CN112675184A - Application of baicalin in preparation of medicine for treating H6N6 subtype avian influenza - Google Patents
Application of baicalin in preparation of medicine for treating H6N6 subtype avian influenza Download PDFInfo
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Abstract
The invention relates to the field of Chinese veterinary medicine, and particularly discloses an effect of baicalin on resisting H6N6 subtype avian influenza virus. The baicalin medicine is safe and effective in resisting H6N6 subtype avian influenza virus, can reduce inflammatory reaction caused by H6N6 subtype avian influenza virus, protects lung injury, prevents mast cells from degranulating and releasing inflammatory mediators, and can be used for treating H6N6 subtype avian influenza.
Description
Technical Field
The invention relates to the field of Chinese veterinary medicine, in particular to application of baicalin in preparation of a medicine for treating avian influenza H6N 6.
Background
With the variation of the H6N6 strain, the H6N6 subtype AIV can infect mammals such as mice, chisels, pigs and the like, and serum-specific antibodies of the H6 subtype AIV are detected even in healthy people, which indicates that the H6N6 subtype avian influenza virus has the capability of infecting the mammals across species barriers and poses potential threats to human health. While the traditional western medicine has the function of resisting influenza virus in clinical application for many years, the virus antigen is mutated or adapted to the function of a self-derived medicine, and escape and drug resistance appear. The traditional Chinese medicine and the compound thereof have unique advantages in resisting influenza viruses and have wide development prospect. Chinese has many antiviral traditional Chinese medicines, such as cassia twig, rhizoma ligustici wallichii, rhizoma atractylodis, folium isatidis, radix sileris, radix scutellariae, rehmannia, houttuynia cordata and the like, wherein some traditional Chinese medicines also have good inhibition effect on avian influenza virus, the effect is not inferior to that of western medicines, and the scutellaria baicalensis has the best effect on resisting H6N6 subtype avian influenza through a chick embryo culture test and a hemagglutination test. Baicalin (Baicalin) is a flavonoid compound separated from radix Scutellariae, and has wide pharmacological effects, especially anti-inflammatory and immunoregulatory effects.
Although the prior art discloses that baicalin has the effects of reducing the total number of white blood cells and inhibiting a bleeding reaction on an inflammatory reaction caused by H6N6 avian influenza, the specific principle is unknown, the application of the baicalin in the preparation of a medicament for treating H6N6 subtype avian influenza is less, and related patents do not disclose baicalin medicaments for treating the H6N6 subtype avian influenza. For example, patent CN201510096401.6 discloses that baicalin has activity of inhibiting neuraminidase from avian influenza virus H7N9, which proves that baicalin has application in preparing anti-avian influenza virus H7N9 drugs. However, it is not directed against H6N6 subtype avian influenza and does not disclose how to specifically inhibit H6N6 subtype avian influenza virus.
In conclusion, the application of the baicalin in preparing the medicine for treating H6N6 subtype avian influenza is lacked at present, and knowing the specific pathogenesis of the baicalin enables people to take medicines according to symptoms better and give play to the baicalin component to treat subtype H6N6 avian influenza diseases better.
Disclosure of Invention
In order to solve the problems, the invention provides application of baicalin in preparing a medicine for treating H6N6 subtype avian influenza, and the medicine is used for treating the H6N6 subtype avian influenza by controlling inflammatory reaction, protecting lung injury and preventing mast cells from degranulation and releasing inflammatory mediators.
One of the purposes of the invention is to provide application of baicalin in preparation of a medicine for treating H6N6 subtype avian influenza, and the specific technical scheme is as follows:
use of baicalin in the preparation of a medicament for treating H6N6 subtype avian influenza, wherein the baicalin reduces the content of avian influenza H6N6 virus by inhibiting the production of neutrophils, reducing the degranulation rate of mast cells in lung tissues and inhibiting the secretion of inflammatory factors, wherein the inflammatory factors are IL-1, IL-2, TNF-alpha, IFN-gamma and 5-HT.
Further, the baicalin can inhibit the activity of HA2 subunit of hemagglutinin and inhibit the replication of avian influenza H6N6 virus genetic material in cells.
Preferably, the baicalin inhibits the production of neutrophils by reducing the genetic material of the H6N6 subtype avian influenza virus.
Further, the reduction in the degranulation rate of mast cells inhibits the secretion of inflammatory factors.
The second purpose of the invention is to provide an application of the baicalin inhibitor, and the specific technical scheme is as follows:
application of baicalin in preparing inhibitor for inhibiting secretion of neutrophil in vivo infected with H6N6 subtype avian influenza is provided.
