Application of quercetin in preparation of medicine for resisting livestock and poultry spleen injury induced by deoxynivalenol

Abstract

The invention discloses an application of quercetin in preparation of a medicine for resisting livestock and poultry spleen injury induced by deoxynivalenol, and belongs to the technical field of veterinary drugs. In-vivo and in-vitro experiments prove that the deoxynivalenol induces endoplasmic reticulum stress to cause oxidative stress, pyroptosis, inflammatory response and mitochondrial dysfunction so as to cause spleen injury. By down-regulating ERS related genes, the quercetin inhibits ROS generation, blocks NLRP3 inflammasome activation and repairs mitochondrial dynamic imbalance, and significantly alleviates Don-induced spleen injury. The invention provides a novel drug target and an application scheme for detoxification of mycotoxin.

Description

Technical Field

[0001] This application belongs to the field of veterinary drug technology, and in particular relates to the application of quercetin in the preparation of drugs for treating deoxynivalenol-induced spleen damage in livestock and poultry. Background Technology

[0002] Deoxynivalenol (Don), commonly known as vomitoxin, is a fungal toxin widely found in cereals that can cause damage to the immune organs of livestock and poultry. The spleen, as the largest immune organ, suffers damage through a mechanism that remains unclear. Current research indicates that Don can induce endoplasmic reticulum stress (ERS), oxidative stress, mitochondrial dysfunction, and inflammatory responses, but effective prevention and treatment methods are lacking. Quercetin (Que) is a widely available plant extract with various biological activities, including antioxidant and anti-inflammatory properties, but its protective effect against Don-induced spleen damage has not been reported. Summary of the Invention

[0003] To overcome the aforementioned deficiencies in the prior art, this application provides the use of quercetin in the preparation of a drug for treating deoxynivalenol-induced spleen damage in livestock and poultry.

[0004] To achieve the above-mentioned objectives, this application provides the following technical solution:

[0005] On the one hand, this application provides the use of quercetin in the preparation of a drug for treating deoxynivalenol-induced spleen damage in livestock and poultry.

[0006] Optionally, the drug uses quercetin as the sole active ingredient or one of its active ingredients;

[0007] The livestock and poultry include broiler chickens.

[0008] Optionally, the effective dosage of the drug is 3 to 5 mg per kilogram of animal body weight.

[0009] Optionally, the effective dosage of the drug is 4 mg per kilogram of animal body weight.

[0010] Optionally, the drug exerts its effects by inhibiting endoplasmic reticulum stress, oxidative stress, mitochondrial dysfunction, pyroptosis, and inflammatory responses.

[0011] Optionally, the drug inhibits endoplasmic reticulum stress by downregulating the expression levels of endoplasmic reticulum stress-related genes GRP78, PERK, ATF-6, and CHOP.

[0012] Optionally, the drug inhibits oxidative stress by upregulating the activity of antioxidant enzymes CAT, GSH-PX, and SOD, and downregulating the expression levels of reactive oxygen species (ROS) and malondialdehyde (MDA).

[0013] Optionally, the drug inhibits mitochondrial dysfunction by downregulating the expression levels of mitochondrial dynamics-related genes MFF, DRP1, and FIS1, and upregulating the expression levels of MFN1, MFN2, and OPA1.

[0014] Optionally, the drug exerts its effect by inhibiting the expression of NLRP3, GSDMD, ASC, Caspase-1, IL-18, IL-1β, and TNF-α.

[0015] Optionally, the dosage form of the drug includes injection, powder for injection, tablet, capsule, powder, oral liquid, granules, pills or suppositories;

[0016] The drug also contains pharmaceutically acceptable excipients.

[0017] Compared with the prior art, this application has the following advantages:

[0018] (1) This application is the first to confirm that quercetin (hereinafter referred to as Que) can alleviate spleen damage in broilers induced by deoxynivalenol (hereinafter referred to as Don), filling the gap in the research on Don toxicity mechanism and natural antidote.

[0019] (2) This application demonstrates through in vitro and in vivo experiments that Don induces endoplasmic reticulum stress (ERS), triggering oxidative stress, pyroptosis, inflammatory responses, and mitochondrial dysfunction, leading to spleen damage. Quercetin significantly alleviates Don-induced spleen damage by downregulating ERS-related genes, inhibiting ROS generation, blocking NLRP3 inflammasome activation, and repairing mitochondrial dynamic imbalance. Quercetin, as a natural compound, has high safety and can be used as a feed additive or veterinary drug to reduce economic losses caused by Don pollution in the livestock industry, thus having broad application value. Attached Figure Description

[0020] To more clearly illustrate the technical solutions of the embodiments of this application, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of this application and should not be regarded as a limitation of the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0021] Figure 1 The broiler chicken condition and necropsy observation results are presented in this application;

[0022] Figure 2 This is the morphological observation result of the broiler spleen in this application;

[0023] Figure 3The H&E staining results of broiler spleens in this application are shown below (Note: Blue arrows point to splenic nodules, orange arrows point to the central artery, yellow arrows point to the periarterial lymphatic sheath, green arrows point to the trabecular arteries, and red arrows point to erythrocytes. The area where WP is located represents white pulp, and the area where RP is located represents red pulp).

[0024] Figure 4 The transmission electron microscopy results of the broiler spleen in this application are shown below (Note: The letter Nuc represents the location of the cell nucleus. For easier observation, the right side is a magnified view of the area within the black box on the left. The red box represents mitochondria, the green box represents the endoplasmic reticulum, and the blue box represents fibrotic deposits).

