to let the grass carp be soaked and infected for 15 min. Then, according to the different concentrations of chlorogenic acid added, this group was further divided into three subgroups:

[0026] 0.1% Chlorogenic Acid Subgroup: 1 ml of 0.1% chlorogenic acid solution (density 0.0001 g / ml) was accurately added to 1 g of feed, and after being fully stirred and mixed evenly, it was fed to the grass carp.

[0027] 0.5% Chlorogenic Acid Subgroup: 1 ml of 0.5% chlorogenic acid solution (density 0.0005 g / ml) was accurately added to 1 g of feed, and after being fully mixed evenly, it was fed to the grass carp.

[0028] 1.0% Chlorogenic Acid Subgroup: 1 ml of 1% chlorogenic acid solution (density 0.001 g / ml) was accurately added to 1 g of feed, and after being fully mixed evenly, it was fed to the grass carp

[0029] The feeding amount and frequency of each subgroup were 1 g each time at fixed time and 2 times a day.

[0030] 3. Acclimation Observation and Sample Collection

[0031] The grass carps in all experimental groups were temporarily raised in an environment with a temperature maintained at about 28°C for 9 days. During the temporary raising period, the behavior dynamics and death conditions of the grass carps were closely observed and detailed records were made. On the 1st, 3rd, 5th, 7th, and 9th days respectively, grass carp samples were collected from each group. The specific operation is as follows: When sampling, the grass carp was dissected aseptically, and its liver, spleen, kidney, intestine, gill, and muscle tissues were carefully taken out, and the taken tissues were quickly put into a cryopreservation tube pre-prepared with 50 mL of tissue protective solution, and immediately placed in a low-temperature environment for refrigerated storage for subsequent experimental analysis.

[0032] 4. ELISA Detection Method

[0033] The enzyme-linked immunosorbent assay (ELISA) method was used to detect relevant indicators of the collected grass carp tissue samples according to the operation instructions of the corresponding kit (Jiangsu enzyme immunoassay). Each link in the experimental operation process was strictly controlled, including sample dilution, reagent addition, incubation time and temperature, etc., to ensure the accuracy and reliability of the detection results.

[0034] Operation Steps

[0035] (1) Take out the required strips from the aluminum foil bag after equilibrating at room temperature for 20 min, and seal the remaining strips with a self-sealing bag and put them back at 4°C.

[0036] (2) Set up standard product wells and sample wells, and add 50 μL of standard products with different concentrations to each standard product well:

[0037] (3) First add 10 μL of the sample to be tested to the sample well, and then add 40 μL of sample diluent. Do not add anything to the blank well.

[0038] (4) Except for the blank well, add 100 μL of the detection antibody labeled with horseradish peroxidase (HRP) to each well of the standard product well and the sample well. Seal the reaction wells with a sealing plate membrane and incubate in a 37°C water bath or incubator for 60 min.

[0039] (5) Discard the liquid, pat dry on the absorbent paper, fill each well with washing solution, let stand for 1 min, discard the washing solution, pat dry on the absorbent paper, and repeat the washing process 5 times (the plate can also be washed with a plate washer).

[0040] (6) Add 50 μL of Substrate A and B to each well and incubate at 37°C in the dark for 15 min

[0041] (7) Add 50 μL of stop solution to each well, and measure the OD value of each well at a wavelength of 450 nm within 15 min. Result Judgment

[0042] Draw a standard curve: In the Excel worksheet, use the standard product concentration as the abscissa and the corresponding OD value as the ordinate to draw the standard product linear regression curve, and calculate the concentration values of each sample according to the curve equation.

[0043] 5. Statistical analysis

[0044] Method: One-way analysis of variance (ANOVA) combined with Tukey's multiple comparison test.

[0045] Significance criteria: *p < 0.05, **p < 0.01, ***p < 0.001.

[0046] Software: GraphPad Prism 9.0, data are expressed as mean ± standard deviation.

[0047] Results

[0048] 1. Comparison results of muscle and intestine of grass carp in different groups on the seventh day of infection are as Figure 1 shown. Pathological damage in the GCRV group: Samples on the 7th day showed significant bleeding points in muscle and intestine (diffuse ecchymosis visible to the naked eye), indicating that the virus caused increased vascular permeability and tissue damage. 0.5% chlorogenic acid group (0.5L): There were no bleeding points visible to the naked eye in muscle and intestine, and there was no obvious difference in tissue morphology from the CK group observed by the naked eye.

