CN116059226A - Application of buddleja saponin IVb in preparation of product for preventing and treating porcine epidemic diarrhea - Google Patents
Application of buddleja saponin IVb in preparation of product for preventing and treating porcine epidemic diarrhea Download PDFInfo
- Publication number
- CN116059226A CN116059226A CN202210804477.XA CN202210804477A CN116059226A CN 116059226 A CN116059226 A CN 116059226A CN 202210804477 A CN202210804477 A CN 202210804477A CN 116059226 A CN116059226 A CN 116059226A
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- ivb
- saponin
- buddleja
- porcine epidemic
- epidemic diarrhea
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Abstract
The invention provides application of buddleja saponin IVb in preparation of a product for preventing and treating porcine epidemic diarrhea, and belongs to the technical field of treatment of porcine epidemic diarrhea. The invention discovers that the buddleja saponin IVb can obviously inhibit the proliferation of the porcine epidemic diarrhea virus in vivo or in vitro for the first time, and the pharmacological effects of the buddleja saponin IVb comprise inhibiting the replication and release of the porcine epidemic diarrhea virus life cycle, inhibiting the activation of NF- κB signal channel induced by the porcine epidemic diarrhea virus and reducing the expression of inflammatory factors caused by the porcine epidemic diarrhea virus infection. The piglet treatment experiment shows that the buddleja saponin IVb can effectively relieve clinical symptoms caused by porcine epidemic diarrhea virus, reduce piglet enterovirus load, provide basis for clinically developing porcine epidemic diarrhea virus treatment medicines, and have important production and application prospects.
Description
Technical Field
The invention belongs to the technical field of treatment of porcine epidemic diarrhea, and particularly relates to application of buddleja officinalis saponin IVb in preparation of a product for preventing and treating porcine epidemic diarrhea.
Background
Porcine epidemic diarrhea (Porcine epidemic diarrhea, PED) is a highly contagious acute infectious disease caused by porcine epidemic diarrhea virus (Porcine epidemic diarrheavirus, PEDV), mainly causes acute enteritis and fatal diarrhea of piglets until dehydration death, and the pathological changes are mainly represented by atrophy and shedding of small intestinal villi. Since PEDV variants are pandemic in swine herds, the frequency of PEDs presents a significant challenge for the control of the disease. Although PEDV vaccines are widely used in our country, they have poor control effects, and there are still cases where PEDV is constantly mutated to avoid immune surveillance of the vaccine or where immune failure is caused by individual animal specificity. Therefore, finding new ways to prevent and control PEDV infection is of practical importance in clinical production. Among them, the search and screening of anti-PEDV natural products is one of the hot spot directions of prevention and control of PEDs in recent years, so research and development of new antiviral drugs are very necessary. Although natural products with anti-PEDV activity, such as quercetin 7-rhamnoside, monolaurate, hypericin, etc., are reported in the art, most of them are in the laboratory verification stage, and the clinical treatment effect is not ideal, so it is not easy to find a natural product for inhibiting PEDV in vitro and in vivo. There is no report on what effect buddleja saponin IVb (Buddlejasaponin IVb) has on PEDV.
Disclosure of Invention
In view of the above, the invention aims to provide the application of the buddleja officinalis saponin IVb in preparing the product for preventing and treating the porcine epidemic diarrhea, and the buddleja officinalis saponin IVb can effectively inhibit the proliferation of the porcine epidemic diarrhea virus both in vivo and in vitro.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides application of buddleja saponin IVb in preparation of a product for preventing and treating porcine epidemic diarrhea.
Preferably, the buddleja saponin IVb inhibits proliferation of porcine epidemic diarrhea virus.
Preferably, the buddleja saponin IVb inhibits replication and release of the porcine epidemic diarrhea virus lifecycle.
Preferably, the buddleoside IVb inhibits NF- κB signaling pathway activation.
Preferably, the buddleja saponin IVb inhibits the activation of NF- κB signaling pathway by down-regulating the expression of p-NF- κ B p65, p-IκBα, up-regulating the expression of IκBα.
Preferably, the buddleja saponin IVb is capable of reducing the expression of inflammatory factors.
Preferably, the inflammatory factors include IL-6, IL-8, IL-1. Beta. And TNF-alpha.
Preferably, the buddleja saponin IVb can effectively relieve clinical symptoms of a body caused by porcine epidemic diarrhea virus and reduce the load of enteroviruses of the body.
The invention also provides a medicament for treating and/or preventing porcine epidemic diarrhea, which comprises an effective amount of buddleja saponin IVb.
Preferably, the percentage content of the buddleja saponin IVb in the medicine is 1% -99%.
The invention has the beneficial effects that:
the invention provides that buddleja saponin IVb can be applied to preparing a product for preventing and treating porcine epidemic diarrhea for the first time. The invention discovers that the buddleja saponin IVb can obviously inhibit the proliferation of the porcine epidemic diarrhea virus in vivo or in vitro for the first time, and the pharmacological effects of the buddleja saponin IVb comprise inhibiting the replication and release of the porcine epidemic diarrhea virus life cycle, inhibiting the activation of NF- κB signal channel induced by the porcine epidemic diarrhea virus and reducing the expression of inflammatory factors caused by the porcine epidemic diarrhea virus infection. The piglet treatment experiment shows that the buddleja saponin IVb can effectively relieve clinical symptoms caused by porcine epidemic diarrhea virus, reduce piglet enterovirus load, provide basis for clinically developing porcine epidemic diarrhea virus treatment medicines, and have important production and application prospects.