[0025] Figure 5 The expression results of endoplasmic reticulum stress-related gene mRNA in broiler spleen tissue for this application (Note: different letters indicate differences between groups);

[0026] Figure 6 The expression results of endoplasmic reticulum stress-related genes and proteins in the spleen tissue of broilers in this application (Note: different letters indicate differences between groups);

[0027] Figure 7 The results of the detection of oxidative stress level in broiler spleen tissue in this application (Note: different letters indicate differences between groups);

[0028] Figure 8 The expression results of mitochondrial dynamics-related gene mRNA in broiler spleen tissue of this application (Note: different letters indicate differences between groups);

[0029] Figure 9 The results of immunofluorescence double staining of broiler spleen tissue for this application (Note: red fluorescence represents GSDMD, green fluorescence represents NLRP3; different letters indicate differences between groups).

[0030] Figure 10 The expression results of pyroptosis-related gene mRNA in broiler spleen tissue of this application (Note: different letters indicate differences between groups);

[0031] Figure 11 The results show the expression of pyroptosis-related gene proteins in the spleen tissue of broilers in this application (Note: different letters indicate differences between groups);

[0032] Figure 12 The expression results of inflammation-related gene mRNA in broiler spleen tissue of this application (Note: different letters indicate differences between groups);

[0033] Figure 13 The expression results of inflammation-related gene proteins in broiler spleen tissue of this application (Note: different letters indicate differences between groups);

[0034] Figure 14 The effect of different concentrations of Don and Que on the survival rate of MDCC-MSB-1 cells in this application (Note: **** indicates differences between groups);

[0035] Figure 15 This application investigates the effect of Que on the survival rate of MDCC-MSB-1 cells after Don exposure (Note: **** indicates differences between groups);

[0036] Figure 16 The expression results of ERS-related gene mRNA and protein in MDCC-MSB-1 cells of this application (Note: different letters indicate differences between groups);

[0037] Figure 17 The results of ROS staining in MDCC-MSB-1 cells for this application (Note: different letters indicate differences between groups);

[0038] Figure 18 The results of oxidative stress detection in MDCC-MSB-1 cells in this application (Note: different letters indicate differences between groups);

[0039] Figure 19 The results of mtROS staining in MDCC-MSB-1 cells for this application (Note: different letters indicate differences between groups);

[0040] Figure 20 JC-1 staining results for MDCC-MSB-1 cells in this application (Note: Different letters indicate differences between groups);

[0041] Figure 21 The expression results of mitochondrial dynamics-related gene mRNA in MDCC-MSB-1 cells of this application (Note: different letters indicate differences between groups);

[0042] Figure 22 The immunofluorescence results of MDCC-MSB-1 cells for this application (Note: different letters indicate differences between groups);

[0043] Figure 23 The expression results of pyroptosis-related genes mRNA and protein in MDCC-MSB-1 cells of this application (Note: different letters indicate differences between groups);

[0044] Figure 24 The expression results of inflammation-related gene mRNA in MDCC-MSB-1 cells of this application (Note: different letters indicate differences between groups);

[0045] Figure 25 The expression results of inflammation-related gene proteins in MDCC-MSB-1 cells of this application (Note: different letters indicate differences between groups);

[0046] Figure 26 The effect of different concentrations of Don and Que on the survival rate of spleen lymphocytes (Note: **** indicates differences between groups);

[0047] Figure 27 This study investigated the effect of Que on the survival rate of spleen lymphocytes after Don exposure (Note: **** indicates differences between groups);

[0048] Figure 28 The results show the expression of ERS-related gene mRNA and protein in splenic lymphocytes in this application (Note: different letters indicate differences between groups);

[0049] Figure 29 The results of ROS staining of spleen lymphocytes in this application (Note: different letters indicate differences between groups);

[0050] Figure 30 The results of oxidative stress detection in splenic lymphocytes in this application (Note: different letters indicate differences between groups);

[0051] Figure 31 The results of mtROS staining of spleen lymphocytes in this application (Note: different letters indicate differences between groups);

[0052] Figure 32 The results of JC-1 staining of spleen lymphocytes in this application (Note: different letters indicate differences between groups);

[0053] Figure 33 The expression results of mitochondrial dynamics-related genes mRNA in spleen lymphocytes in this application (Note: different letters indicate differences between groups);

[0054] Figure 34 The results show the expression of pyroptosis-related genes mRNA and protein in spleen lymphocytes in this application (Note: different letters indicate differences between groups);

[0055] Figure 35 The expression results of splenic lymphocyte inflammation-related genes mRNA and protein in this application (Note: different letters indicate differences between groups);

[0056] Figure 36 The effect of 4-PBA treatment on the survival rate of MDCC-MSB-1 cells after Don exposure (Note: *** indicates differences between groups);

[0057] Figure 37 The results of this application on the effect of 4-PBA treatment on ERS of MDCC-MSB-1 cells (Note: different letters indicate differences between groups);

[0058] Figure 38 The results of the effect of 4-PBA treatment on ROS generation in MDCC-MSB-1 cells in this application (Note: different letters indicate differences between groups);

[0059] Figure 39 The results of this application on the effect of 4-PBA treatment on oxidative stress in MDCC-MSB-1 cells (Note: different letters indicate differences between groups);

