CN115671095A - Application of mycophenolic acid in inhibiting echinococcosis hepatica - Google Patents
Application of mycophenolic acid in inhibiting echinococcosis hepatica Download PDFInfo
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- CN115671095A CN115671095A CN202110824550.5A CN202110824550A CN115671095A CN 115671095 A CN115671095 A CN 115671095A CN 202110824550 A CN202110824550 A CN 202110824550A CN 115671095 A CN115671095 A CN 115671095A
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Abstract
The invention provides an application of mycophenolic acid in inhibiting echinococcosis hepatica, wherein the mycophenolic acid can inhibit the action of inosine 5' -monophosphate dehydrogenase in echinococcus granulosus and/or echinococcus multilocularis, block the synthesis of purine nucleotides, and cause the echinococcus granulosus and/or echinococcus multilocularis to undergo apoptosis.
Description
Technical Field
The invention relates to the field of anti-echinococcosis drugs, and in particular relates to application of mycophenolic acid in inhibiting echinococcosis hepatica.
Background
Echinococcosis, also known as echinococcosis, is a parasitic disease of zoonosis caused by the parasitosis of echinococcus larvae in human or animal bodies. The advanced areas of livestock husbandry such as Tibet, xinjiang, ningxia, gansu, inner Mongolia and Qinghai in China are echinococcosis epidemic areas, and the threatened population is nearly 6600 ten thousand. Infection by echinococcosis can cause damage to organs and tissues such as liver, lung, brain and bones, and the patient loses work ability. Meanwhile, the echinococcosis also has the characteristics of infectivity, latency and difficult detection of early infection, and if the echinococcosis is incompletely killed, the echinococcosis can bring a heavy burden of 'poverty caused by diseases' to the people in the epidemic area.
Echinococcosis in china mainly includes Cystic Echinococcosis (CE) caused by the parasitism of the larval Echinococcosis (Eg) in human tissues and organs, and Alveolar Echinococcosis (AE) caused by the parasitism of the larval Echinococcosis in the ovate Echinococcosis (Em). If the patient with alveolar echinococcosis is not treated, the 10-year mortality rate can reach 94 percent, which is also called as 'insect cancer'.
The inventor provides a method for screening the gene segment of the hydatid hepatica, an amplification primer and a kit in a patent ZL 202010854980.7, the screening method eliminates the influence of a human genome and a genetic relationship tapeworm genome, fundamentally avoids false positive caused by the human gene or the genetic relationship tapeworm gene existing in the DNA of a tissue to be detected, the primer designed aiming at the screened gene segment has high accuracy and specificity, and the misdiagnosis probability of false positive or false negative results is remarkably reduced.
However, the prior art lacks a drug capable of effectively inhibiting echinococcosis hepatica. At present, the main medicine for clinically treating echinococcosis is albendazole. Zhang Imamine et al, in the analysis of the 38 cases of adverse reactions in children caused by albendazole, indicate that albendazole needs to be taken for a long time and has large adverse reactions, and do not suggest that albendazole be used for treating echinococcosis. Zhengrongyuan et al also pointed out the same problem in the report on reevaluation of safety after albendazole was marketed.
Therefore, there is a need to find other suitable drugs capable of inhibiting the growth of hydatid as drugs capable of effectively inhibiting echinococcosis.
Disclosure of Invention
The invention aims to provide application of mycophenolic acid in inhibiting echinococcosis hepatica, alkaline phosphatase tests verify that mycophenolic acid can effectively kill echinococcus granulosus, trypan blue staining is utilized to evaluate the killing effect of mycophenolic acid on echinococcus granulosus prototheca, and the mycophenolic acid can effectively kill echinococcus granulosus prototheca and echinococcus polyporum in vitro, so that the mycophenolic acid can be used as a medicament for inhibiting echinococcosis hepatica possibly, and the problems that albendazole needs to be taken for a long time, has large adverse reaction and low cure rate in the prior art are solved.
Specifically, the purpose is realized by the following technical scheme:
application of mycophenolic acid in inhibiting growth of liver infusorian.
Mycophenolic acid, also known as mycophenolic acid, [ E-4-methyl-6- (1, 3-dihydro-7-methyl-4-hydroxy-6-methoxy-3-oxo-5-isobenzofuranyl) -4-hexenoic acid ] having the formula: c 17 H 20 O 6 It is a milky white powder. At present, the application research of mycophenolic acid mainly surrounds the derivative Mycophenolate Mofetil (MMF), and the MMF is widely applied to preventing and treating acute rejection of transplanted organs at home and abroad as a main immunosuppressant. However, the use of mycophenolic acid in inhibiting echinococcosis hepatica is still blank.
In the continuous research, the inventor screens the existing mature drugs to obtain the drug capable of inhibiting the growth of the echinococcosis hepatica, and further obtains the potential drug capable of inhibiting the echinococcosis hepatica or the main component of the echinococcosis resistant drug.
Experiments show that the mycophenolic acid has the effect of killing echinococcus granulosus and echinococcus multocida in vitro.
