CN113244243A

CN113244243A - Application of oleanolic acid in preparation of medicine for treating herpes encephalitis

Info

Publication number: CN113244243A
Application number: CN202110644918.XA
Authority: CN
Inventors: 任哲; 王巧利; 叶翠芳; 蒋毓洲
Original assignee: Jinan University
Current assignee: Jinan University
Priority date: 2021-06-09
Filing date: 2021-06-09
Publication date: 2021-08-13

Abstract

The invention belongs to the technical field of medicines, and particularly discloses application of oleanolic acid in preparation of a medicine for treating herpes-type encephalitis. According to the invention, through in-vitro and in-vivo experiments such as a CCK8 method, a plaque reduction method and the like, UL47 is found to be a potential target point of the oleanolic acid for exerting the anti-HSV-1 effect for the first time, and the oleanolic acid can exert the antiviral effect by inhibiting inflammatory factors, and researches show that the oleanolic acid has significant anti-HSV-1 activity on common HSV-1 strains, can inhibit the inflammatory reaction induced by HSV-1, can significantly improve the viral encephalitis caused by the infection of mice with HSV-1, and has good anti-HSV-1 activity in vivo. The discovery of the invention provides a new medicine which can effectively treat herpes encephalitis and is not easy to generate drug resistance for patients, and also provides a new idea for the later-stage further experimental study of determining whether the direct target of oleanolic acid is UL47 or not and the molecular mechanism of signal pathway of oleanolic acid which plays the role of resisting HSV-1 through UL 47.

Description

Application of oleanolic acid in preparation of medicine for treating herpes encephalitis

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to application of oleanolic acid in preparation of a medicine for treating herpes-type encephalitis.

Background

Herpes simplex virus type 1 (HSV-1) is a double-stranded DNA virus, and belongs to the Alphaheresvirinae subfamily together with another member of the Herpes simplex virus family, namely Herpes simplex virus type 2 (HSV-2). HSV-1 can cause various diseases including viral encephalitis, viral dermatitis, and stromal keratitis, and furthermore, people with low immunity can cause disseminated infection after infection with HSV-1, thereby generating pneumonia and hepatitis. Among the above-mentioned various diseases caused by HSV-1, two diseases, HSV-1 encephalitis (HSE) and HSV-1 stromal keratitis (HSK) are most representative. HSE is an inflammation of the brain parenchyma associated with neurological dysfunction that damages the central nervous system and leads to death of the patient due to brain injury. Encephalitis is common in patients with normal immune responses, with an incidence of 2-4 cases per 100 million people, distributed worldwide. While HSK caused by HSV-1 infection can affect almost every eye tissue and is a major cause of blindness in developed countries. Among these infections, herpes viruses mainly adopt a survival strategy that establishes chronic latent infections in the host. Thus, herpes viruses are highly persistent pathogens with seropositivity rates approaching 100% in the adult population^[9]. Current clinical anti-HSV-1 drugs are based on a range of nucleoside analogues, such as acyclovir, valacyclovir and famciclovir, and the mechanism of their antiviral action is primarily inhibition of viral DNA polymerase.

Currently, the main effective drugs for resisting the herpes virus type 1 are Acyclovir (Acylovir, ACV) which is an Acyclovir analogue, and valacyclovir, penciclovir and famciclovir which are homologous compounds of the Acyclovir. When ACV enters a cell infected with HSV-1, the viral protein Thymidine Kinase (TK) protein interacts with it causing it to phosphorylate, eventually forming a triphosphate state by cellular kinase action, and the triphosphate is incorporated into the replicating DNA strand by viral polymerase (pol) instead of guanosine triphosphate, resulting in chain end termination and inhibition of viral replication, however, as acyclovir is increasingly inundated, HSV-1 is increasingly resistant to it. Particularly for patients with low immune function, the therapeutic effect of ACV is obviously weakened. Therefore, there is a need to develop a drug which can effectively treat the herpes virus type 1 and is not easy to generate drug resistance.