[0060] Figure 40 The results of the effect of 4-PBA treatment on immunofluorescence staining of MDCC-MSB-1 cells in this application (Note: different letters indicate differences between groups);

[0061] Figure 41 The results of this application on the effect of 4-PBA treatment on the expression of pyroptosis-related genes mRNA in MDCC-MSB-1 cells (Note: different letters indicate differences between groups);

[0062] Figure 42 The results of this application on the effect of 4-PBA treatment on the expression of pyroptosis-related gene proteins in MDCC-MSB-1 cells (Note: different letters indicate differences between groups);

[0063] Figure 43 The results of the effect of 4-PBA treatment on inflammation of MDCC-MSB-1 cells in this application (Note: different letters indicate differences between groups). Detailed Implementation

[0064] The present application is further illustrated below with reference to specific embodiments. The following descriptions are merely a few embodiments of the present application and are not intended to limit the present application in any way. Although the present application discloses preferred embodiments as follows, they are not intended to limit the present application. Any modifications or variations made by those skilled in the art without departing from the scope of the technical solution of the present application using the disclosed technical content are equivalent to equivalent implementation cases and all fall within the scope of the technical solution.

[0065] Unless otherwise specified, the raw materials used in the embodiments of this application are all purchased commercially and used directly without any special treatment.

[0066] Unless otherwise specified, the analytical methods in the embodiments all adopt conventional instrument or equipment settings and conventional analytical methods.

[0067] Example 1

[0068] I. Materials and Reagents

[0069] Experimental animals: 1-day-old white-feathered broiler chickens (purchased from Harbin Shanhe Poultry Industry, Heilongjiang Province).

[0070] Main reagents: Don (Maclean), Que (Aladdin), 4-PBA (MCE), ROS detection kit (Nanjing Jiancheng), etc.

[0071] II. Experimental Procedure

[0072] 1. In vivo experiments

[0073] Grouping and Dosing:

[0074] 80 broiler chickens were randomly divided into 4 groups:

[0075] Control group: 1 mL corn oil was administered by gavage.

[0076] Don group: 10 mg / kg Don corn oil solution was administered by gavage.

[0077] Que group: 4 mg / kg Que corn oil solution was administered by gavage.

[0078] Don+Que group: 10 mg / kg Don + 4 mg / kg Que corn oil solution were administered by gavage.

[0079] Sample collection: Broilers were euthanized 30 days later, and spleen tissue was taken for pathological and molecular testing.

[0080] 2. In vitro experiments

[0081] Cell culture: MDCC-MSB-1 cells and spleen lymphocytes were treated with Don (2 μM / 1 μM) and Que (5 μM), respectively, and pretreated with 4-PBA (1 mM) as an ERS inhibitor.

[0082] III. Experimental Results and Analysis

[0083] 1. Broiler condition and necropsy observation

[0084] The broiler gavage model was conducted for 30 days. On day 14, six broilers in the Don group showed loss of appetite, drooping their heads in the corner of the cage, and were lethargic. By day 21, all broilers in the Don group exhibited varying degrees of anorexia, drooling, open-mouth breathing, facial swelling, cyanosis of the comb and abdomen, and ruffled or even fallen feathers. At this time, broilers in the Don+Que group showed reduced feed intake and some were lethargic, but no drooling or cyanosis was observed. From day 28 to 30, broilers in the Don group died daily. After day 30, four broilers in the Don+Que group showed drooling and cyanosis of the comb, but to a lesser degree than in the Don group. Other broilers showed similar symptoms to those in the Don group, but to a significantly lesser degree, and their overall condition improved compared to the Don group, with no deaths observed. Throughout the gavage period, broilers in the Con and Que groups showed normal feed and water intake and no abnormalities in their mental state. During necropsy, varying degrees of swelling and congestion were observed in the abdominal organs of broilers in the Don group. Swelling was less pronounced in the Don+Que group, while no abnormalities were found in the organs of broilers in the Con and Que groups. These results are shown in [the table below]. Figure 1 .

[0085] 2. Results of the detection of the effects of Don and Que on the spleen of broilers

[0086] (1) Results of the effects of Don and Que on the morphology of broiler spleen

[0087] Morphological changes of the spleen, such as Figure 2 As shown, the spleen tissue in the Con and Que groups was smooth, regularly shaped, relatively small, and a normal dark red color, with elasticity to the touch. The spleen in the Don group was enlarged, congested, and deep red, with visible yellow infarct foci; it was firm to the touch, brittle, and easily broken. Although the spleen in the Don+Que group was also enlarged, it also showed scattered infarct foci, but to a lesser extent than in the Don group, and its color was similar to normal tissue. These results suggest that Don exposure causes morphological damage to the spleen tissue, while the addition of Que has a mitigating effect.

[0088] (2) Results of the effects of Don and Que on the microstructure of broiler spleen

[0089] H&E staining was used to detect pathological damage to the spleen of broilers. The staining results are as follows: Figure 3As shown in the image, blue arrows point to splenic nodules, orange arrows to the central artery, yellow arrows to the periarterial lymphatic sheath, green arrows to the trabecular arteries, and red arrows to erythrocytes. The area marked WP represents white pulp, and the area marked RP represents red pulp. Based on the staining results, the white and red pulp boundaries in the spleen tissue of the Con and Que groups are clear, and the splenic nodules are structurally intact with clear boundaries. The central artery and periarterial lymphatic sheath are tightly arranged and structurally intact, with lymphocytes evenly distributed. In the Don group, however, the boundary between white and red pulp is difficult to distinguish; the two structures are mixed together, and structures such as the central artery are not observed. In the Don group, only thickened trabecular arteries and aggregated erythrocytes are visible in the center of the image, and the overall structure appears very loose. The Don+Que group shows some improvement compared to the Don group, exhibiting a looser overall structure, with the splenic nodule structure observable, the white and red pulp roughly identifiable, the central artery and periarterial lymphatic sheath more tightly arranged, and occasional erythrocyte aggregation. These results indicate that Don can cause pathological damage to the spleen of broilers, while Que can alleviate this damage.