Drawings
FIG. 1 is an IC of Buddlejasaponin IVb 50 CC for inhibiting PEDV 50 Wherein the left panel shows Buddlejasaponin IVb as CC inhibiting PEDV 50 IC with Buddlejasaponin IVb on right 50 ；
FIG. 2 shows the results of a Buddlejasaponin IVb assay for inhibition of replication of PEDV on Vero cells, wherein A is a real-time fluorescent quantitative PCR analysis of the PEDV content in Vero cells and B is TCID of the PEDV content in the supernatant of Vero cell culture 50 Analysis, wherein C and D are Western blot analysis of PEDV content in Vero cells, and E and F are IFA analysis of PEDV in Vero cells;
FIG. 3 is an assay for Buddlejasaponin IVb inhibiting replication of PEDV on IPEC-J2 cells, wherein A is a real-time fluorescent quantitative PCR analysis of PEDV content within IPEC-J2 cells; b is TCID of PEDV content in IPEC-J2 cell culture supernatant 50 Analyzing; c is CCK8 to evaluate the activity of the cells;
FIG. 4 shows the inactivation effect of Buddlejasaponin IVb on PEDV, A is an inactivation assay flow chart; b is real-time fluorescence quantitative PCR analysis of PEDV content; c is Western blot analysis of PEDV content; d is a protein analysis histogram of PEDV content;
FIG. 5 shows the adsorption effect of Buddlejasaponin IVb on PEDV, A is an adsorption experimental flow chart; b is real-time fluorescence quantitative PCR analysis of PEDV content; c is Western blot analysis of PEDV content; d is a protein analysis histogram of PEDV content;
FIG. 6 is a flowchart showing the effect of Buddlejasaponin IVb on entry of PEDV, A is a flowchart showing an entry experiment; b is real-time fluorescence quantitative PCR analysis of PEDV content; c is Western blot analysis of PEDV content; d is a protein analysis histogram of PEDV content;
FIG. 7 shows the replication effect of Buddlejasaponin IVb on PEDV, A is a replication experimental flow chart; b is real-time fluorescence quantitative PCR analysis of PEDV content; c is Western blot analysis of PEDV content; d is a protein analysis histogram of PEDV content;
FIG. 8 is a graph showing the effect of Buddlejasaponin IVb on the release of PEDV, A is a release experimental flow chart; b is real-time fluorescence quantitative PCR analysis of PEDV content; c is Western blot analysis of PEDV content; d is a protein analysis histogram of PEDV content;
FIG. 9 shows the effect of Buddlejasapnion IVb on inflammatory response of PEDV-infected Vero cells, A being the expression level of IL-6; b is the expression level of IL-8; c is the expression level of IL-1 beta; d is the expression level of TNF- α;
FIG. 10 shows Buddlejasaponin IVb inhibition of PEDV activation of the Vero-induced NF- κB signaling pathway, A shows Western blotting analysis of NF- κ B p65, p-NF- κ B p65, ikBα and p-IkBα expression; b is a protein gray analysis histogram of NF- κ B p 65; c is a protein gray analysis histogram of p-NF- κ B p 65; d is a protein gray analysis histogram of IκBα; e is a protein gray analysis histogram of p-IκBα;
FIG. 11 is a flow chart of an animal experiment;
FIG. 12 shows clinical symptoms of a piglet infected with PEDV, A shows the survival rate of the piglet; b is a piglet clinical score;
FIG. 13 shows the content of virus mRNA in the feces of a piglet infected with PEDV and intestinal tissues, and A shows the content of PEDV in the feces of a piglet; b is the content of PEDV in intestinal tissues of piglets;
fig. 14 shows general pathological changes and histopathological changes of intestinal tracts of piglets infected with PEDV, a shows intestinal tissues of experimental piglets, B shows intestinal pathological sections of experimental piglets, C shows scores of intestinal tissues of experimental piglets, and D shows scores of intestinal pathological sections of experimental piglets;
FIG. 15 shows the inflammatory factor levels in intestinal tissues of PEDV-infected piglets, A-D being the detection of mRNA expression of IL-6, IL-8, IL-1. Beta. And TNF-alpha. In each group of intestinal tissues using RT-qPCR, respectively;
the above figures represent significant differences, where P <0.05; * P <0.01; * P <0.001; ns: is not significant.
Detailed Description
The invention provides application of buddleja saponin IVb in preparation of a product for preventing and treating porcine epidemic diarrhea.
The present invention is not particularly limited as to the specific kind of the product, and the kind of the product preferably includes a drug, a kit or an agent. The specific source of the buddleja saponin IVb is not particularly limited, and the buddleja saponin IVb can be prepared by adopting products which are commercially available in the field. In the present invention, the inhibition of PEDV by buddleoside IVb is dose-dependent and its selectivity index SI>10, in particular, IC of buddleja saponin IVb 50 6.943 mu M, CC 50 SI (CC) was calculated using the screening index at 84.56 μm 50 /IC 50 ) =12.18, capable of significantly inhibiting replication of PEDV. In the present invention, the buddleja saponin IVb preferably plays a role in inhibiting proliferation of porcine epidemic diarrhea virus by inhibiting replication and release of porcine epidemic diarrhea virus life cycle.