The third purpose of the invention is to provide an application of the baicalin inhibitor, and the specific technical scheme is as follows:
application of baicalin in preparing inhibitor for inhibiting mast cell degranulation rate in lung tissue infected with H6N6 subtype avian influenza is provided.
The fourth purpose of the invention is to provide an application of the baicalin inhibitor, and the specific technical scheme is as follows:
application of baicalin in preparing inhibitor for inhibiting secretion of inflammatory factor 5-HT in vivo infected with H6N6 subtype avian influenza is provided.
Further, said inhibition of the secretion of inflammatory factor 5-HT in vivo in avian influenza infected with subtype H6N6 is achieved by inhibiting the mast cell degranulation rate in lung tissue.
The invention has the advantages that: the key inflammatory factor of the baicalin in the treatment of H6N6 subtype avian influenza is researched, the treatment aim is achieved by inhibiting the generation of neutrophils and the degranulation rate of mast cells in lung tissues, and a specific thought and a key control factor are provided for the treatment of the H6N6 subtype avian influenza.
Drawings
FIG. 1 is a flow chart of research on baicalin resisting avian influenza H6N6 virus.
FIG. 2 is a graph showing the results of in vivo AIV-resistant test of Chinese herbs in chicken embryo (n-6).
FIG. 3 is a graph of the total number of leukocytes in each group of mice (10)9·L-1，n＝6)。
FIG. 4 is a graph showing the number of neutrophils in each group of mice (10)9·L-1，n＝6)。
Fig. 5 is a graph of the percentage of neutrophils in each group of mice (%, n-6).
FIG. 6 lymphocyte counts (10) for each group of mice9·L-1，n＝6)。
Figure 7 percentage of lymphocytes in each group of mice (%, n-6).
FIG. 8 viral content (. mu.L) in lung tissue of mice in each group-1，n＝6)。
FIG. 9 virus content (. mu.L) in tracheal tissue of mice in each group-1，n＝6)。
FIG. 10 spleen tissue virus content (. mu.L) of each group of mice-1，n＝6)。
FIG. 11 Lung mast cell number (pieces/mm)2，n＝6)。
Fig. 12 lung mast cell degranulation (%, n ═ 6).
FIG. 13 serum IL-1 content (pg. mL) in each group of mice-1，n＝6)。
FIG. 14 serum IL-2 content (pg. mL) in each group of mice-1，n＝6)。
FIG. 15 serum TNF-. alpha.content (pg. mL) in each group of mice-1，n＝6)。
FIG. 16 serum IFN-. gamma.content (pg.mL) in each group of mice-1，n＝6)。
FIG. 17 serum 5-HT content (ng. mL) of each group of mice-1，n＝6)。
Detailed Description
The present invention will be described in further detail with reference to the following examples and drawings, it being understood that the present invention is not limited to the particular examples described herein, but is capable of modification in various forms and details, and can be modified within the spirit and scope of the invention.
DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION
1. Experimental Material
10 Chinese medicinal materials such as cassia twig, szechuan lovage rhizome, swordlike atractylodes rhizome, indigowoad leaf, divaricate saposhnikovia root, baical skullcap root, rehmannia root, heartleaf houttuynia herb, honeysuckle flower, fineleaf schizonepeta herb and the like are purchased from Chinese herbal medicine branches of Tongji Tang in Guiyang city; baicalin (Shanxi Yuning Biotechnology Co., Ltd., lot number: YN-20180512), Ostat phosphate (Yichang Changjiang medicine Co., Ltd., lot number: H20055532); 9-day-old non-immunized chick embryos, provided by scientific research chicken farms at Guizhou university. 108 male and female halves (22.17 + -2.24 g) of Kunming-line white mice (6-8 weeks old) were purchased from Chongqing Tengxin Biotechnology Ltd. An H6N6 subtype avian influenza virus (107.66EID50) which is derived from Guizhou province animal epidemic disease and veterinary public health key laboratories.
2. Screening of Chinese medicinal materials for resisting avian influenza H6N6 virus (shown in figure 1)
Screening of traditional Chinese medicines for resisting avian influenza H6N6 virus: randomly and averagely dividing 9-day-old chick embryos into 12 groups (a negative control group, a virus positive group, a cinnamon twig group, a ligusticum wallichii group, a rhizoma atractylodis group, a folium isatidis group, a radix sileris group, a scutellaria baicalensis group, a rehmannia glutinosa group, a houttuynia cordata group, a honeysuckle group and a schizonepeta tenuifolia group), wherein each group contains 6 chick embryos, and the chick embryos of the negative control group are injected with physiological saline 0.2 mL/chick embryos; the injection concentration of the virus control group chick embryo is 100EID500.2 mL/piece of the N6 subtype AIV virus; 10 experimental groups of Chinese medicine chick embryo are injected with Chinese medicinal decoction and 100EID501:1 of virus/0.2 mL/piece. After inoculation, the embryos are placed in a constant temperature box at 37.8 ℃ for incubation for 72h, dead embryos within 24h are discarded, allantoic fluid of the dead embryos after 24h and live embryos after 72h is harvested, and Hemagglutination (HA) titer is measured. The results of the AIV resistance test of the traditional Chinese medicines in chick embryos are shown in figure 2, and the scutellaria baicalensis is judged to be the best traditional Chinese medicine for resisting avian influenza H6N6 virus through HA titer.