Patent document CN105418721A discloses a chemical modification of oleanolic acid with anti-tumor activity and a preparation method thereof, wherein a series of structural analogs with biological activity are obtained by the chemical structural modification and modification of natural product oleanolic acid. Pharmacological experiments prove that the oleanolic acid chemical modifier has a good inhibition effect on human cervical cancer Hela cells, human liver cancer HepG2 cells and gastric cancer BGC-823 cells, and is superior to a parent compound oleanolic acid, but the effect of the oleanolic acid in treating herpes encephalitis is not disclosed.

Patent document CN102838651A discloses a class of oleanolic acid derivatives, their preparation methods and uses, providing an oleanolic acid derivative with a new structure and its salts, and also relates to the preparation methods of the above compounds and their salts, and discloses pharmaceutical compositions using the compounds as active ingredients and their applications as hypoglycemic drugs, but does not disclose that oleanolic acid has the effect of treating herpes encephalitis.

Disclosure of Invention

The invention aims to provide application of oleanolic acid in preparing a medicament for treating herpes encephalitis, and in vitro experiments prove that the oleanolic acid can obviously inhibit HSV-1 activity, HSV-1-induced inflammatory reaction and obviously improve viral encephalitis caused by infection of mice with HSV-1, and in vivo experiments prove that the oleanolic acid also has good anti-HSV-1 activity in organisms.

In order to achieve the purpose, the invention adopts the following technical scheme: application of oleanolic acid in preparing medicine for treating herpes encephalitis is provided.

Preferably, said oleanolic acid exerts antiviral activity via UL 47.

Preferably, said oleanolic acid exerts an antiviral effect by inhibiting inflammatory factors.

Preferably, the medicament for treating the herpes encephalitis is an oral preparation.

Preferably, the oral formulations include tablets, capsules, powders, granules and film-coated tablets.

Compared with the prior art, the invention has the following beneficial effects:

according to the invention, through in-vitro and in-vivo experiments such as a CCK8 method, a plaque reduction method and the like, UL47 is found to be a potential target point of the oleanolic acid for exerting the anti-HSV-1 effect for the first time, and the oleanolic acid can exert the antiviral effect by inhibiting inflammatory factors, and researches show that the oleanolic acid has significant anti-HSV-1 activity on common HSV-1 strains, can inhibit the inflammatory reaction induced by HSV-1, can significantly improve the viral encephalitis caused by the infection of mice with HSV-1, and has good anti-HSV-1 activity in vivo.

Drawings

FIG. 1 is a schematic representation of the toxicity of oleanolic acid to various cells.

FIG. 2 is a schematic representation of the anti-HSV-1 activity of oleanolic acid on different cells.

FIG. 3 is a schematic of oleanolic acid dosing at different time points on Vero cells.

FIG. 4 is a schematic diagram of the direct inactivation, adsorption and penetration of HSV-1 by oleanolic acid.

FIG. 5 is a schematic diagram showing the effect of oleanolic acid on HSV-1 gene.

FIG. 6 is a schematic representation of the inhibition of HSV-1 induced inflammatory responses by oleanolic acid.

FIG. 7 is a schematic diagram showing the experimental results of the copy number of the viral genes for reducing the infection of mouse encephalovirus by oleanolic acid.

FIG. 8 is a schematic diagram showing the experimental results of immunohistochemical analysis of oleanolic acid to reduce viral infection in mouse brain.

FIG. 9 is a schematic representation of the inhibition of HSV-1 induced inflammatory responses by oleanolic acid.

Detailed Description

The present invention will be described in further detail with reference to the following examples. It should not be understood that the scope of the above-described subject matter of the present invention is limited to the following examples.

The Vero cells were purchased from american type culture collection center (ATCC). The culture condition is DMEM medium containing 10% of ilex purpurea Hassk serum and 1% of double antibody;

the culture medium of the mouse neuroma blast (Neuro-2a) is a DMEM culture medium containing 10% Australia fetal bovine serum and 1% double antibody;

the culture medium used by the human glioma blast cells (SH-SY5Y) is a DMEM culture medium containing 10% Australia fetal bovine serum and 1% double antibody;

the culture medium used by the mouse microglia (BV2) is DMEM culture medium containing 10% Australia fetal bovine serum and 1% double antibody.