Specifically, it was found through the alkaline phosphatase test that after mycophenolic acid had been exposed to the alkaline phosphatase for 36 hours and 120 hours, the concentration of alkaline phosphatase in the culture supernatant was significantly increased to a greater extent than that of the blank control group and the albendazole experimental group used in the prior art, which indicated that more echinococcus multilocularis in the mycophenolic acid group was killed, resulting in more alkaline phosphatase in the cell wall being released into the culture supernatant.
The killing effect of mycophenolic acid on echinococcus granulosus protocoid is analyzed by trypan blue staining, and the survival rate of the echinococcus granulosus after 24 hours of mycophenolic acid treatment is about 60%, compared with the survival rate of nearly 80% in an albendazole experimental group, the killing efficiency is further improved, and the survival rate of the echinococcus granulosus is basically 0% by the sixth day. Thus, mycophenolic acid can effectively kill echinococcus granulosus and echinococcus multocida in vitro, and can be used as a medicine or a medicine component for mainly or auxiliarily killing the hydatid.
Further, mycophenolic acid kills echinococcus granulosus and echinococcus multilocularis in a different way than albendazole. By analysis, it is possible that mycophenolic acid blocks the synthesis of purine nucleotides by inhibiting the action of inosine 5' -monophosphate dehydrogenase in echinococcus granulosus or echinococcus multilocularis, resulting in apoptosis of echinococcus granulosus or echinococcus multilocularis. Based on this principle, mycophenolic acid exhibits a stronger killing power in vitro compared to albendazole.
The invention also provides a medicament for inhibiting the echinococcosis hepatica, and the formula of the medicament contains mycophenolic acid. Mycophenolic acid is used as the main component of the medicine and has the function of inhibiting the growth of the liver infusorian.
Further, the medicine also comprises pharmaceutically acceptable auxiliary materials. Preferably, the excipient may be starch or powdered sugar.
Further, the medicament blocks the synthesis of purine nucleotides by inhibiting the action of inosine 5' -monophosphate dehydrogenase in echinococcus granulosus and/or echinococcus multilocularis, resulting in apoptosis of echinococcus granulosus and/or echinococcus multilocularis.
The invention also provides a medicament for inhibiting the growth of the liver infusorian, and the formula of the medicament contains mycophenolic acid.
Further, the medicine also comprises pharmaceutically acceptable auxiliary materials. Preferably, the adjuvant may be starch or powdered sugar.
Further, the medicament blocks the synthesis of purine nucleotides by inhibiting the action of inosine 5' -monophosphate dehydrogenase in echinococcus granulosus and/or echinococcus multilocularis, resulting in apoptosis of echinococcus granulosus and/or echinococcus multilocularis.
In conclusion, the invention has the following advantages and beneficial effects: mycophenolic acid can inhibit the action of inosine 5' -monophosphate dehydrogenase in echinococcus granulosus and/or echinococcus multilocularis, block the synthesis of purine nucleotides, and cause the echinococcus granulosus and/or echinococcus multilocularis to generate apoptosis, and alkaline phosphatase test and trypan blue staining test verify that mycophenolic acid can effectively kill the echinococcus granulosus and echinococcus multilocularis in vitro, so the mycophenolic acid can be used as a drug or a drug component for mainly or auxiliarily killing echinococcus hepatica, and a reference is provided for developing anti-echinococcus drug replacing albendazole.
Drawings
The accompanying drawings, which are included to provide a further understanding of the embodiments of the invention and are incorporated in and constitute a part of this application, illustrate embodiment(s) of the invention and together with the description serve to explain the principles of the invention. In the drawings:
FIG. 1 is a graph showing the effect of alkaline phosphatase assay on Echinococcus multilocularis in the Mycophenolic acid group (Mycophenoic acid), albendazole group (Albendazole) and control group (DMSO, 0.2% dimethylsulfoxide) in the control group in an embodiment of the present invention, where the abscissa is treatment time and the ordinate is absorbance A 405 A value;
FIG. 2 shows Trypan blue staining analysis of the killing effect on Echinococcus granulosus prototheca in the mycophenolic acid group, albendazole group and control group according to an embodiment of the present invention, wherein the abscissa is the treatment time and the ordinate is the survival rate of the prototheca.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail below with reference to examples and accompanying drawings, and the exemplary embodiments and descriptions thereof are only used for explaining the present invention and are not meant to limit the present invention.
The technology related to the invention is a conventional technical means of molecular cloning, wherein related enzymes, primers, reagents and reaction conditions can be reasonably selected according to the experience of a person skilled in the art without description, related reagent consumables belong to common products sold in the market, and related detection means and instruments are well known and skilled in the art.
In the present invention, the data was analyzed using Prism version 9 (GraphPad Software) statistical Software.
[ example 1 ]
The killing effect of the medicine on the echinococcus multilocularis is detected by an alkaline phosphatase experiment.
The alkaline phosphatase test can detect the content of the alkaline phosphatase released into the supernatant of the culture solution after the cyst wall of the echinococcus multilocularis is damaged, thereby evaluating the effect of the medicine on resisting the echinococcus multilocularis.