Determination of safety: low dose baicalin group (0.014 mg. only)-1·d-1) Middle-dose baicalin treatment group (0.028 mg. only)-1·d-1) High dose baicalin treatment group (0.056 mg. only)-1·d-1) And ultra-high dose baicalin treatment group (0.112 mg. only)-1·d-1) Injected into mice without causing significant clinical symptoms, after sacrifice, in miceNo lesions were found in heart, liver and kidney tissues, indicating that baicalin is safe for mice.
108 6-8 week old Kunming mice were randomly and evenly divided into 6 groups: negative control group, virus positive group, western medicine control group, low dose baicalin group, middle dose baicalin group, and high dose baicalin group, all the doses are obtained by performing body surface area conversion according to traditional Chinese medicine pharmacological experiment method (Lieqiu, 1991) in combination with literature. The mice of each group are kept in isolation under the same condition, and are fed with food and water freely. Treatment was started 3d after challenge, and samples were collected by jugular phlebotomy at 3d, 6d and 9d after treatment (6 mice were taken in each group). Subsequently, obtaining the virus copy number of a corresponding experimental group through fluorescent quantitative PCR; observing tissue damage and the number of mast cells through pathological sections; the number change and the morphological distribution of the blood cells and the number of inflammatory factors (IL-1, IL-2, TNF-alpha, IFN-gamma and 5-HT) were obtained by blood routine, ELISA.
1. Index of total number of leukocytes
When 3d, 6d and 9d are treated, as shown in fig. 3, the total number of leukocytes in the negative control group, the western medicine control group and the high-dose baicalin group is remarkably lower than that in the virus positive group (P is less than 0.01); when the treatment is carried out for 6d and 9d, the total number of white blood cells in the low-dose and medium-dose baicalin groups is very obviously lower than that in the virus positive group (P is less than 0.01), and the total number of white blood cells in the high-dose and medium-dose baicalin groups is very obviously lower than that in the low-dose and medium-dose baicalin groups (P is less than 0.01) (Table 1).
TABLE 1 Total leukocyte count (10) in each group of mice9·L-1，n＝6)
Note that the data in the same column are marked with different lower case letters to show significant difference (P < 0.05); the capital letters with different shoulder marks show obvious difference (P is less than 0.01); shoulder marks with the same letter or no letter designation indicate no significant difference (P > 0.05). The same applies below.
2. Neutrophil index
(1) When 3d, 6d and 9d were treated, as shown in fig. 4, the numbers of neutrophils were significantly higher in the virus-positive group than in the negative control group and all the treatment groups (P < 0.01); the number of neutrophils in the high-dose baicalin group is extremely obvious or obviously lower than that in the low-dose and medium-dose baicalin group (P is less than 0.01 or P is less than 0.05), and the difference with a western medicine control group is not obvious (P is more than 0.05) when the high-dose baicalin group is treated for 3d (Table 2).
TABLE 2 neutrophil counts (10) in each group of mice9·L-1，n＝6)
(2) When 3d and 6d are treated, as shown in fig. 5, the percentage of neutrophils in the high-dose baicalin treated group is extremely lower than that in the virus positive group (P is less than 0.01), and the difference between the high-dose baicalin treated group and the western medicine control group is not significant (P is more than 0.05); the percentage of neutrophils in the medium-dose and high-dose baicalin group was significantly lower than that in the virus-positive group (P < 0.05) at treatment time 9d (Table 3).
Table 3 percent neutrophil for each group of mice (%, n ═ 6)
3. Lymphocyte index
(1) When 3d, 6d and 9d are treated, as shown in fig. 6, the number of lymphocytes in the high-dose baicalin group is remarkably higher than that in the virus positive group (P is less than 0.01), and the difference with a western medicine control group is not remarkable (P is more than 0.05); the lymphocyte numbers of the low and medium dose baicalin treatment groups were significantly higher than those of the virus positive group (P < 0.01) in both 6d and 9d treatment (Table 4).