The herpes simplex virus I type F strain (HSV-1/F) is offered as a gift by hong Kong university, and is inoculated and amplified in Vero cells, the culture conditions are DMEM culture medium containing 2% of ilex purpurea Hassk serum and 1% of double antibody, and the DMEM culture medium is frozen at-80 ℃;

the Oleanolic acid (Oleanolic acid) is purchased from Shanghai Allandin Biotech Co., Ltd, and has a product number of O110088-1 g.

Example 1 drug toxicity test

First, experiment method

Taking Vro cells as an example, the CCK8 method is used for detecting drug toxicity of Vero cells:

(1) vero cells were plated 12 hours in advance at 8X10⁴Cells were seeded in 96-well plates at cell/ml density and placed in a cell incubator overnight;

(2) observing the cell state under a microscope, starting a drug toxicity test after confirming that the cells grow to a single layer, preheating a cell maintenance solution in advance, carrying out ultraviolet irradiation for 30min on a biological safety cabinet, diluting oleanolic acid mother liquor with the cell maintenance solution in the biological safety cabinet, wherein the dilution concentrations are respectively 50 mu M, 25 mu M, 12.5 mu M, 6.25 mu M, 3.125 mu M, 1.0625 mu M and 0.63125 mu M, and setting a DMSO control group and a blank cell control group;

(3) taking out a 96-well plate in a cell culture box, throwing off an original culture medium, sucking out a large amount of liquid residues by using a 200ul liquid transfer gun, adding prepared medicine diluent and DMSO diluent into the 96-well plate, adding 6 multiple wells per concentration, setting the remaining 12 multiple wells as cell contrast, adding a cell maintenance solution in the air, marking, and putting the cell maintenance solution into the cell culture box for continuous culture;

(4) after culturing for 48h, carrying out a drug toxicity test on Vero cells by using a CCK8 reagent, firstly diluting a CCK8 reagent by using a cell maintenance solution in a biological safety cabinet to ensure that the concentration of the reagent is 5%, then spin-drying the culture medium on a 96-well plate, absorbing and removing a large amount of liquid residues by using a 200-microliter pipette gun if the liquid residues exist, adding the diluted CCK8 reagent into the 96-well plate in the shade, wrapping each hole by 100 microliter, and putting the 96-well plate into a cell culture box to be continuously cultured for 2h in a dark place;

(5) after 2 hours, the 96-well plate was taken out from the cell incubator, the microplate reader was opened, the absorbance was measured at a wavelength of 450nm, and the half-toxic concentration of the drug ((IC50) and the cell viability were calculated from the measured values.

The calculation formula is as follows: cell viability (%). gtoreq (medicated a 450/cell control a450) × 100%

(6) The method is the same when testing toxicity of DMSO and oleanolic acid on N2A cells, BV2 cells and SY5Y cells.

Second, experimental results

As shown in FIG. 1, the survival rate of cells after oleanolic acid acts on different cells for different time periods is respectively detected by a CCK8 detection method. According to the calculation of absorbance, the toxicity of oleanolic acid to different cells is different, and the cell survival rate is in a descending trend along with the increase of the drug concentration. Among them, it can be seen from the analysis data that fig. 1A shows that IC50 is 48.05 μ M when oleanolic acid acts on Vero cells for 72 hours, and the cell viability reaches 90% when the concentration is 25 μ M. Meanwhile, FIG. 1B, FIG. 1C and FIG. 1D show that after oleanolic acid is applied to SY5Y, BV2 and N2A cells for 24 hours, its IC50 values are 67.8. mu.M, 71.35. mu.M and 58.36. mu.M, respectively, and the non-toxic concentration of oleanolic acid is 25. mu.M for these three cells.