[0090] (3) Results of the effects of Don and Que on the ultrastructure of broiler spleen

[0091] The ultrastructure of broiler spleen was observed using transmission electron microscopy, and the results are as follows: Figure 4 As shown. The letter Nuc represents the location of the cell nucleus. For easier observation, the right side is a magnified view of the left side. The red box represents mitochondria, the green box represents the endoplasmic reticulum, and the blue box represents fibrotic deposits. Based on the electron microscopy results, the nuclear structure was intact, the nuclear membrane was clear, the mitochondrial cristae were neatly arranged and normal in morphology, and the endoplasmic reticulum membrane structure was intact in the Con and Que groups. In the Don group, nuclear condensation, membrane rupture, leakage of nuclear contents, mitochondrial swelling, cristae breakage or vacuolation, and high swelling of the endoplasmic reticulum were observed, along with a large amount of fibrous deposits. In the Don+Que group, some mitochondrial structures were abnormal, the swelling of the endoplasmic reticulum was reduced (orange box), and the amount of fibrous deposits was reduced. These results indicate that Don causes damage to the ultrastructure of the broiler spleen, and Que has a mitigating effect.

[0092] (4) Results of the effects of Don and Que on ERS-related genes in broiler spleen

[0093] The effects of Don and Que on the expression of ERS-related gene mRNA in broiler spleen are as follows: Figure 5As shown. Compared with the Con group, the expression levels of GRP78, ATF-6, and CHOP in the Que group were slightly decreased, but there was no significant difference (P>0.05). The expression levels of GRP78, PERK, ATF-6, and CHOP in the Don group were significantly increased compared with the Con group (P<0.05). Compared with the Don group, the expression of all genes in the Don+Que group was significantly decreased (P<0.05), and although the expression levels of PERK and CHOP were higher than those in the Con group, there was no significant difference (P>0.05). In addition, protein levels were also detected in this application ( Figure 6 Similar to the mRNA expression results, the expression levels of GRP78, ATF-6, and CHOP in the Don group were 201.67%, 210.67%, and 166.3% higher than those in the Con group, respectively. In the Don+Que group, these expression levels were 31.24%, 42.14%, and 37.8% lower than those in the Don group (P<0.05), while the expression levels of ATF-6 and CHOP showed no significant difference compared to the Con group (P>0.05). This indicates that Don exposure leads to ERS in broiler spleens, while the addition of Que can alleviate ERS levels.

[0094] (5) Results of the effects of Don and Que on oxidative stress levels in broiler spleen

[0095] The effects of Don and Que on the activity of antioxidant enzymes and total antioxidant capacity in broiler spleen are as follows: Figure 7 As shown in the figure. The relevant indicators were detected using reagent kits from the Nanjing Jiancheng Research Institute. Compared with the Con group, the Que group showed significantly increased GSH-PX and SOD activities (P<0.05), while CAT activity and T-AOC levels were significantly higher than the Con group (P>0.05), and MDA production was significantly lower than the Con group (P>0.05). Compared with the Con group, the Don group showed significantly increased MDA production, but all other indicators were significantly lower (P<0.05). Compared with the Don group, the Don+Que group showed significantly lower MDA production, significantly increased CAT, GSH-PX, and SOD activities, and significantly increased T-AOC levels (P<0.05). This indicates that Don exposure leads to oxidative stress in the spleen of broilers, while the addition of Que can alleviate oxidative stress levels.

[0096] (6) To investigate the Don exposure-induced mitochondrial dysfunction in broiler spleen tissue and the protective role of Que in this process, this application examined the mRNA expression of genes related to mitochondrial dynamics, such as... Figure 8As shown in the bar chart, the expression of mitochondrial fragmentation-related genes MFF, FIS1, and DRP1 was significantly upregulated in the Don group, and significantly downregulated in the Don+Que group compared to the Don group (P<0.05). For mitochondrial fusion-related genes MFN1, MFN2, and OPA1, their expression was increased in the Que group compared to the Con group, but the expression of the first two genes did not differ significantly from the Con group (P>0.05), while the expression of OPA1 was significantly higher than that in the Con group (P<0.05). Compared to the Don group, the expression of all three genes was significantly increased in the Don+Que group (P<0.05). This indicates that Don exposure leads to an imbalance in mitochondrial fusion and fragmentation in the broiler spleen, and the addition of Que can effectively alleviate this imbalance.