TABLE 4 lymphocyte counts (10) for each group of mice9·L-1，n＝6)
(2) As shown in fig. 7, the percentage of lymphocytes in the high-dose baicalin group was significantly higher than that in the virus-positive group (P < 0.01) when 3d, 6d and 9d were treated, and the difference from the western medicine control group was not significant (P > 0.05); the lymphocyte percentage was significantly higher in both the low and medium dose baicalin treated groups than in the virus positive group (P < 0.01) for 6d and 9d treatments (Table 5).
Table 5 percentage of lymphocytes in each group (%, n ═ 6)
4. Histopathological observation
Histology shows that a small amount of red blood cells are scattered in normal lung tissues of a mouse, a small amount of granulocytes are occasionally seen in the mouse, when the time is 3-9 days, a positive lung tissue has a large amount of bleeding, exfoliated epithelial cells, inflammatory cell infiltration and fibrin secretion, when the time is 3-9 days, the phenomena of bleeding, inflammatory exudate, inflammatory cell infiltration and the like of the lung of a western medicine control group, a low-dose baicalin group and a medium-dose baicalin group are reduced to different degrees compared with those of the positive lung tissue, and no obvious damage is found in histological observation of trachea and spleen.
4.1 replication of the Virus in Lung tissue
As shown in FIG. 8, the virus content in lung tissue of the high-dose baicalin-treated group was significantly lower than that of the virus-positive group (P < 0.05) at 3d and 6d, and the difference from the western medicine control group was not significant (P > 0.05). When the treatment lasts for 9 days, the virus content of lung tissues of the medium-dose baicalin group and the high-dose baicalin group is obviously lower than that of the virus positive group (P is less than 0.05), and the difference with a western medicine control group is not obvious (P is more than 0.05) (Table 6).
TABLE 6 Lung tissue disease in groups of miceToxic content (μ L)-1，n＝6)
4.2 replication of the Virus in tracheal tissue
As shown in fig. 9, the virus content in tracheal tissue was lower than that in the virus positive control group in each of the treatment groups at 3d, 6d, and 9d, but the difference was not significant (P > 0.05) (table 7).
TABLE 7 virus content (μ L) in tracheal tissue of mice in each group-1，n＝6)
4.3 replication of the Virus in spleen tissue
As shown in FIG. 10, the virus content in spleen tissues of each treatment group was lower than that of the virus positive control group in the treatment of 3d, 6d and 9d, but the difference was not significant (P > 0.05) (Table 8).
TABLE 8 spleen tissue Virus content (. mu.L) of mice in each group-1，n＝6)
4.4 Lung mast cell degranulation Rate
As shown in fig. 11 and 12, the virus-positive group mast cell MC degranulation rate was significantly higher than that of the negative control group (P < 0.01) when 3d, 6d and 9d were treated; when the medicine is used for treating 6 days and 9 days, the MC degranulation rate of the medium-dose and high-dose baicalin groups is remarkably lower than that of a virus positive group (P is less than 0.05), and the difference with a western medicine control group is not remarkable (P is more than 0.05) (Table 9).
TABLE 9 Lung mast cell degranulation (%, n ═ 6)
5. Inflammatory cell outcome
5.1 IL-1 results
As shown in FIG. 13, the serum IL-1 of the mice in the virus positive group was significantly higher than that in the negative control group (P < 0.01) when 3d, 6d and 9d were treated. When the mice are treated for 3 days, the IL-1 content in the serum of the mice in the low-dose baicalin group, the medium-dose baicalin group and the high-dose baicalin group is not obviously different from that in the virus positive group (P is more than 0.05). When the treatment is carried out for 6 days and 9 days, the IL-1 content in the serum of the mice in the western medicine control group, the low-dose baicalin group, the medium-dose baicalin group and the high-dose baicalin group is remarkably lower than that in the virus positive group (P is less than 0.01) (Table 10).
TABLE 10 serum IL-1 content (pg. mL) in each group of mice-1，n＝6)
5.2 IL-2 results
As shown in fig. 14, the content of IL-2 in the virus positive group, western medicine control group, low dose baicalin group, medium dose baicalin group, and high dose baicalin group was very significant or significantly higher than that in the negative control group (P < 0.01 or P < 0.05) when 3d, 6d, and 9d were treated, and the difference between the low, medium, and high dose baicalin group and the western medicine control group was not significant (P > 0.05) (table 11).