Example 2 combination of antiviral action

First, experiment method

1.1 detection of comprehensive anti-HSV-1F strain activity of oleanolic acid d by using plaque reduction experiment (PRA) method

(1) Vero cells were plated 12 hours in advance at 3.2X10⁵Cells were seeded in 24-well plates at cell/ml density and placed in a cell incubator overnight

(2) Observing the cell state under a microscope, starting a plaque reduction experiment when the confluence degree of cells in a plate is confirmed to reach 100%, taking a cell maintenance solution in advance, diluting the oleanolic acid to 50 mu M, 25 mu M, 12.5 mu M, 6.25 mu M and 3.125 mu M, mixing the diluted oleanolic acid with 100PFU/ml virus solution in the same volume, and blowing and uniformly mixing the mixture by using a 1ml pipette;

(3) taking out a 24-hole plate in a cell culture box, sucking and removing a culture medium by using a 1ml pipette gun, then taking a small amount of PBS (phosphate buffer solution) for rinsing twice, completely sucking the PBS, sequentially adding a medicament-virus mixed solution into the holes, wherein each hole is 250 mu l, and a virus control group and a cell control group are arranged at the same time, and each group has 3 multiple holes;

(4) placing the 24-hole plate in an incubator for adsorption for 2 hours, shaking once every 30min, and shaking for four times;

(5) diluting the drug to the same concentration by using a covering solution, removing a virus adsorption solution after adsorption for 2 hours is finished, taking a small amount of PBS (phosphate buffer solution) for rinsing twice, then adding the covering solution with the same drug concentration, and putting the covering solution into a cell culture box for continuous culture;

(6) observing the cytopathic condition under a microscope every 24 hours, sucking and removing the covering liquid after the cytopathic condition is not changed any more, and taking a small amount of PBS for rinsing twice;

(7) the PBS was removed by aspiration, and 4% paraformaldehyde fixing solution was added to the plate, 400. mu.l per well, and fixed for 20 min;

(8) removing the fixing solution by suction, rinsing a small amount of PBS twice, completely sucking the PBS, adding 1% crystal violet staining agent into a 24-hole plate, and staining for 30min, wherein each hole is 300 mu l;

(9) recovering 1% of crystal violet staining solution after dyeing is finished, and cleaning residual crystal violet staining solution by using tap water;

(10) and (3) inversely placing the cleaned 24-hole plate on a balcony for natural air drying, and after air drying, recording the number of the plaques and calculating the plaque inhibition rate.

1.2 detection of Virus Titers after drug action

Taking SY5Y cell as an example, virus titer detection after drug action is carried out

(1) SY5Y cells were plated 12 hours in advance at 1.5X10⁵The cells are paved in a 12-hole plate per ml, and placed in a culture box for overnight culture;

(2) observing the cell state under a microscope, sucking and removing the culture medium, taking a small amount of PBS for rinsing twice, sucking and removing the PBS, and sucking out residual liquid by using a 200 mu l pipette;

(3) preparing drug diluents with the concentrations of 50 mu M and 25 mu M by using a cell maintenance solution, simultaneously diluting a virus stock solution to MOI of 10, uniformly mixing the drug diluents with the same volume to reduce the drug concentration by half, and adding the prepared virus-drug mixed solution into a 12-hole plate with 500 mu l per hole;

(5) adsorbing for 2 hours, sucking away the culture medium, taking a small amount of PBS for rinsing twice, sucking away the PBS, completely sucking away residual liquid by using a 200 mu l pipette gun, preparing a medicine diluent with equal concentration, and adding the medicine diluent into a 12-hole plate for continuous culture;

(6) culturing for 12 hr and 24 hr, respectively placing 12-well plate into-80 deg.C ultra-low temperature refrigerator, repeatedly freezing and thawing for three times, and collecting supernatant;

(7) with reference to the comprehensive antiviral effect, the collected supernatant was added to a 96-well plate previously plated with Vero cells, and the virus titer after drug action was detected and calculated.

Second, experimental results

As shown in FIG. 2, the oleanolic acid in FIG. 2A has a significant inhibitory effect on the formation of plaque, and the effect shows a strong concentration dependence, and the statistical data shows that the plaque inhibition rate is higher than 90% when the concentration of oleanolic acid is 20 μ M.