Further research on the action mechanism of drunk fish saponin IVb for inhibiting porcine epidemic diarrhea virus proliferation shows that drunk fish saponin IVb preferably plays a role in inhibiting NF- κB signal pathway activation, more preferably plays a role in inhibiting NF- κB signal pathway activation by down-regulating the expression of p-NF- κ B p65 and p-IκBα and up-regulating the expression of IκBα. In the present invention, the buddleoside IVb is preferably also capable of reducing the expression of inflammatory factors, preferably including IL-6, IL-8, IL-1. Beta. And TNF-alpha.
In-vivo experiments are also carried out, and particularly, drunk-fish saponin IVb is adopted for treating piglets, and the results show that compared with a PEDV virus-fighting non-treatment control group, piglets in a 0.5mg/kg Buddlejasaponin IVb treatment group only show moderate clinical symptoms, mild injury, lower viral load and lower intestinal inflammation response. And the 1mg/kg treatment group has minimal piglet clinical symptoms, the intestinal lesions, the viral load and the like are almost the same as those of the negative control group, and the intestinal inflammatory factor level is low. The results prove that the buddleja saponin IVb can effectively relieve the clinical symptoms of the organism caused by porcine epidemic diarrhea virus and reduce the load of the enterovirus of the organism.
The invention also provides a medicament for treating and/or preventing porcine epidemic diarrhea, which comprises an effective amount of buddleja saponin IVb.
In the medicine, the medicine also preferably comprises pharmaceutically acceptable auxiliary materials. The source and the type of the auxiliary materials are not particularly limited, and the conventional pharmaceutical auxiliary materials in the field can be utilized. In the medicine, the buddleja saponin IVb is taken as an effective component, the percentage content of the buddleja saponin IVb in the medicine is preferably 1% -99%, and the purity of the buddleja saponin IVb is preferably more than or equal to 99.9%.
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
In the following examples, conventional methods are used unless otherwise specified.
Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
In the following experiments of the invention, african green monkey kidney cells (Vero), pig small intestine epithelial cells (IPEC-J2), PEDV AH-2018-HF1 (Genebank: MN 315264.1) were all kept in Anhui agricultural university animal infectious disease laboratory; the required media and reagents are shown in Table 1; the main solution preparation comprises the following steps: (1) Phosphate Buffered Saline (PBS): 0.1g KCl and 0.1g KH are respectively weighed by halving in electronic days 2 PO 4 ，1.5g Na 2 HPO 4 ·12H 2 O,4g NaCl in 500mL distilled water, stirring uniformly with a glass rod, adjusting the pH to 7.0, placing in an autoclave at 121 ℃ for 20min, and cooling for standby. (2) PBST buffer: 0.1g KCl and 0.1g KH are respectively weighed by halving in electronic days 2 PO 4 ，1.5gNa 2 HPO 4 ·12H 2 O,4g NaCl is added in 500mL distilled water, stirred by a glass rod until the solvent is dissolved, 250 mu L Tween 20 is added, and the mixture is uniformly mixed for later use. (3) 5 XSDS-PAGE running buffer: the 5g SDS,15.1g Tris,94g glycine is weighed into 900mL of distilled water by an electronic day, stirred by a glass rod until the solvent is dissolved, and then the PH is regulated to be approximately equal to 8.3, and the water is added to be constant to 1L for standby. (4) 1 XSDS-PAGE running buffer: 100mL of 5 XSDS-PAGE running buffer was added to 400mL of distilled water and mixedAnd (5) homogenizing for later use. (5) 1 Xtransfer buffer: 3.05g of Tris and 14.4g of glycine are weighed separately in 800mL of distilled water by electronic days, stirred by a glass rod until the solvent is dissolved, 200mL of methanol solution is added, and the mixture is placed in a refrigerator at 4 ℃ for storage. (6) citrate buffer: 40mM citric acid, 10mM potassium chloride, 135mM sodium chloride, pH was adjusted to 3.0.
TABLE 1 Main Medium and reagents
All experiments according to the invention were independently repeated at least 3 times. The grayscale scan of Westernblotting was done by Image J software. Data were statistically analyzed using GraphPad Prism 6.0 software for Student's t-tests, and differences for each group were compared, with P <0.05 indicating significant differences, P <0.01 and P <0.001 indicating significant differences.
Example 1
Cell resuscitation: after the Vero cells are taken out of the liquid nitrogen tank, the Vero cells are immediately placed into a water bath kettle with the temperature of 37 ℃, and the freezing and storing pipe is slowly shaken to enable the cells to be completely thawed, and then 75% alcohol is used for wiping the surface of the freezing and storing pipe, so that pollution is avoided. Placing the frozen tube in a centrifuge and balancing, centrifuging at 2000r/min for 2min, discarding frozen solution, adding 1mL of DMEM soft and heavy suspension containing 10% FBS, transferring into cell bottle, supplementing 5mL of culture solution containing 10% FBS, adding CO at 37deg.C 2 Culturing in an incubator.
Cell passage: when the fusion degree of Vero cells reaches more than 90%, subculturing is carried out, cell culture solution is discarded, PBS is used for cleaning for three times, a proper amount of pancreatin is added, then the cells are put into a cell culture box for digestion for 1min, the pancreatin is discarded and cell bottles are gently patted after the cells are dispersed and rounded, a proper amount of DMEM containing 10% FBS is added for blowing to prepare cell suspension, the cell suspension is packaged into new cell bottles, and DMEM containing 10% FBS is supplemented, and the cell suspension is put into the cell culture box for culturing.