[0097] (7) Results of the effects of Don and Que on mid-spleen mortality-related indicators in broiler chickens

[0098] To investigate Don exposure-induced pyroptosis in broiler spleen tissue and the alleviating role of Que, this application used multiple methods for validation. First, immunofluorescence was performed on spleen tissue sections, with double staining for NLRP3 and GSDMD. The staining results are shown below. Figure 9 Red fluorescence represents GSDMD, and green fluorescence represents NLRP3. It can be seen that the fluorescence intensity of GSDMD in the Que group was not significantly different from that in the Con group (P>0.05), while the fluorescence intensity of NLRP3 was significantly lower than that in the Con group (P<0.05). The fluorescence intensities of both NLRP3 and GSDMD in the Don+Que group were upregulated compared to the Con group, and significantly downregulated compared to the Don group (P<0.05). Subsequently, the transcriptional levels of pyroptosis-related genes were detected. Figure 10 The results showed that, compared with Con, the mRNA levels of GSDMD and Caspase-1 in the Que and Don+Que groups were not significantly different from those in Con (P>0.05), while the expression of NLRP3, ASC, IL-1β, and IL-18 in the Don+Que group was significantly higher than that in the Con group and significantly lower than that in the Don group (P<0.05), with ASC showing the highest relative transcriptional level in the Don group. Similar to the results of transcriptional levels, this study performed a Western blot assay to detect protein expression levels. Figure 11Compared with Con, the protein levels of NLRP3, ASC, and IL-18 in the Que and Don+Que groups were not significantly different from those in Con (P>0.05). However, the expression levels of GSDMD, IL-1β, and Caspase-1 in the Don+Que group were significantly higher than those in the Con group and significantly lower than those in the Don group (P<0.05), with GSDMD showing the highest expression level in the Don group. In conclusion, Don exposure leads to pyroptosis of the spleen in broilers, while the addition of Que can alleviate the degree of pyroptosis.

[0099] (8) Results of the effects of Don and Que on inflammation-related markers in broiler spleen

[0100] Pyroptosis is an inflammatory cell death process; therefore, this application also investigated the effects of Don and Que on inflammation-related markers in broiler spleens. qRT-PCR and Western blotting were used to detect some inflammatory genes. Figure 12 , Figure 13 The results showed that Don exposure significantly upregulated the mRNA levels of TNF-α, IL-2, IL-6, and IFN-γ, while the Don+Que group showed the opposite trend compared to the Don group (P<0.05). Western blot analysis detected the expression levels of some proteins. Similar to transcriptional levels, TNF-α and IL-6 expression was increased in the Don group and significantly different from the other three groups (P<0.05), while the expression levels in the other three groups showed no significant difference (P>0.05). In conclusion, Don exposure leads to spleen inflammation in broilers, while the addition of Que can alleviate the inflammation.

[0101] 3. Results of the detection of the effects of Don and Que on MDCC-MSB-1 cells

[0102] (1) To determine the optimal exposure concentration of Don and the optimal therapeutic concentration of Que in MDCC-MSB-1 cells, this application used the CCK8 kit to determine the reference dosages of Don and Que. The results are as follows: Figure 14 As Don concentration increased (0, 1, 2, 4, 8, 16, 32, 64, 128 μM), cell viability decreased, reaching only 53.49% at 8 μM. Cell viability was 75.28% at 2 μM, therefore 2 μM was chosen as the Don exposure concentration. In exploring the safe concentration range of Que, the study found that at lower concentrations, cell viability was not dose-dependent on Que concentration (0, 0.156, 0.312, 0.625, 1.25, 2.5, 5 μM). However, when the Que concentration reached 10 μM, cell viability significantly decreased (P < 0.05). In the results of co-treatment with Don and Que (…), Figure 15Ultimately, the treatment concentration of Que was chosen to be 5 μM, at which point the cell survival rate reached its highest level.

[0103] (2) Results of the effects of Don and Que on ERS in MDCC-MSB-1 cells

[0104] The transcriptional levels and protein expression of ERS-related genes in MDCC-MSB-1 cells exposed to Don and Que were analyzed, and the following results were obtained ( Figure 16 At the transcriptional level, the levels of GRP78, PERK, ATF-6, and CHOP were significantly increased in the Don group (P<0.05), with CHOP transcription increasing nearly 5-fold. There were no significant differences in gene expression between the Con and Que groups, and PERK expression in the Don+Que group was also not significantly different from the Con group (P>0.05). Compared to the Don group, all genes in the Don+Que group were significantly decreased (P<0.05). Protein expression results were similar to those at the transcriptional level. GRP78, ATF-6, and CHOP protein expression were significantly higher in the Don group than in the Con group, and significantly decreased in the Don+Que group compared to the Don group (P<0.05). GRP78 decreased to the point where it was not significantly different from the Con group, and CHOP decreased to the point where it was not significantly different from the Que group (P>0.05). Therefore, this application demonstrates that Don induces ERS in MDCC-MSB-1 cells, and the addition of Que has the opposite effect.

[0105] (3) The ROS production level in MDCC-MSB-1 cells was detected using the DCFH-DA fluorescent probe, and the results are as follows: Figure 17 As shown in the figure. The intensity of green fluorescence represents the production of ROS. It can be seen that the fluorescence intensity in the Don group is higher than that in the Con and Que groups. The fluorescence intensity in the Don+Que group is 30.58% lower than that in the Don group, but higher than that in the Con and Que groups (P<0.05). The results of intracellular antioxidant index detection are as follows: Figure 18 Compared with the Con group, all indicators except MDA in the Don group decreased significantly, with T-AOC showing the largest decrease. In the Don+Que group, all indicators except MDA showed an upward trend compared with the Don group (P<0.05). This demonstrates that Don can induce oxidative stress in MDCC-MSB-1 cells, while Que has a mitigating effect.