Meanwhile, in order to further investigate whether oleanolic acid has antiviral effect on other cells, the virus titer detection after the drug effect is carried out on other cells, taking BV2 cells as an example, firstly, a cell maintenance solution containing HSV-1(MOI 10) and high-concentration oleanolic acid (25 muM) and a cell maintenance solution containing HSV-1(MOI 10) and low-concentration oleanolic acid (12.5 muM) are respectively added into BV2 cells planted in a 6-well plate for 12h, after 2 hours of virus adsorption, the culture medium is sucked away, a cell maintenance solution containing corresponding concentration oleanolic acid is added, and the culture is continued to 24 hours in the culture box. And after the culture is finished and the 6-hole plate is frozen and thawed for three times, the titer detection is carried out, and the result shows that the oleanolic acid in the figure 2B still presents good antiviral activity on the BV2 cells. In a similar way, the same detection is carried out on SH-SY5Y cells and N2A cells, and as shown in figures 2C and 2D, oleanolic acid still shows good anti-HSV-1 activity on the two cells.

The results are combined to show that the oleanolic acid has good anti-HSV-1 activity on various cells.

Example 3 detection of action time Point of drug

First, experiment method

(1) Vero cells were plated 12 hours in advance at 3.2X10⁵Cells were seeded in 24-well plates at cell/ml density and placed in a cell incubator overnight;

(2) observing the cell state under a microscope, starting to detect the drug action time point when the confluence degree of cells in the plate reaches 100% is confirmed, sucking away the culture medium, taking a small amount of PBS to rinse twice, sucking up the PBS, adding 50 PFU/hole virus diluent, setting eight groups of 0h, 2h, 4h, 6h, 8h, 10h, 12h and 24h according to different time points, adding 125 mul of the drug-containing culture medium into the 0h group, and adding 125 mul of cell maintenance solution into the rest groups;

(3) placing the 24-hole plate into a cell culture box for adsorbing for 2 hours, and shaking once every 30min for 4 times;

(4) after 2 hours, taking out the 24-hole plate from the cell incubator, sucking away the virus diluent, taking a small amount of PBS for rinsing twice, sucking up the PBS, adding the drug-containing covering liquid with equal concentration into the 0h group and the 2h group, adding 500 mu l of cell maintenance liquid into the rest groups, and putting the cells into the incubator for continuous culture;

(5) after 2 hours, taking out the 24-hole plate, changing the cell maintenance liquid of the 4h group into the drug-containing covering liquid with the equal concentration, and repeating the steps until the cell maintenance liquid of the 24h group is changed into the drug-containing covering liquid with the equal concentration;

(6) and (3) putting the 24-hole plate into a cell culture box to continue culturing until the cytopathic condition is not changed any more, dyeing according to a method in comprehensive antiviral action, recording the number of the plaques and calculating the plaque inhibition rate.

Second, experimental results

As shown in figure 3, in order to deeply understand the antiviral mechanism of oleanolic acid, the specific time point of the oleanolic acid playing antiviral action after HSV-1 infected cells is determined, and the action time point of oleanolic acid by using Vero cells is selected for research. By adding drugs at different time points between 0 and 24 hours, the inhibition effect of oleanolic acid on the plaque within 0 to 4 hours is obviously better than that of the subsequent time points as shown in figure 3A. It is therefore believed that oleanolic acid exerts antiviral effects at an early stage immediately after HSV-1 infection.

Example 4 Effect of Oleanolic acid on direct inactivation, adsorption and puncture of viruses

First, experiment method

1.1 direct inactivation of viruses by drugs

(2) observing the cell state under a microscope, starting to detect the direct inactivation of the drug to the virus when confirming that the confluence degree of cells in the plate reaches 100%, firstly diluting a virus stock solution to 50 PFU/hole by using a cell maintenance solution, mixing the virus stock solution with a drug stock solution, transferring the virus-drug mixed solution into a sterilized 2ml EP tube, and putting the tube into a cell incubator to incubate for 2 hours;

(3) setting 3 multiple holes of each group of the drug adding group, the cell control group and the virus control group, and taking out from the cell culture box after the incubation is finished;

(4) removing the culture medium by aspiration, rinsing a small amount of PBS twice, completely aspirating the PBS, and adding the virus-drug mixed solution into the 24-well plate according to the volume of 250 mul per well;

(5) placing the 24-hole plate in a cell culture box for adsorbing for 2 hours, shaking once every 30min, and shaking for four times;

(6) after 2 hours, taking out the 24-well plate from the cell culture box, sucking and discarding the maintenance liquid, taking a small amount of PBS for rinsing twice, sucking up the PBS, and adding 500 mu l of covering liquid into the 24-well plate;

(7) and (3) putting the 24-hole plate into a cell culture box to continue culturing until the cytopathic condition is not changed any more, dyeing according to a method in comprehensive antiviral action, recording the number of the plaques and calculating the plaque inhibition rate.