After the degree of fusion of Vero reached 80-90%, buddlejasaponin IVb at various concentrations (1, 5, 10, 20, 30, 50, 100. Mu.M) was added to the cell culture broth, and DMSO was added as a negative control. Incubation for 24h in a cell incubator, operating according to the instructions of the enhanced CCK8 cell activity kit: adding 10 mu L of CCK8 working solution into each hole, placing the mixture into a cell culture box for incubation for 2 hours, and reading OD (optical density) by using an enzyme-labeled instrument 450 Cell activity was calculated according to the formula.
Cell viability (%) = (OD experimental well-OD blank well)/(OD control well-OD blank) ×100. Finally, the concentration of 50% of cells that produced cytotoxicity (CC was calculated using GraphPadPrism 6.0 software 50 )。
Vero cells were incubated with Buddlejasaponin IVb (1, 5, 10, 20, 30 μm) at various concentrations for 1h, while DMSO groups were established for negative control, PEDV (0.01 MOI) was inoculated, and after incubation for 1h, DMEM maintenance solution was changed and incubated for 24h at 37 ℃. Performing an indirect immunofluorescence test, and randomly recording three photos by using a fluorescence microscope; measuring the fluorescence value intensity by using imageJ software; finally, 50% Inhibitory Concentration (IC) of each natural product against PEDV was calculated using GraphPad Prism 6.0 software 50 ) The results were recorded.
Medicament CC 50 Antiviral IC 50 The value and the screening index SI are important indexes for measuring the toxicity and antiviral effect of the medicine. IC (integrated circuit) 50 The values represent drug concentrations effective to inhibit 50% of the cell-infected virus. SI screening index=cc 50 /IC 50 Is a parameter for evaluating the excellent effect of the drug, and the larger the value is, the better the antiviral effect of the drug is.
The results are shown in FIG. 1: buddlejasaponin IVb inhibition of PEDV shows a dose-dependent relationship and is Budddlejasaponin IVb CC 50 84.56. Mu.M, IC 50 6.943 mu M, selectivity index SI>10, the inhibition of PEDV was most pronounced and dose-dependent inhibition of PEDV infection.
Example 2
Effect of different concentrations Buddlejasaponin IVb on PEDV
Vero cells were treated with 5. Mu.M, 10. Mu.M and 20. Mu.M Buddlejasaponin IVb, respectively (treated with DMSO (1. Mu.L))Vero cells as control) for 1h, PEDV (0.01 MOI) was added to infect the cells for 1h. After washing the cells three times with PBS, the culture solution containing 5. Mu.M, 10. Mu.M and 20. Mu.M of Buddlejasaponin IVb or DMSO (1. Mu.L) was added back to the corresponding wells. 20h after infection, RT-qPCR, western blot and TCID are adopted 50 And indirect immunofluorescence detection of PEDV virus content.
The RT-qPCR method comprises the following steps:
extraction of total RNA: (1) The culture medium was discarded, the cells were washed with PBS and blotted well, 350. Mu. LTRK lysate was added to each well, left at room temperature for 5min, shaken several times during which time all cells and lysates were aspirated to RNA Homogenizer Spin Column, and centrifuged at maximum speed for 2min. (2) The centrifuged sample was transferred to an RNase-free EP tube, and an equal amount of 70% absolute ethanol was added thereto and vortexed. (3) The mixed solution is moved to
RNA Mini Column,12000r/min was centrifuged for 1min. And (3) passing the centrifuged liquid through a column again, centrifuging for 1min at 12000r/min, and discarding the waste liquid. (4) To->
500. Mu.L of RNAWash Buffer I was added to RNAMini Column, and the mixture was centrifuged at 12000r/min for 1min, to discard the waste liquid. (5) To the direction of
Adding 500 mu LRNAWash Buffer II (containing absolute ethanol) into RNAMini Column, centrifuging at 12000r/min for 1min, and discarding the waste liquid. And (6) repeating the step (5), and fully airing the adsorption column. (7) Will->
RNAMini Column was transferred to a new EP tube, 30. Mu.L DEPC water was added dropwise to the center of the Column membrane, and the mixture was left stand for 5min and centrifuged at 12000r/min for 2min to obtain sample RNA.
Synthesis of cDNA: the cDNA synthesis system and conditions were as follows: (1) genomic DNA removal: the mixture shown in Table 2 was prepared in a centrifuge tube of RNase-free, and gently stirred and mixed with a pipette. 42 ℃ for 2min.
TABLE 2 genomic DNA removal
(2) A reverse transcription reaction system shown in Table 3 was prepared, and the mixture was gently stirred and mixed with a pipette at 50℃for 15min and 85℃for 5s. The cDNA product was stored at-80℃until use.
TABLE 3 cDNA Synthesis reaction System
RT-qPCR: relative fluorescence quantitative detection was performed using the SYBE Green dye method. The primer sequences of RT-qPCR are shown in Table 4, the reaction system of RT-qPCR is shown in Table 5, and the reaction conditions are as follows: 15min at 95 ℃, 2min at 95 ℃, 30s at 60 ℃ and 40 cycles.
