New use of corydalis edulis in preventing and treating human cytomegalovirus infection
阅读说明：本技术 紫堇灵在防治人巨细胞病毒感染中的新用途 (New use of corydalis edulis in preventing and treating human cytomegalovirus infection ) 是由 毛根祥 王三应 徐小刚 万晓青 苏慧丽 张婧 孙川 代继桓 邢文敏 于 2020-12-10 设计创作，主要内容包括：本发明属生物技术领域,本发明提供了紫堇灵在制备防治人巨细胞病毒感染的药物中的新用途。实验表明,紫堇灵具有明显的抗HCMV的作用。紫堇灵(1-20μM)单独处理人巨细胞病毒HCMV的宿主细胞-人胚肺二倍体成纤维细胞WI-38没有表现明显的细胞毒性,紫堇灵可以显著改善HCMV感染形成的细胞病变效应,抑制HCMV特征性立即早期蛋白IE1/2和早期蛋白UL44的表达,有效抑制HCMV的DNA复制,具有制备抗HCMV药物的应用前景。(The invention belongs to the technical field of biology, and provides a new application of corydalis edulis in preparing a medicament for preventing and treating human cytomegalovirus infection. Experiments show that the corynolen has obvious HCMV resistance effect. The host cell of the human cytomegalovirus HCMV, namely the human embryonic lung diploid fibroblast WI-38, treated by the corynollin (1-20 mu M) alone has no obvious cytotoxicity, the corynollin can obviously improve the cytopathic effect formed by HCMV infection, inhibit the expression of HCMV characteristic immediate early protein IE1/2 and early protein UL44, effectively inhibit the DNA replication of the HCMV, and has application prospect in preparing anti-HCMV medicaments.)
1. Application of corydalis edulis in preparing medicament for preventing and treating human cytomegalovirus infection.
2. Use according to claim 1, characterized in that corynoline is used for preventing and treating human cytomegalovirus infection by inhibiting the expression of HCMV immediate early protein IE1/2 and early protein UL 44.
3. The use as claimed in claim 1, wherein the form of corydalis edulis is comprised of a salt form thereof in combination with a pharmaceutically acceptable carrier.
4. The use of claim 1, wherein the medicament for preventing and treating human cytomegalovirus infection is in the form of liquid, granule, tablet, granule, capsule, sustained release agent, drop pill or injection.
Technical Field
The invention belongs to the field of biological medicines, and particularly relates to a new application of corydalis edulis in preventing and treating human cytomegalovirus infection.
Background
Human Cytomegalovirus (HCMV) belongs to the sub-family of herpes virus beta, is generally susceptible to people, and carries virus for life after infection, after infection is relieved, the virus remains in the body for a long time to form latent infection, and in patients with low body immunity or organ transplantation, secondary infection is easy to occur, so that injuries of a nervous system, a liver, a respiratory system, a blood system and the like are easily caused, and even the life is threatened in serious cases; meanwhile, in pregnant women, secondary infection of HCMV is a main pathogen causing congenital diseases of newborn, and causes multiple organ and multiple system diseases such as icterohepatitis, mental retardation, blindness, deafness and the like. Therefore, the positive and effective treatment of the cytomegalovirus infection of the human is an important means for improving the birth population quality and improving the life quality of the old with low immunity. At present, no approved vaccine is available on the market, clinically used symptomatic medicines are mainly antiviral medicines such as foscarnet, cidofovir, fomivirsen and the like, but the medicines have obvious side effects of bone marrow inhibition, nephrotoxicity, electrolyte disorder and the like, and can also generate drug resistance, so that the clinical application of the medicines is limited.
The corynolamine is an alkaloid derived from corynolamine buchneri of papaveraceae, and the application of the corynolamine in preparing a medicament for preventing and/or treating HCMV is not provided so far.
Disclosure of Invention
The invention adopts commercial corydaline to carry out a series of biological experiments, and provides a theoretical basis for the new application of the corydaline in preventing and treating human cytomegalovirus infection.
The invention provides a new application of corydalis edulis in preparing a medicament for preventing and treating human cytomegalovirus infection.
The corynoline can inhibit the expression of HCMV immediate early protein IE1/2 and early protein UL44, thereby playing a role in resisting HCMV infection.
The corynoline form comprises a salt form and a pharmaceutically acceptable carrier combination form.
The preparation form of the medicament for preventing and treating human cytomegalovirus infection is liquid preparation, granule, tablet, electuary, capsule, sustained-release agent, dripping pill or injection.