1.2 antiviral adsorption of drugs

(2) observing the cell state under a microscope, starting to detect the antiviral adsorption of the medicine when the confluence degree of cells in the plate reaches 100%, precooling a 24-pore plate in a refrigerator at 4 ℃ for 1 hour, and precooling PBS (phosphate buffer solution);

(3) diluting the virus stock solution to 50 PFU/hole by using the cell maintenance solution, and uniformly mixing the virus stock solution with the drug mother solution;

(4) after the pre-cooling is finished, removing the culture medium by suction, taking a small amount of pre-cooled PBS for rinsing twice, completely sucking the PBS, adding virus-medicine mixed liquid, and simultaneously setting 3 compound holes in each group of the medicine adding group, the cell control group and the virus control group;

(5) adsorbing the 24-hole plate in a refrigerator at 4 ℃ for 2 hours, shaking once every 30min, and shaking for four times;

(6) after 2 hours, taking out the 24-hole plate from a refrigerator at 4 ℃, sucking and discarding the cell maintenance solution, taking a small amount of PBS for rinsing twice, sucking up the PBS, and adding a covering solution into the holes;

1.3 antiviral penetration of drugs

(2) observing cell state under microscope, and detecting antiviral puncture effect of the medicine when cell confluency in plate reaches 100%

(3) Diluting the virus stock solution to 50 PFU/hole with the cell maintenance solution, blowing, beating and mixing uniformly;

(4) after the pre-cooling is finished, sucking away the culture medium, taking a small amount of pre-cooled PBS for rinsing twice, sucking up the PBS, adding virus diluted solution, putting the 24-hole plate in a refrigerator at 4 ℃ for adsorbing for 2 hours, shaking once every 30min, and shaking for four times in total;

(5) taking a proper amount of cell maintenance liquid, diluting the drug mother liquor, blowing and uniformly mixing;

(6) after 2 hours, taking out the 24-hole plate from a refrigerator at 4 ℃, sucking away the cell maintenance liquid, taking a small amount of PBS to rinse twice, sucking up the PBS, simultaneously setting 3 multiple holes for each group of a medicine adding group, a cell control group and a virus control group, adding the medicine-containing cell maintenance liquid into the medicine adding group, and incubating for 10 minutes in a cell incubator;

(7) after the incubation, the 24-well plate was taken out from the cell incubator, the maintenance solution was aspirated and discarded, 300ul of acidic PBS (PH 3.0) was added thereto, and after 1min, the acidic PBS (PH 3.0) was aspirated;

(8) adding alkaline PBS (pH 11.0), neutralizing for 1min, and sucking out alkaline PBS (pH 11.0);

(9) rinsing a small amount of PBS twice, completely sucking the PBS, and adding 500 mu l of covering liquid into a 24-well plate;

(10) and (3) putting the 24-hole plate into a cell culture box to continue culturing until the cytopathic condition is not changed any more, dyeing according to a method in the comprehensive anti-virus effect, recording the number of plaques and calculating the plaque inhibition rate.

Second, experimental results

The results are shown in FIG. 4. In order to further explore the way that oleanolic acid immediately and early resists HSV-1 infection, the inhibition effect of oleanolic acid on two stages of HSV-1 adsorption and puncture is detected through a plaque reduction experiment. It can be seen from fig. 4B and 4C that oleanolic acid has no significant effect on both stages.

Similarly, the direct inactivation of HSV-1 by oleanolic acid was examined by plaque reduction experiments, and it can be seen from the results in FIG. 4A that oleanolic acid has no significant effect on the direct inactivation of HSV-1. Therefore, the oleanolic acid does not influence the adsorption and puncture stages of HSV-1, and has no direct inactivation effect on HSV-1.