TABLE 4 RT-qPCR primer sequences
TABLE 5 RT-qPCR reaction System
The Westernblot method comprises the following steps:
preparation of protein samples: (1) RIPA and PMSF were thoroughly mixed in a ratio of 100:1 and placed on ice for use. (2) Cells were washed with pre-chilled PBS, 100. Mu.L of RIPA protein lysate was added to each well of a 24-well plate and lysed on ice for 15min, during which time the cells were shaken 1-2 times. (3) Collecting cell lysate, centrifuging at 12000r/min at 4deg.C for 5min, and collecting supernatant. (4) Protein concentration was measured by BCA method, and protein concentrations of different groups were adjusted to be uniform. (5) Taking the same amount of protein sample, adding appropriate amount of 5×loading Buffer, and boiling in boiling water for 7 min.
SDS-PAGE electrophoresis: (1) And (3) correctly assembling the gel plate, preparing separating gel according to the size of the target protein, and preparing concentrated gel after the separating gel is solidified. (2) And (3) assembling the gel in an electrophoresis tank, adding electrophoresis liquid, and gently pulling out the comb. And (5) sequentially loading the protein Marker and the denatured protein, and carrying out protein electrophoresis. The electrophoresis condition is that the constant pressure is about 80V, and when the gel enters the separation gel, the constant pressure is about 100V and about 1.5 h.
Transferring: and after electrophoresis, the gel plate is disassembled, the PVDF film is sheared into a proper size, methanol is used for activation, the transfer pad, the filter paper, the SDS film and the PVDF film are assembled according to a sandwich structure to form a sandwich, and bubbles are gently removed. The transfer plate was placed in a transfer tank and transferred at a constant current of 350mA for 90min.
Closing: after the film transfer is finished, the PVDF film is placed on a sealing liquid containing 5% of skimmed milk powder upwards from the front surface, and is placed on a horizontal shaking table for sealing for 1h.
Incubating primary antibodies: at the end of blocking, the corresponding antibodies were diluted to the corresponding concentrations with PBST as per instruction, PVDF membranes were placed face up and incubated for 2h at room temperature or overnight at 4 ℃.
Incubating a secondary antibody: after the primary antibody incubation was completed, the membranes were washed 3 times for 10min each with PBST. HRP-labeled secondary antibody was diluted in PBST as required by the instructions, PVDF membrane was placed face up and incubated for 1h at room temperature. After the incubation, the incubation was completed, washed 3 times for 10min each with PBST.
Color development: and uniformly adding the ECL luminous solution on the PVDF film, and putting the PVDF film into an exposure instrument for exposure.
TCID 50 Is determined by:
(1) Vero cells were grown according to 2X 10 4 The wells/wells were seeded in 96-well plates and allowed to grow to confluence with monolayers. (2) Diluting PEDV virus solution with serum-free DMEM at 10 times ratio to obtain 10 diluted solutions -1 -10 -8 8 dilutions. (3) The culture medium in the cells was discarded, washed three times with PBS, and 100. Mu.L of 10 was added, respectively -1 -10 -8 Dilutions of virus were used and negative controls were made with serum-free DMEM, and each group was repeated 8 times. Placing the mixture into a cell culture box for incubation for 1h. (4) The virus solution was discarded, washed three times with PBS, and 100. Mu.L of serum-free DMEM containing an appropriate amount of pancreatin was added. Incubation was continued for 72h. (5) Observe andcounting the number of virus positive holes, and calculating the virus TCID by using a Reed-Muench method 50 。
Indirect Immunofluorescence (IFA) detection method:
(1) Vero cells were seeded into cell culture plates and allowed to grow to an abundance of about 85% -90%. (2) After treatment of natural products and PEDV infection with Vero, incubation was continued for a certain period of time, and the culture was discarded and washed three times with PBS for 5min each. (3) 4% paraformaldehyde was added, and after fixing the cells at room temperature for 30min, the cells were washed with PBS 3 times for 5min each. (4) 0.1% Triton X-100 was added, and the mixture was allowed to pass through at room temperature for 8-10min, and then washed with PBS 3 times for 5min each. (5) Blocking was performed for 1h at room temperature using 5% BSA, and washing was performed 3 times for 5min each with PBS. (6) The polyclonal serum was added and incubated with diluted anti-PEDV at room temperature for 1h, washed 3 times with pbs for 5min each. (7) Diluted goat anti-rabbit FITC-labeled secondary antibody was added and incubated at room temperature for 1h in the dark, washed 3 times with PBS for 5min each. (8) 100 mu L/well of DAPI dye solution was added, stained 5min at room temperature in dark, and washed 3 times with PBS for 5min each.
The results are shown in figure 2, where PEDV content gradually decreased with increasing Buddlejasaponin IVb dose, indicating Buddlejasaponin IVb was able to inhibit PEDV replication in a dose dependent manner.
Example 3
Buddlejasaponin IVb Effect on proliferation of PEDV on IPEC-J2 cells
After IPEC-J2 cells were treated with 5. Mu.M, 10. Mu.M and 20. Mu.M Buddlejasaponin IVb or DMSO (1. Mu.L), respectively, for 1h, the cells were infected with PEDV (0.01 MOI) added. After washing the cells three times with PBS, the culture solution containing 5. Mu.M, 10. Mu.M and 20. Mu.M of Buddlejasaponin IVb or DMSO (1. Mu.L) was added back to the corresponding wells. 20h after infection, RT-qPCR and TCID were used 50 PEDV virus content was measured and cell viability was assessed using CCK8 kit. Wherein RT-qPCR, TCID 50 The detection method is the same as in example 2.