TABLE 11 serum IL-2 content (pg. mL) in each group of mice-1，n＝6)
5.3 TNF-. alpha.results
As shown in fig. 15, when 3d, 6d and 9d were treated, the TNF- α content in the western medicine control group, low, medium and high dose baicalin group was significantly lower than that in the virus positive group (P < 0.01), and the difference between the high dose baicalin group and the western medicine control group was not significant (P > 0.05) (table 12).
TABLE 12 serum TNF- α content (pg. mL) in each group of mice-1，n＝6)
5.4 IFN-. gamma.results
As shown in fig. 16, when 3d, 6d, and 9d were treated, the levels of INF- γ in the sera of the virus-positive mice were significantly higher than those of the negative control group (P < 0.01), significantly lower than those of the western medicine control group, the medium-dose baicalin group, and the high-dose baicalin group (P < 0.01), and significantly different from those of the low-dose baicalin group (P > 0.05) (table 13).
TABLE 13 IFN-. gamma.content (pg.mL) in serum of mice of each group-1，n＝6)
Results of 5.55-HT
As shown in fig. 17, in 3d treatment, the 5-HT content in the high-dose baicalin group was significantly lower than that in the virus-positive group (P < 0.01), the difference from the western medicine control group was not significant (P > 0.05), and the medium-dose baicalin group was significantly lower than that in the virus-positive group (P < 0.05); when 6d and 9d are treated, the 5-HT content of the low, medium and high dose baicalin groups is extremely lower than that of the virus positive group (P is less than 0.01). The difference between the medium and high dose baicalin groups and the western medicine control group is not significant (P is more than 0.05) (Table 14).
TABLE 14Serum 5-HT content (ng. mL) of each group of mice-1，n＝6)
6. Conclusion
From the above experimental data and figures, it can be seen that: baicalin inhibits the increase of the total number of leukocytes in a mouse by inhibiting the production of inflammatory factors. Baicalin can inhibit the invasion of influenza cells into the body by inhibiting the activity of hemagglutinin HA2 subunit and reducing the replication of virus genetic material in cells, which is also the reason for the reduction of the number of neutrophils.
The lung of the mouse with the high-dose baicalin group can remarkably reduce the lung bleeding, epithelial cell shedding and inflammatory cell infiltration compared with the lung of the mouse with the virus positive group. In particular, the mast cell degranulation rate of the medium-dose baicalin group and the high-dose baicalin group are both significantly lower than that of the virus positive group, which indicates that the baicalin can effectively inhibit MC degranulation in lung tissues of the affected mice.
The baicalin can effectively inhibit the increase of IL-1, TNF-alpha, IFN-gamma and 5-HT of a patient mouse, and when 6d and 9d are treated, the content of 5-HT in a low-dose baicalin group, a medium-dose baicalin group and a high-dose baicalin group is extremely lower than that in a virus positive group, and the content of 5-HT is indirectly inhibited by inhibiting MC degranulation, and meanwhile, the secretion of IL-1, TNF-alpha and IFN-gamma is also inhibited.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Claims (8)
1. Use of baicalin in the preparation of a medicament for treating H6N6 subtype avian influenza, wherein the baicalin reduces the content of avian influenza H6N6 virus by inhibiting the production of neutrophils, reducing the degranulation rate of mast cells in lung tissues and inhibiting the secretion of inflammatory factors, wherein the inflammatory factors are IL-1, IL-2, TNF-alpha, IFN-gamma and 5-HT.
2. The use of claim 1, wherein baicalin inhibits the activity of hemagglutinin HA2 subunit and inhibits the replication of avian influenza H6N6 virus genetic material in cells.
3. The use of claim 2, wherein said baicalin inhibits neutrophil production by reducing the genetic material of avian influenza virus subtype H6N 6.
4. The use according to claim 1, wherein said reduction of mast cell degranulation inhibits the secretion of inflammatory factors.
5. Application of baicalin in preparing inhibitor for inhibiting secretion of neutrophil in vivo infected with H6N6 subtype avian influenza is provided.
6. Application of baicalin in preparing inhibitor for inhibiting mast cell degranulation rate in lung tissue infected with H6N6 subtype avian influenza is provided.
7. Application of baicalin in preparing inhibitor for inhibiting secretion of inflammatory factor 5-HT in vivo infected with H6N6 subtype avian influenza is provided.
8. The use according to claim 7, wherein said inhibition of the secretion of inflammatory factor 5-HT in vivo in avian influenza infected with subtype H6N6 is achieved by inhibiting the mast cell degranulation rate in lung tissue.
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| 李卫鹏，等: "黄芩苷对H6N6 禽流感患鼠血液指标的影响", 《中国兽药杂》 * |
| 李卫鹏: "黄芩苷对小鼠感染H6N6禽流感的治疗效果", 《贵州大学硕士学位论文》 * |
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