Example 5 detection of the copy number of viral genomic DNA (taking SY5Y cells as an example)

First, experiment method

(3) preparing drug diluents with the concentrations of 50 mu M and 25 mu M by using a cell maintenance solution, simultaneously diluting a virus stock solution to MOI 1, uniformly mixing the drug diluents with the same volume to reduce the drug concentration by half, and adding the prepared virus-drug mixed solution into a 12-hole plate with 500 mu l per hole;

(7) extracting a virus genome by using a virus DNA extraction kit;

(8) the extracted viral DNA was diluted with DEPC water, typically 5-fold, and then the amount of viral DNA was detected by Real Time PCR.

Second, experimental results

The results of the experiment are shown in FIG. 5. Because the oleanolic acid has no obvious influence on the adsorption and puncture functions of the virus at the immediate early stage, but the oleanolic acid has obvious inhibition effect on the virus, the virus gene related to HSV-1 at the immediate early stage is detected. From the results of fig. 5A, 5B and 5C, it can be seen that oleanolic acid has a significant inhibitory effect on UL47 on different cells. It can therefore be hypothesized that oleanolic acid exerts antiviral activity by inhibiting UL 47.

Example 6 Effect of Oleanolic acid on infection of mice with HSV-1 to cause encephalitis

First, experiment method

1.1 establishment of model of mouse encephalitis infected by HSV-1

(1) Purchasing SPF-grade BABL/c mice of 4 weeks old in the animal center of Guangdong province, and placing the mice in an SPF-grade mouse feeding room for feeding;

(2) after adaptive feeding for one week, randomly grouping according to body weight, wherein each group comprises 18 animals and 5 animals;

(3) stopping adaptive feeding when the average weight of the mice reaches about 25g, molding, and performing nasal drip for 2x10 in the model group and the dosing group⁶PFU virus, placebo given the same volume of DMEM medium;

(4) recording the weight change of the mouse every day, and performing intragastric administration according to the weight of the mouse, wherein the two administration doses are 50mg/kg and 25mg/kg respectively, and meanwhile, performing intragastric administration according to the dose of 20mg/kg by taking ACV as a positive drug;

(5) the mice are sacrificed on the 9 th day after infection, brain tissues are taken out, physiological saline is used for rinsing to remove residual blood, filter paper is used for sucking, the whole brain is placed in 4% paraformaldehyde for fixation for subsequent experiments such as immunohistochemistry, and other brain tissues are divided into three parts of Olfactory Bulb (OB), Cerebral Cortex (CX), Pons, Medulla oblongata and Cerebellum (Pons, Medulla oblongata, Cerebellum and P/M/C) according to requirements, placed in an imported EP (No RNase) tube, placed on ice and then transferred to the 80 ℃ below zero for storage.

1.2 histopathological experiments

Taking out a tissue sample to be sliced, soaking the tissue sample in 4% paraformaldehyde, fixing for 3-4 hours, putting the tissue sample into an embedding box for dehydration, and embedding the tissue sample by using paraffin, wherein bubbles are prevented from being generated in the embedding process; and (3) slicing by using a paraffin slicer, carrying out antibody incubation after the slicing is finished, staining cell nuclei by using hematoxylin, and finally, photographing and recording under a microscope after the sealing is finished.

1.3 mice were bled

(1) Firstly, anesthetizing a mouse by intraperitoneal injection of phenobarbital sodium (20 mu l/mouse), and grabbing the mouse after the mouse is completely anesthetized;

(2) slightly pressing the skin beside the eyeball to ensure that the eyeball is congested and protruded;

(3) using a surgical instrument to cut mouse whiskers to prevent hemolysis, then using forceps to grasp the protruding eyeball and quickly pick it up, allowing blood to flow from within the orbit into a pre-prepared 1.5ml ep tube;

(4) after blood sampling is finished, carrying out neck dislocation on the mouse to kill the mouse so as to take brain tissue subsequently;

(5) standing the blood of the mouse at room temperature for 2h, opening a centrifugal machine during standing for precooling, after standing, sucking the upper serum, transferring the upper serum into a clean EP tube, centrifuging for 20min at 4 ℃ and 3000rpm, sucking the supernatant, transferring the supernatant into the clean EP tube again, and storing the supernatant in an ultra-low temperature refrigerator at-80 ℃.