The results are shown in FIG. 3, where PEDV content gradually decreased with increasing Buddlejasaponin IVb dose, indicating that Buddlejasaponine IVb was able to inhibit replication of PEDV on IPEC-J2 cells in a dose-dependent manner.
Example 4
Buddlejasaponin IVb Effect on the replication cycle of PEDV
Detection of inactivation of viral particles
Will contain a: PEDV (0.01 MOI) + Buddlejasaponin IVb (10 μm); b: PEDV (0.01 MOI) +dmso; c: DMSO and D: buddlejasaponin IVb (10. Mu.M) four groups of culture solutions were placed in an incubator at 37℃for 3 hours and 5 hours, respectively. The mixtures of group A and C or B and D were inoculated into Vero cells cultured in 24-well plates, incubated at 37℃for 1 hour, and then cultured for 11 hours with fresh DMEM instead of the culture supernatant. And extracting cell genes, and detecting the content of PEDV virus by adopting RT-qPCR and Western blot. Wherein the RT-qPCR, westernblot assay was as in example 2. The results are shown in figure 4, where Buddlejasaponin IVb +pedv groups were statistically not significantly different from dmso+pedv. Buddlejasaponin IVb does not inactivate PEDV directly to PEDV.
Detection of effects on viral adsorption phase
Vero cells were pretreated with Buddlejasaponin IVb or DMSO in an incubator at 37 ℃ for 1h, then Buddlejasaponin IVb or a mixture of DMSO and PEDV (1 MOI) was used instead of the above culture, incubated at 4 ℃ for 15min, 30min, 60min, and then washed three times with pre-chilled PBS, and the PEDV virus content was detected with RT-qPCR, westernblot. Wherein the RT-qPCR and Western blot detection method is the same as in example 2. As shown in FIG. 5, buddlejasaponin IVb did not affect the adsorption phase of PEDV.
Detection of effects on the viral entry phase
Vero cells were infected with PEDV (1 MOI) for 1h at 4 ℃. PBS was washed three times, replaced with fresh medium containing Buddlejasaponin IVb or DMSO, and then incubated at 37 ℃ for the indicated times 30min, 1h, 2h (i.e., 1.5hpi, 2hpi, 3 hpi). Cells were washed with citrate buffer (ph=3) to remove non-internalized virus. The PEDV virus content was detected by RT-qPCR, westernblot. Wherein the RT-qPCR and Western blot detection method is the same as in example 2. As shown in FIG. 6, buddlejasaponin IVb did not affect the entry phase of PEDV.
Detection of effects on viral replication phase
PEDV (1 MOI) was inoculated into Vero cells, incubated in an incubator at 37 ℃ for 1h, washed three times with PBS, fresh broth was added, at 4h, the cell broth was changed to fresh broth containing Buddlejasaponin IVb or DMSO, and after the cells were placed in the incubator at 37 ℃ for further incubation for 2h, 4h, 6h (i.e., 6hpi, 8hpi, 10 hpi), washed three times with PBS, and the PEDV virus content was detected by RT-qPCR, western blot. Wherein the RT-qPCR, westernblot assay was as in example 2. The results are shown in figure 7, buddlejasaponin IVb significantly inhibited the replication phase of PEDV.
Detection of effects on the viral Release phase
Vero cells were infected with PEDV (1 MOI) at 37 ℃ for 1h with fresh DMEM instead of medium. At 10hpi, cells were washed three times with PBS and the medium was replaced with fresh DMEM containing Buddlejasaponin IVb or DMSO. The cells were further cultured at 37℃for 0.5, 1 and 2 hours (i.e.10.5 hpi, 11hpi, 12 hpi), at which time the supernatant was harvested. The PEDV virus content was detected by RT-qPCR, western blot. Wherein the RT-qPCR, westernblot assay was as in example 2. The results are shown in figure 8, buddlejasaponin IVb significantly inhibited the release phase of PEDV.
Example 5
Vero cells were inoculated with 0.01moi pedv and incubated at 37 ℃ for 1h, the nutrient solution was changed to fresh nutrient solution containing Buddlejasaponin IVb (or DMSO), and after further incubation for 24h total cellular RNA was extracted. Meanwhile, an LPS positive control group, i.e. a control group treated with 1. Mu.g/mL LPS (or DMEM) by Vero cells, was established. Intracellular IL-1β, IL-6, IL-8 and TNF- α mRNA levels were detected by RT-qPCR. The RT-qPCR assay was the same as in example 2, except that the primer sequences used are shown in Table 6.
TABLE 6 RT-qPCR primer sequences
As shown in FIG. 9, both LPS and PEDV infection induced an increase in IL-1. Beta., IL-6, IL-8 and TNF-. Alpha.as positive stimulators. Buddlejasaponin IVb alone did not cause changes in intracellular inflammatory factor levels, but Buddlejasaponin IVb treatment significantly reduced the surge in IL-1β, IL-6, IL-8 and TNF- α stimulated by LPS and was effective in reducing the intracellular elevation of IL-1β, IL-6, IL-8 and TNF- α induced by infection with PEDV, indicating that Buddlejasaponin IVb was able to reduce the inflammatory response induced by infection with PEDV.