The results of the biological experimental study show that:
1. the corydalis yanhusuo alone treated HCMV host cells-human embryonic lung diploid fibroblasts WI-38 show no obvious cytotoxicity at 0-20 mu M;
2. the corynoline has good anti-HCMV effect, and particularly shows that the expression of immediate early protein IE1/2 and early protein UL44 is obviously inhibited; while inhibiting nucleic acid replication of HCMV.
The invention has the advantages and beneficial effects that a series of biological experiments find and prove that the corynolamine has obvious anti-HCMV effect under the condition of no obvious cytotoxicity dose, is safe and effective, and is expected to be developed into a drug for preventing and treating HCMV.
Drawings
FIG. 1: the MTT method measures the influence of the corynoline on the WI-38 cell viability.
Wherein: ordinate-cell viability (setting control group to 1)
The abscissa: 1-Control group (Control)
2-Violin (1. mu.M) alone for 2 days
3-Violin (2. mu.M) alone for 2 days
4-Violin (5. mu.M) alone for 2 days
5-Violin (10. mu.M) alone for 2 days
6-Violin (20. mu.M) alone for 2 days
FIG. 2: western-blot method for detecting the influence of corydalis on HCMV immediate early protein IE1/2 and early protein UL44 (GAPDH is an internal reference)
Wherein in FIG. 2A: 1-HCMV (MOI 0.01) was inoculated for 1 day
2-Violin (10. mu.M) + HCMV (MOI 0.01) for 1 day
3-DMSO + HCMV (MOI 0.01) was inoculated for 1 day
4-Phosphosodium formate (200. mu.g/ml) + HCMV (MOI 0.01) was inoculated for 1 day
5-HCMV (MOI 0.01) was inoculated for 4 days
6-Violin (10. mu.M) + HCMV (MOI 0.01) for 4 days
7-DMSO + HCMV (MOI 0.01) were inoculated for 4 days
8-Phosphosodium formate (200. mu.g/ml) + HCMV (MOI 0.01) was inoculated for 4 days
9-HCMV (MOI 0.01) were inoculated for 7 days
10-Violin (10. mu.M) + HCMV (MOI 0.01) for 7 days
11-DMSO + HCMV (MOI 0.01) were inoculated for 7 days
12-Phosphosodium formate (200. mu.g/ml) + HCMV (MOI 0.01) was inoculated for 7 days
Wherein FIG. 2B: 1-HCMV (MOI 0.01)
2-DMSO+HCMV(MOI 0.01)
3-Violalin (10. mu.M) + HCMV (MOI 0.01)
4-Phosphosodium formate (200. mu.g/ml) + HCMV (MOI 0.01)
P <0.5, P <0.05, P <0.001, compared to control group
FIG. 3: IFA method for detecting influence of corydaline on expression of HCMV immediate early protein IE1/2
Wherein: 1-DMSO + HCMV (MOI 0.01) was inoculated for 7 days
2-Violin (1. mu.M) + HCMV (MOI 0.01) for 7 days
3-Violin (5. mu.M) + HCMV (MOI 0.01) for 7 days
4-Violin (10. mu.M) + HCMV (MOI 0.01) for 7 days
5-Violin (20. mu.M) + HCMV (MOI 0.01) for 7 days
6-Phosphosodium formate (200. mu.g/ml) + HCMV (MOI 0.01) was inoculated for 7 days
FIG. 4: qPCR method for detecting influence of corynolen on HCMV DNA amplification
Wherein: 1-HCMV (MOI 0.01) was inoculated for 1 day
2-DMSO + HCMV (MOI 0.01) was inoculated for 1 day
3-Violin (10. mu.M) + HCMV (MOI 0.01) for 1 day
4-Phosphosodium formate (200. mu.g/ml) + HCMV (MOI 0.01) was inoculated for 1 day
5-HCMV (MOI 0.01) was inoculated for 4 days
6-DMSO + HCMV (MOI 0.01) were inoculated for 4 days
7-Violin (10. mu.M) + HCMV (MOI 0.01) for 4 days
8-Phosphosodium formate (200. mu.g/ml) + HCMV (MOI 0.01) was inoculated for 4 days
9-HCMV (MOI 0.01) were inoculated for 7 days
10-DMSO + HCMV (MOI 0.01) were inoculated for 7 days
11-Violin (10. mu.M) + HCMV (MOI 0.01) for 7 days
12-Phosphosodium formate (200. mu.g/ml) + HCMV (MOI 0.01) was inoculated for 7 days
P <0.5, P <0.001, compared to HCMV group
Detailed Description
The human embryonic lung diploid fibroblast strain WI-38 and the Human Cytomegalovirus (HCMV) Towne strain related to the present invention are both from American Type Culture Collection (ATCC).