1.4 Effect experiment of Oleanolic acid on infection of mice with HSV-1 to cause encephalitis

The administration by gavage was started at a fixed time per day on the second day after the molding, and according to the experimental requirements, Ctrl group, high dose group (50mg/kg), low dose group (25mg/kg), ACV group (20mg/kg) and encephalitis model group were set. After the ninth day after the administration, the mice were subjected to an eyeball bleeding and cervical dislocation treatment and the mental state of the mice was observed and analyzed before the mice were sacrificed.

Plotting by Graphpad prime 5; all data were analyzed using SPSS10.0 statistical software, and two comparisons were Student's two-tailed t-test, p<0.5 is a significant difference, denoted as x; p is a radical of<0.1 is the most significant difference, denoted as x. All experimental data to

And (4) showing.

Second, experimental results

As shown in figure 6, at 6h and 9h of drug action, oleanolic acid has obvious inhibitory effect on virus-induced IL6, TNF-alpha and NOS 2.

As shown in FIG. 7, compared with Ctrl group mice, HSV group mice have no light hair and have ulcerated swollen eyes, and the mental state of the HSV group mice is obviously different from that of Ctrl group mice, thereby proving that the molding is successful. Subsequently, we performed viral gene copy number (as shown in fig. 7B, fig. 7C) and immunohistochemical analysis (as shown in fig. 8D, fig. 8E, fig. 8F, fig. 8G, fig. 8H) after killing mice, and the analysis results of fig. 7B and fig. 7C showed that the viral gene copy number was significantly down-regulated in the high oleanolic acid dose group (50mg/kg) and the low oleanolic acid dose group (25mg/kg) relative to the HSV group; by immunohistochemical analysis, the number of virus particles in the high oleanolic acid dose group (50mg/kg) and the low oleanolic acid dose group (25mg/kg) was significantly reduced compared to the HSV group.

Meanwhile, we also tested the gene expression levels of IL6, TNF- α, and NOS2 in the brain, as shown in FIG. 9. FIGS. 9A, 9B and 9C show that the genes of IL6, TNF-alpha and NOS2 in mouse brain were inhibited in either the high oleanolic acid dose group (50mg/kg) or the low oleanolic acid dose group (25 mg/kg).

From the above experiments, it can be shown that the invention proves that oleanolic acid has significant anti-HSV-1 virus activity through in vitro and in vivo experiments such as drug toxicity experiments, virus titer detection experiments, plaque reduction experiments and the like, and plays an antiviral role immediately in the early stage after HSV-1 infection, and simultaneously obtains the conclusion that oleanolic acid can play an antiviral role by inhibiting UL47 or inhibiting the expression of inflammatory factors by detecting virus genes immediately related to HSV-1 in the early stage, thereby providing a new idea for researching a signal pathway molecular mechanism for determining whether the direct target of oleanolic acid is UL47 and whether the oleanolic acid plays an anti-HSV-1 role through UL47 in the later stage of further experimental exploration.

The foregoing embodiments are merely illustrative of the principles and utilities of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or change the above-mentioned embodiments without departing from the spirit and scope of the present invention. Accordingly, it is intended that all equivalent modifications or changes which may be accomplished by those skilled in the art without departing from the spirit and scope of the present invention as set forth in the appended claims.

Claims

1. Application of oleanolic acid in preparing medicine for treating herpes encephalitis is provided.

2. The use according to claim 1, wherein said oleanolic acid exerts antiviral activity via UL 47.

3. The use of claim 1, wherein said oleanolic acid exerts an antiviral effect by inhibiting inflammatory factors.

4. The use according to claim 1, characterized in that the medicament for the treatment of herpes encephalitis is an oral formulation.

5. The use according to claim 4, wherein the oral formulation comprises tablets, capsules, powders, granules and film-coated tablets.