Example 6
The effect of Buddlejasaponin IVb on PEDV-induced NF- κb signaling pathway activation was studied by Western-blot. Western blot detection method is the same as in example 2. As a result, as shown in FIG. 10, the expression levels of p-NF-. Kappa.Bp 65 and p-IκBα, which were infected with PEDV, were significantly increased, and the expression level of IkBα was decreased. In contrast, buddlejasaponin IVb treatment down-regulated protein expression of p-NF- κ B p65, p-IκBα and up-regulated IκBα expression. The following is indicated: buddlejasaponin IVb inhibits PEDV-induced activation of NF- κB signaling pathways.
Example 7
The 20 3-day-old piglets were randomly divided into 4 groups of 5 piglets. (1) positive control group (vecile): PEDV infection and therapeutic treatment with drug dilutions (no Buddlejasaponin IVb); (2) 0.5mg/kg drug treatment group: PEDV infection and treatment with 0.5mg/kg drug; (3) 1mg/kg drug treatment group: PEDV infection and treatment with 1mg/kg drug; (4) negative control group (Mock): DMEM and drug dilutions (without Buddlejasaponin IVb) were treated therapeutically. Wherein the PEDV infection is carried out in the form of oral administration of piglets, the dosage is 1mL of virus liquid for each piglet, and the virus titer is 1 multiplied by 10 6 TCID 50 /mL. After 12h, intramuscular injection treatment is carried out by using the medicine, the time interval is once every 6h, the clinical manifestations of the piglet, such as the morbidity, the mortality and the like are recorded, and the piglet manure is collected for later viral load measurement. All piglets were euthanized 3d after challenge, and intestinal tissue was collected to determine viral load and inflammatory factors and analyzed for histopathology. The animal experiment flow chart is shown in fig. 11.
Clinical symptom scoring method
The piglets are fed separately after the toxin is removed, and the clinical symptoms of the piglets, including fur, skin, feeding, diarrhea, vomiting, mental states and the like are observed every day. The clinical condition of piglets was scored daily. The scoring items are mental status, diarrhea and vomiting. Each term has a score of 0-4, a score of 0, a symptom of 1, a symptom of 2, a symptom of 3, and a death of 4. And scoring each index every day after the virus attack, counting the sum of the indexes, and finally carrying out statistical analysis on the average value of each group.
Visual inspection of intestinal lesions, including thinning of the intestinal wall, air blowing in the intestinal tract and bleeding of the intestinal wall. The scoring was performed according to the above three items, each of which has a score of 0 to 4, a normal state of 0 score, a symptom of extremely light of 1 score, a symptom of slightly 2 score, a symptom of 3 score, and a symptom of extremely heavy of 4 score. Finally, the average value of each group is statistically analyzed.
And (3) carrying out scoring statistics according to intestinal villus states of intestinal tissues of piglets, wherein the score is an integer between 0 and 4, the normal state is 0 score, the intestinal villus is extremely slightly broken into 1 score, the slight break into 2 score, the serious break into 3 score and the extremely serious break into 4 score. Finally, the average value of each group is statistically analyzed.
Preparation of pathological tissue sections
(1) Drawing materials: after the test is finished, the piglets are killed, and the whole observation and photographing are carried out. And fixing intestinal tissues of piglets for subsequent experiments. (2) fixing: tissue pieces were fixed in fresh 4% paraformaldehyde for 24h. (3) trimming and flushing: trimming the fixed tissue into tissue blocks with neat sections. The tissue is rinsed with running tap water for more than 12 hours to remove the paraformaldehyde in the tissue. (4) dehydration: tissue pieces were placed in sequence in 75%, 85%, 90%, 95% and 100% gradient alcohol. The specific procedures are as follows: 3h of 75% alcohol, 2h of 85% alcohol, 1.5h of 90% alcohol, 1.5h of 95% alcohol, 40min of absolute alcohol I and 40min of absolute alcohol II. (5) transparent and waxing: the wax box is opened at the same time when dehydration starts, and wax is melted. After complete dehydration of the tissue mass, it was immersed in xylene for transparency. Placing the transparent tissue blocks in a 60 ℃ incubator, and dipping the transparent tissue blocks in wax for 3 hours. (6) embedding: placing the waxed tissue blocks into a 70 ℃ incubator for preheating for 15min, pouring paraffin which is melted in advance in the 70 ℃ incubator into an embedding frame made of iron, neatly placing the tissue blocks, and marking. (7) slicing: the sections were cut (thickness: 3 μm) with a tissue slicer. (8) spreading: sticking the cut tissue slices by using a writing brush, putting the tissue slices into water at 44 ℃, spreading the slices, ensuring that the slices have no wrinkles, and taking out the slices by using a glass slide. (9) baking the slices: the slide with the slice attached was placed in a 37℃incubator for 2 hours. (10) HE staining: the staining sequence was as follows: 15min of dimethylbenzene I, 15min of dimethylbenzene II, 3-5min of absolute ethyl alcohol, 3-5min of 95% alcohol, 3-5min of 85% alcohol, 3-5min of 75% alcohol, 4-6min of distilled water, 3-5min of hematoxylin staining solution, 3-4 times of dipping and washing by tap water until water does not change blue, 5s of 1% hydrochloric acid alcohol, 10-15min of flowing tap water washing, 2-4s of 90% alcohol, 3-6s of 1% eosin staining solution, 3s of 95% alcohol I, 1-2min of 95% alcohol II, 2-4min of absolute ethyl alcohol I, 5min of absolute ethyl alcohol II, and 20-30min of dimethylbenzene I. (11) sealing piece: neutral resin was dropped on the tissue section, and then covered with a cover slip. The slices were baked in a 37 ℃ incubator. (12) observation: tissue sections were observed under an optical microscope.