Example 1 Effect of treatment with Violin alone on WI-38 cell proliferation Activity
Cell viability was measured by the MTT method as follows: the human embryonic lung diploid fibroblast WI-38 of PD30 was seeded in 96-well cell culture plates at a cell density of 3000 cells per well, and after 24h of culture, different concentrations of violaxyl were added, 3 parallel wells were provided for each concentration, and a drug-free blank control and a cell-free solvent control were provided. After 48 hours in the incubator, 20. mu.l of MTT (5mg/ml, prepared in serum-free DMEM medium) was added to each well at 37 ℃ with 5% CO2Culturing for 4h, completely sucking the liquid in the cell culture well, adding 150 μ l DMSO into each well, shaking gently on a shaking table for 10min to dissolve the crystal completely, measuring the light absorbance at 570nm, and calculating relative cell activity, wherein the cell activity of the blank control group without drug is 100%. The results of the experiment are shown in FIG. 1.
Example 2 Violin treatment and inoculation of HCMV
Human embryonic lung fibroblast WI-38 cells using PD30 in 2X 10 medium containing 10% FBS4/cm2The cell amount of (2) is plated on a six-well cell culture plate, after 24 hours, the culture medium of 0.2% FBS is replaced to continue culturing for 48 hours, and the cell cycle is synchronized with G0/G1 by a serum starvation method, so that HCMV infection is facilitated. Thereafter, inoculation of HCMV virus (Towne strain) was performed at an inoculation dose of 0.01MOI (multiplicity of infection), and the culture was continued for a prescribed period of time for relevant detection. When the anti-HCMV activity of the corydaline is detected, the corydaline with a certain concentration is added into a culture medium 2 hours ahead of time, then the HCMV (MOI 0.01) is inoculated, and further the changes of viral protein expression, DNA amplification and the like are observed.
Example 3Western-blot detection of the Effect of Violin on HCMV Virus protein expression
To confirm the anti-HCMV effect of corynolamine, we examined the effect of corynolamine on the expression of HCMV immediate early protein IE1/2 and early protein UL44, cell culture and corynolamine treatment and HCMV vaccination as described in example 2 above. Setting a control group which is not subjected to any drug treatment and only inoculated with HCMV; solvent DMSO treatment, HCMV inoculated control group, HCMV inoculated drug control group treated with HCMV and clinically sodium foscarnet (200. mu.g/ml), and corydalis edulis (10. mu.M) treated and HCMV inoculated experimental groups at different times. 1, 4 and 7 days after HCMV inoculation, supernatant is discarded, cells are washed with PBS three times, cell samples are collected after the cells are lysed by intermediate-strength RIPA lysate, protein concentration is measured by a BCA kit, and Western-blot detection is carried out by adopting corresponding IE1/2 and UL44 antibodies, and the result shows that the violaxalin can obviously inhibit the expression of HCMV virus proteins IE1/2 and UL44 (fig. 2A and 2B).
Example 4 Indirect immunofluorescence assay (IFA) to determine the Effect of Violin on HCMV viral protein expression
As described in example 2 above, 7 days after drug treatment and HCMV inoculation, the supernatant was discarded, the cells were washed three times with PBS, fixed with pre-cooled methanol acetone (1:1) at-20 ℃ for 20 minutes, then the fixing solution was discarded, and left to air-dry in a fume hood. The conventional IFA procedure with an antibody against IE1/2 protein, followed by photographing under an inverted fluorescence microscope, showed that corynoline was able to significantly reduce the expression of HCMV immediate early protein IE1/2 and was dose-dependent (fig. 3).
Example 5qPCR determination of the Effect of Violin on HCMV DNA copy number
Cell culture, drug treatment and HCMV inoculation treatment were as described in example 2 above. The copy number of HCMV DNA was determined by qPCR method, 10ng of total DNA was taken according to the iQ SYBR Green Supermix kit (Bio-Rad) kit instructions, and the primers used were: upstream 5-TCTGCCAGGACATCTTTCTC-3 ' (SEQ ID NO.1) and downstream 5'-GTGACCAAGGCCACGACGTT-3' (SEQ ID NO. 2). Amplification conditions: at 95 ℃ for 5min, (95 ℃ for 5sec, 60 ℃ for 30 sec). times.40 cycles, run throughThe results of the calculation show that the corynoline can obviously inhibit the copy number of the virus DNA (figure 4).
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 and improvements made within the spirit and principle of the present invention are included in the scope of the present invention.