[0106] (4) Results of the effects of Don and Que on mitochondrial dysfunction in MDCC-MSB-1 cells

[0107] To investigate whether Don can cause mitochondrial dysfunction in MDCC-MSB-1 cells and the protective effect of Que, this application examined the production level of mt-ROS, changes in mitochondrial membrane potential, and mRNA expression levels of genes related to mitochondrial fusion and cleavage. Figure 19 The results showed that Don could promote the generation of mt-ROS (red fluorescent label). The fluorescence intensity of the Don+Que group was significantly lower than that of the Don group, but higher than that of the Con group (P<0.05). Figure 20 This shows changes in mitochondrial membrane potential. In the Don group, the strongest green fluorescence, representing a low potential, and the weakest red fluorescence, representing a high potential, were observed. There was no significant difference in red and green fluorescence intensity between the Con and Que groups (P>0.05). In the Don+Que group, red fluorescence was higher than in the Don group but lower than in the Con group, while green fluorescence was lower than in the Don group but higher than in the Con group. At the transcriptional level ( Figure 21 The levels of MFF, FIS1, and DRP1 in the Don group were significantly higher than those in the Con group, while MFN1, MFN2, and OPA1 were significantly lower than those in the Con group (P<0.05). The addition of Que showed an improving trend, which indicates that Que effectively alleviates Don-induced mitochondrial dysfunction.

[0108] (5) Results of the effects of Don and Que on pyroptosis in MDCC-MSB-1 cells

[0109] To investigate the effects of Don and Que on pyroptosis in MDCC-MSB-1 cells, this application conducted various experiments for analysis. Figure 22 Immunofluorescence was used to detect the expression of NLRP3 (red) and GSDMD (green). Under uniform cell distribution, the red fluorescence in the Don group was approximately 4-fold stronger than that in the Con group, and the green fluorescence was approximately 10-fold stronger (P<0.05). The NLRP3 fluorescence intensity in the Que group was lower than that in the Con group, but this was not statistically significant (P>0.05). Compared with the Don group, the fluorescence intensity of both NLRP3 (red) and GSDMD (green) was significantly lower in the Don+Que group. Furthermore, the expression levels of genes involved in inflammasome assembly and activation related to pyroptosis were detected. Figure 23The results showed that Don exposure significantly increased the expression levels of inflammasome assembly-related genes (NLRP3, ASC, Caspase-1) at the mRNA level. Additionally, the expression levels of key pyroptosis genes GSDMD, IL-18, and IL-1β were also upregulated (P<0.05). Compared to the Don group, all genes were significantly downregulated in the Don+Que group, with IL-1β and GSDMD decreasing to levels not significantly different from the Con and Que groups (P>0.05). Consistent with these results, Western blotting showed that the expression of NLRP3, ASC, Caspase-1, GSDMD, IL-18, and IL-1β was significantly increased in the Don group, while all genes were significantly decreased in the Don+Que group compared to the Don group. These results indicate that Que can alleviate Don-induced pyroptosis.

[0110] (6) Results of the effects of Don and Que on inflammation in MDCC-MSB-1 cells

[0111] qRT-PCR and WB methods were used to detect some inflammatory genes. Figure 24 , Figure 25 The bar chart results showed that Don exposure upregulated the mRNA levels of TNF-α, IL-2, IL-6, and IFN-γ (P<0.05). Que intervention reversed this change; in the Don+Que group, IL-6 mRNA levels returned to control levels, and similarly, IL-6 protein levels returned to a level indistinguishable from control (P>0.05). In conclusion, Que can alleviate some of the inflammatory responses induced by Don.

[0112] 4. Results of the detection of the effects of Don and Que on splenic lymphocytes

[0113] (1) To determine the optimal therapeutic concentrations of Don and Que in splenic lymphocytes, the CCK8 assay was also used to determine the reference doses of Don and Que. The results are as follows: Figure 26 With increasing Don concentration (0, 0.25, 0.5, 1, 2, 4, 8, 16, 32 μM), the survival rate of spleen lymphocytes decreased, reaching only 47.01% at 8 μM. At 1 μM, the cell survival rate was 73.84%, therefore 1 μM was chosen as the Don exposure concentration. In exploring the safe concentration range of Que, the study found that, consistent with MDCC-MSB-1 cells, cell survival was not affected when the Que concentration was ≤5 μM (P>0.05). Therefore, in the results of Don and Que co-treatment (…), Figure 27 Ultimately, the treatment concentration of Que was still chosen to be 5 μM, at which point the cell survival rate was 95.21%.

[0114] (2) Results of the effects of Don and Que on splenic lymphocyte ERS

[0115] Similarly, this study detected the mRNA level and protein expression of ERS-related genes in splenic lymphocytes. Figure 28 At the transcriptional level, there were no significant differences in the expression of GRP78, PERK, ATF-6, and CHOP between the Con and Que groups (P>0.05). In the Don+Que group, the expression of all genes was significantly lower than that in the Don group (P<0.05). ATF-6 expression was more than three-fold increased in the Don group. The expression of some proteins was similarly analyzed: GRP78 showed no significant difference between the Con, Que, and Don+Que groups; ATF-6 showed no significant difference between the Con and Que groups; and CHOP showed no significant difference between the Con and Que groups, and between the Que and Don+Que groups (P>0.05). However, the protein expression of all three proteins in the Don+Que group was significantly lower than that in the Don group (P<0.05). This indicates that Don induces splenic lymphocyte ERS, and Que can alleviate the level of ERS.