The results of the piglet clinical observation are as follows:
the survival rate and clinical symptoms of piglets are observed every day 0-72 hours after virus infection, and the results show that: after virus inoculation, the piglets in the Vehicle group show obvious diarrhea, and simultaneously show clinical symptoms such as vomiting, inappetence, somnolence, rough hair, dyspnea, periocular edema and the like, 48-72 hours after virus inoculation, 5 pigs in the positive control group die successively, 0.5mg/kg treatment group shows slight clinical symptoms, and 1mg/kg treatment group has no obvious clinical symptoms in the whole infected piglets. The Mock piglets have no diarrhea and vomiting during the experimental process, have no obvious clinical symptoms and survive healthily. As can be seen by comprehensive clinical scoring (FIG. 12), the 0.5mg/kg and 1mg/kg treated piglets showed a significant relief in symptoms compared to the Vehicle group. The above results are sufficient to demonstrate that Buddlejasaponin IVb can significantly alleviate the characteristic symptoms caused by PEDV infection.
Piglet viral load:
3d after virus infection, piglets are killed, the fecal and enterovirus content of each piglet is detected by RT-qPCR, the RT-qPCR detection method is the same as that of example 2, and the result is shown in figure 13: the PEDV virus content in the faeces and intestinal tissues of piglets of the treatment group of 0.5mg/kg and 1mg/kg is significantly lower than that of the Vehicle group and the dose dependency is reduced. The above results demonstrate that Buddlejasaponin IVb can significantly reduce replication of PEDV in piglets.
Intestinal pathological changes of piglets:
and 3d, killing piglets after the toxin is attacked, observing the intestinal lesions of each group of piglets by naked eyes, and performing statistical scoring. The results are shown in fig. 14, and intestinal eye observation of the Mock group piglets is completely normal without obvious lesions. The intestinal tissue of the piglet in the Vehicle group has thin intestinal wall and obvious intestinal lesions. Whereas Buddlejasaponin IVb treatment group had significantly lighter intestinal lesions than Vehicle group and was dose dependent. Statistics of the macroscopic lesions score of the intestinal tract show that the lesions score of the Vehicle group is significantly higher than that of the blank control group, and that the 0.5mg/kg and 1mg/kg groups are significantly lower than those of the Vehicle group. The observed results of the intestinal histopathological changes are: the intestinal tissue intestinal villi of the piglets of the Vehicle group are severely broken. The intestinal tissue lesions of piglets in the treatment groups of 0.5mg/kg and 1mg/kg Buddlejasaponin IVb were significantly milder than Vehicle. Statistics of intestinal tissue microscopic lesions scores show that the scores of intestinal tissue lesions in the treatment groups of 0.5mg/kg and 1mg/kg Buddlejasaponin IVb are significantly lower than those of the toxicity attack control group.
Piglet intestinal tissue inflammatory factor levels:
extracting RNA of intestinal tissues of each group of piglets, detecting the mRNA levels of inflammatory factors IL-1 beta, IL-6, IL-8 and TNF-alpha in the intestinal tissues by adopting an RT-qPCR method, and obviously increasing the levels of four inflammatory factors in the intestinal tissues of the positive group of piglets by adopting the RT-qPCR detection method as shown in the result of the RT-qPCR detection method in the example 2, wherein the result is shown in figure 15; whereas the inflammatory factors in the intestinal tissues of piglets after Buddlejasapnion IVb treatment decreased in a dose-dependent manner, the mRNA levels of IL-1β, IL-6, IL-8 and TNF- α in the intestinal tissues of piglets in the treatment group of 0.5mg/kg and 1mg/kg Buddlejasapnion IVb were significantly lower than those in the challenge control group, indicating that Buddlejasapnion IVb was effective in alleviating inflammation caused by PEDV.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (10)
1. Application of buddleja officinalis saponin IVb in preparing a product for preventing and treating porcine epidemic diarrhea is provided.
2. The use according to claim 1, wherein the buddleja saponin IVb inhibits proliferation of porcine epidemic diarrhea virus.
3. The use according to claim 1, wherein said buddleja saponin IVb inhibits replication and release of the porcine epidemic diarrhea virus lifecycle.
4. The use according to claim 1, wherein the buddleoside IVb inhibits NF- κb signaling pathway activation.
5. The use according to claim 4, wherein said buddleja saponin IVb inhibits the activation of NF- κb signaling pathway by down-regulating the expression of p-NF- κbp65, p-ikbα, up-regulating the expression of ikbα.
6. The use according to claim 1, wherein the buddleja saponin IVb is capable of reducing the expression of inflammatory factors.
7. The use of claim 6, wherein the inflammatory factors comprise IL-6, IL-8, IL-1 β and TNF- α.
8. The use according to claim 1, wherein the buddleja saponin IVb is effective in alleviating clinical symptoms of the body caused by porcine epidemic diarrhea virus and reducing the body enterovirus load.
9. A medicament for treating and/or preventing porcine epidemic diarrhea, wherein the medicament comprises an effective amount of buddleja saponin IVb.
10. The medicament according to claim 9, wherein the percentage of buddleja saponin IVb in the medicament is 1-99%.
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| CN116920073A (en) * | 2023-09-15 | 2023-10-24 | 青岛汇丰动物保健品有限公司 | Application of polypeptide in diarrhea treatment and medicine containing polypeptide |
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