[0049] 2. Data table of inflammatory factor concentrations

[0050] Table 1 TNF-α concentration (pg / mL, mean ± SD)

[0051]

[0052] Table 2 IL-1β concentration (pg / mL, mean ± SD)

[0053]

[0054] Table 3 IL-16 concentration (pg / mL, mean ± SD)

[0055]

[0056]

[0057] Symbol description:

[0058] ●▲: Significantly higher than the CK group (p < 0.05)

[0059] ·*: Significantly decreased compared with the GCRV group (p < 0.05)

[0060] ·**: Significantly decreased compared with the GCRV group (p < 0.01)

[0061] ·▲▲: Significantly higher than the CK group (p < 0.01)

[0062] 3. Heatmap visualization analysis

[0063] The results are as Figure 2 shown below:

[0064] GCRV group: On the 3rd day, IL-16 (319.4 pg / mL) and on the 7th day, TNF-α (287 pg / mL) showed dark red color, reflecting the peak of inflammation during the active virus period.

[0065] 0.5L group: On the 7th day, a blue area was formed (TNF-α: 178 pg / mL, IL-16: 217 pg / mL), indicating a significant inhibitory effect.

[0066] 1L group: On the 9th day, a red patch suddenly appeared in IL-16 (475 pg / mL), showing a toxicity reversal.

[0067] 4. Significant comparison

[0068] Table 4 Significant analysis on the 7th day

[0069]

[0070] Conclusion

[0071] 1. Verification of the best anti-inflammatory regimen

[0072] 0.5% chlorogenic acid group (0.5L):

[0073] Time window: The 3rd - 7th day after infection is the critical protection period. The inhibition rates of TNF-α and IL-16 reach 38.2% (p = 0.0012) and 47.1% (p = 0.00009) respectively, and there is no significant difference compared with the blank group (CK) (p > 0.05)

[0074] Dose specificity: 0.5% is the threshold concentration. Below this dose (0.1L), the effect is weak (inhibition rate < 20%), and above this dose (1L), it causes toxicity reversal.

[0075] Inflammatory factor inhibition (TNF-α↓38.2%, IL-16↓47.1%) is highly correlated with pathological protection (r = 0.92, p < 0.001), confirming that chlorogenic acid reduces tissue damage by regulating the inflammatory response.

[0076] It is speculated that chlorogenic acid achieves pathological protection through the following pathways: inhibiting the cytokine storm, that is, chlorogenic acid may block the apoptosis of vascular endothelial cells induced by TNF-α (via the Caspase-3 pathway); downregulating the expression of matrix metalloproteinase (MMP-9) mediated by IL-16, reducing the degradation of the basement membrane, and maintaining vascular integrity; at the same time, chlorogenic acid may also scavenge ROS to inhibit tissue leakage related to oxidative stress.

[0077] 2. Analysis of the toxicity mechanism of high dose

[0078] 1% Chlorogenic Acid Group (1L):

[0079] Time-dependent toxicity: Pro-inflammatory factor rebound occurred on the 9th day (TNF-α↑23.6%, p = 0.031; IL-16↑29.7%, p = 0.007), possibly due to over-activation of the ROS / NLRP3 pathway.

[0080] Dose-effect paradox: IL-16 was abnormally increased by 25.2% on the early stage (the 1st day) (p = 0.004), suggesting caution should also be taken for short-term use at high doses.

[0081] 3. Virus-host interaction pattern

[0082] The peak value of IL-16 on the 3rd day (319.4 pg / mL) was synchronized with the peak of virus replication (r = 0.89, p < 0.01), which could be used as a biomarker for the infection process; TNF-α spontaneously decreased (↓22.3%) on the 5th day in the GCRV group, which might reflect the strategy of the virus to inhibit the early immune response of the host.

Claims

1. The use of chlorogenic acid in the preparation of a product for preventing and treating GCHV viral inflammation, characterized in that: The chlorogenic acid is used as an active ingredient in the product.

2. The use according to claim 1, characterized in that: The product described is a pharmaceutical product.

3. The use according to claim 1, characterized in that: The mass fraction of the chlorogenic acid in the product is 0.01-0.05%.

4. The use according to claim 3, characterized in that: The mass fraction of the chlorogenic acid in the product is 0.05%.

5. The use according to claim 1, characterized in that: The GCHV viral inflammation is caused by at least one of the inflammatory factors TNF-α and IL-16.

6. The use of chlorogenic acid in the preparation of a product for preventing and treating GCHV virus, characterized in that: The chlorogenic acid is used as an active ingredient in the product.

7. The use according to claim 6, characterized in that: The product described is a pharmaceutical product.

8. The use according to claim 6, characterized in that: The mass fraction of the chlorogenic acid in the product is 0.01-0.05%.

9. The use according to claim 8, characterized in that: The mass fraction of the chlorogenic acid in the product is 0.05%.

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