[0116] (3) Results of the effects of Don and Que on oxidative stress in splenic lymphocytes

[0117] To investigate whether Don can induce oxidative stress and Que-relieving effects in splenic lymphocytes, this application examined the production of ROS in splenic lymphocytes, such as... Figure 29 As shown in the figure, the fluorescence intensity in the Don group was significantly higher than that in the Con group, the fluorescence intensity in the Don+Que group was significantly lower than that in the Don group, and the fluorescence intensity in the Que group was significantly lower than that in the Con group (P<0.05). The results of the oxidative stress kit are as follows. Figure 30 As shown, the levels of CAT, GSH-PX, SOD, and T-AOC were lowest in the Don group, while MDA was highest. In the Don+Que group, all indicators showed the opposite trend to those in the Don group (P<0.05). However, there was no statistically significant difference between the Con group and the Que group except for T-AOC (P>0.05). This indicates that Don induces oxidative stress in splenic lymphocytes, while Que can enhance the antioxidant capacity of splenic lymphocytes.

[0118] (4) Similarly, in order to verify whether Don can cause mitochondrial dysfunction in spleen lymphocytes and the role of Que in this process, this application used a spleen lymphocyte model to detect the expression of mt-ROS, JC-1 and mitochondrial dynamics-related genes. Figure 31The results showed that, under uniform labeling of live mitochondria (green fluorescence), the Don group produced the most mt-ROS (red fluorescence), followed by the Don+Que group, and the difference between the two groups was statistically significant (P<0.05). The Con and Que groups showed the weakest red fluorescence, with no significant difference (P>0.05). JC-1 results indicated that ( Figure 32 In the Don group, green fluorescence was concentrated, and the membrane potential showed a local decrease, while the red fluorescence intensity was the lowest among the four groups (high potential). There was no significant difference in red and green fluorescence intensity between the Con and Que groups (P>0.05). Compared with the Don group, the green fluorescence in the Don+Que group was significantly weakened, and the red fluorescence was significantly enhanced (P<0.05). To further investigate the damage, the mRNA level was finally detected. Figure 33 The bar chart shows that, compared with the Con group, the Don group had significantly higher levels of MFF, FIS1, and DRP1, and significantly lower levels of MFN1, MFN2, and OPA1. Compared with the Don group, the Don+Que group had significantly lower levels of MFF, FIS1, and DRP1, and significantly higher levels of MFN1, MFN2, and OPA1 (P<0.05). These results suggest that Que can improve Don-induced splenic lymphocyte mitochondrial dysfunction, bringing all indicators back to normal.

[0119] (5) Results of the effects of Don and Que on pyroptosis of splenic lymphocytes

[0120] To investigate the effects of Don and Que on pyroptosis of splenic lymphocytes, this application measured the expression levels of pyroptosis-related genes. Figure 34 The results showed the expression and quantification of mRNA and proteins. At the mRNA level, the transcription levels of NLRP3, ASC, Caspase-1, GSDMD, IL-18, and IL-1β in the Don group were significantly higher than those in the Con group, with Caspase-1 showing the most significant increase, approximately 7-fold (P<0.05). All indicators in the Don+Que group were significantly lower than those in the Don group, with the expression levels of NLRP3, ASC, and IL-18 decreasing to a level not significantly different from the Con group (P>0.05). Protein expression results showed that the expression of genes involved in inflammasome assembly (NLRP3, ASC, Caspase-1), a key factor triggering pyroptosis, was significantly increased in the Don group. The expression of these genes was significantly decreased in the Don+Que group compared to the Don group (P<0.05). No statistically significant differences were found in the expression of any gene between the Con and Que groups (P>0.05). Therefore, this demonstrates that Don induces pyroptosis of splenic lymphocytes, and Que plays an inhibitory role in this process.

[0121] (6) Results of the effects of Don and Que on splenic lymphocyte inflammation

[0122] qRT-PCR and WB methods were used to detect some inflammatory genes. Figure 35 Transcriptional results showed that, compared with the Con group, the expression of TNF-α, IL-2, IL-6, and IFN-γ in the Que group was not significantly different (P>0.05), while the expression of all indicators was significantly increased in the Don group. The expression of all genes in the Don+Que group was significantly downregulated compared with the Don group (P<0.05). At the protein level, the expression of TNF-α in the Don+Que group showed a decreasing trend compared with the Don group, and it was observed to recover to a level that was not statistically significant compared with the control (P>0.05). In conclusion, it can be considered that Que can reverse the partial inflammatory response of splenic lymphocytes induced by Don.

[0123] 5. To investigate the key role of ERS in Don-induced MDCC-MSB-1 cell damage, MDCC-MSB-1 cells were treated with 4-PBA before Don treatment. To determine the optimal concentration of 4-PBA, the CCK8 assay was used to detect the survival rate of MDCC-MSB-1 cells treated with different concentrations of 4-PBA (0, 0.125, 0.25, 0.5, 1, 2 mM). Figure 36 As shown, compared with the Don exposure group alone, cell survival gradually increased when the concentration of 4-PBA was 0.125-1 mM, reaching 86.36% at 1 mM (P<0.05), while cell survival decreased to 77.78% at 2 mM. Therefore, the intervention concentration of 4-PBA was finally selected as 1 mM.

[0124] Results of the effect of 4-PBA treatment on ERS of MDCC-MSB-1 cells:

[0125] Changes in ERS-related genes in MDCC-MSB-1 cells were detected using qRT-PCR and Western blotting. Figure 37 The results showed no significant difference in the mRNA and protein expression levels of GRP78, PERK, and ATF-6 between the Con group and the 4-PBA group (P>0.05), while the expression of CHOP protein in the 4-PBA group was significantly lower than that in the Con group (P<0.05). Compared with the Don group, the expression levels of all gene mRNAs in the Don+4-PBA group were significantly downregulated. Among them, the mRNA level of ATF-6 was not significantly different from that in the Con group according to statistical analysis, and the expression of GRP78, ATF-6, and CHOP proteins was also significantly downregulated (P<0.05). Therefore, 4-PBA treatment can effectively inhibit the degree of Don-induced ERS in MDCC-MSB-1 cells.

[0126] To investigate the effect of Don-induced ERS on oxidative stress in MDCC-MSB-1 cells, this study performed ROS staining ( Figure 38 ) and detection of antioxidant indicators ( Figure 39 The amount of ROS generated in the 4-PBA group was lower than that in the Con group, but the difference was not statistically significant (P>0.05). The amount of ROS generated in the Don+4-PBA group was significantly lower than that in the Don group (P<0.05). The levels of antioxidant enzymes CAT, GSH-PX, SOD, and total antioxidant capacity T-AOC in the Don+4-PBA group were significantly higher than those in the Don group, while the amount of MDA generated was significantly lower (P<0.05). Therefore, it is easy to see that 4-PBA inhibition of ERS can effectively alleviate the degree of Don-induced oxidative stress in MDCC-MSB-1 cells.

[0127] To investigate the effect of Don-induced ERS on pyroptosis of MDCC-MSB-1 cells, this application performed immunofluorescence staining ( Figure 40 ) and detection of pyroptosis-related genes ( Figure 41 , Figure 42 The results were analyzed as follows: In the 4-PBA group, the red fluorescence of NLRP3 was weaker than that in the Con group, and the fluorescence intensity in the Don+4-PBA group was halved compared to the Don group; all these differences were statistically significant (P<0.05). The green fluorescence of GSDMD was significantly weaker in the Don+4-PBA group than in the Don group (P<0.05). The detection of pyroptosis-related genes showed that the transcriptional levels and protein expression of NLRP3, ASC, Caspase-1, GSDMD, IL-18, and IL-1β in the Don+4-PBA group were significantly lower than those in the Don group (P<0.05). However, the mRNA level of NLRP3 and the protein expression levels of GSDMD, ASC, and IL-18 were not significantly different from those in the Con group (P>0.05). Therefore, 4-PBA inhibition of ERS can effectively alleviate Don-induced pyroptosis in MDCC-MSB-1 cells.

[0128] To investigate the effect of Don-induced ERS on inflammation in MDCC-MSB-1 cells, this application examined the expression of some inflammatory genes. The results are as follows: Figure 43 As shown, the mRNA and protein levels of TNF-α and IL-6 were downregulated in the Don+4-PBA group compared to the Don group, while IL-6 and IFN-γ were downregulated at the mRNA level (P<0.05). This indicates that 4-PBA inhibition of ERS can partially alleviate Don-induced inflammation in MDCC-MSB-1 cells.

[0129] The above description is merely a few embodiments of this application and is not intended to limit this application in any way. Although this application discloses preferred embodiments as described above, it is not intended to limit this application. Any changes or modifications made by those skilled in the art without departing from the scope of the technical solution of this application using the disclosed technical content are equivalent to equivalent implementation cases and fall within the scope of the technical solution.

Claims

1. Application of quercetin in the preparation of drugs for treating deoxynivalenol-induced spleen damage in livestock and poultry.

2. The application according to claim 1, characterized in that, The drug has quercetin as its sole active ingredient or one of its active ingredients; The livestock and poultry include broiler chickens.

3. The application according to claim 1, characterized in that, The effective dosage of the drug is 3-5 mg per kilogram of livestock or poultry body weight.

4. The application according to claim 3, characterized in that, The effective dosage of the drug is 4 mg per kilogram of animal body weight.

5. The application according to claim 1, characterized in that, The drug works by inhibiting endoplasmic reticulum stress, oxidative stress, mitochondrial dysfunction, pyroptosis, and inflammatory responses.

6. The application according to claim 5, characterized in that, The drug inhibits endoplasmic reticulum stress by downregulating the expression levels of endoplasmic reticulum stress-related genes GRP78, PERK, ATF-6, and CHOP.

7. The application according to claim 5, characterized in that, The drug inhibits oxidative stress by upregulating the activity of antioxidant enzymes CAT, GSH-PX, and SOD, and downregulating the expression levels of reactive oxygen species (ROS) and malondialdehyde (MDA).

8. The application according to claim 5, characterized in that, The drug inhibits mitochondrial dysfunction by downregulating the expression levels of mitochondrial dynamics-related genes MFF, DRP1, and FIS1, and upregulating the expression levels of MFN1, MFN2, and OPA1.

9. The application according to claim 5, characterized in that, The drug works by inhibiting the expression of NLRP3, GSDMD, ASC, Caspase-1, IL-18, IL-1β, and TNF-α.

10. The application according to claim 1, characterized in that, The dosage forms of the drug include injections, powder injections, tablets, capsules, powders, oral liquids, granules, pills, or suppositories. The drug also contains pharmaceutically acceptable excipients.

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