(1) Experimental Material
The echinococcus multicavis is obtained from abdominal cavity of Kandelia parviflora infected with echinococcus multicavis, the feeder cells are human liver cancer cell strain QGY-7701, and the culture medium is DMEM culture medium.
(2) Experimental method
Washing Echinococcus multilocularis twice with Hanks solution, placing Echinococcus multilocularis into culture flask coated with human liver cancer cell QGY-7701, adding DMEM culture medium, and 5% CO 2 After 2 months of culture at 37 ℃, vesicles with the diameter of 1-5 mm are collected. Washed with Hanks solution, resuspended in DMEM medium, and added to 24-well culture plates, each well containing 2ml of 25-35 vesicles.
The experimental groups included the mycophenolic acid group, the albendazole group and the 0.2% DMSO control group. Each drug was provided with 2 parallel wells at a final concentration of 40. Mu. Mol/L, repeated 2 times. 5% of CO 2 After incubation at 37 ℃ for 36 hours and 120 hours, 170. Mu.L of alkaline phosphatase reaction solution was added to each well of a 96-well plate, 30. Mu.L of the supernatant was added thereto and mixed, and the mixture was incubated at 37 ℃ for 30 minutes and then the absorbance A was measured 405 The value is obtained.
(3) Results of the experiment
Table 1 shows A of alkaline phosphatase in culture supernatants of three experiments performed on the mycophenolic acid group, the albendazole group and the control group after 36 hours and 120 hours of treatment 405 The value is obtained.
Table 1:
as shown in FIG. 1, after the treatment for 36 hours, A of alkaline phosphatase was found in the culture supernatant of the mycophenolate group 405 The value is obviously higher than that of the albendazole group and the control group, and reaches 0.334 +/-0.033, while the values of the albendazole group and the control group are respectively 0.246 +/-0.019 and 0.115 +/-0.009. Alkaline phosphatase A in the supernatant of the mycophenolic acid group after 120 hours of action 405 The value reaches 0.566 +/-0.043, which is obviously higher than the albendazole group and the control group and is respectively 0.424 +/-0.038 and 0.116 +/-0.010. It can be seen that mycophenolic acid kills echinococcosis poly-chamber more strongly than albendazole after 36 hours and 120 hours of treatment.
[ example 2 ]
Trypan blue staining was used to analyze the killing effect of the drug on echinococcus granulosus protocoid.
Trypan blue can be used to stain and count surviving protoheads, and then analyze the viability of protoheads over the incubation period. The initial survival rate of the procephalic segment is greater than 90%.
(1) Experimental Material
Echinococcus granulosus is extracted from cyst fluid of naturally infected echinococcus granulosus-infected sheep liver vesicles in RPMI 1640 medium containing 25mmol/L Hepes, 2mmol/L L-glutamic acid, 100U/mL penicillin, and 100. Mu.g/mL streptomycin.
(2) Experimental methods
Standing the cyst fluid to naturally settle the echinococcus granulosus protocephalus, washing with Hanks fluid for 3 times, then re-suspending, taking 200 mu L of re-suspension, staining with 0.4% trypan blue and counting the live protocephalus.
Procephalic nodules were cultured in RPMI 1640, and 1mL of culture suspension was added to each well of a 24-well plate, and the number of procephalic nodules was about 100.
The experimental groups included the mycophenolic acid group, the albendazole group and the 0.2% DMSO control group. Each drug was provided with 2 parallel wells at a final concentration of 40. Mu. Mol/L, repeated 2 times. 5% of CO 2 Incubating at 37 deg.C for 1 day, 2 days, 3 days, 4 daysAfter 5, 6 and 7 days, the survival of protocephalic segments was calculated by staining with 0.4% trypan blue and calculating live protocephalic segments.
(3) Results of the experiment
Table 2 shows the survival rates of echinococcus granulosus in the mycophenolic acid group, albendazole group and control group after 1 to 7 days of treatment, which were performed in three experiments, respectively.
TABLE 2
As shown in FIG. 2, the Echinococcus granulosus survival rate was 58.33. + -. 4.922% after 1 day of mycophenolic acid treatment, and almost all of the primary knots in the mycophenolic acid group died after 6 days. The survival rate of the protocephalad of the albendazole group is gradually reduced within one week, and 6.667 +/-2.517% of the protocephalad still survives by the 7 th day. It can be seen that mycophenolic acid kills echinococcus granulosus more efficiently than albendazole.
In conclusion, the in-vitro killing effect of mycophenolic acid on echinococcus multilocularis and echinococcus granulosus is stronger than that of albendazole adopted in the prior art, and meanwhile, mycophenolic acid is already used for a human body as a mature drug, so that the mycophenolic acid is expected to be used as a substitute for albendazole for a drug or a drug component for mainly or auxiliarily killing echinococcus hepatica.
The above-mentioned embodiments are intended to illustrate the objects, technical solutions and advantages of the present invention in further detail, and it should be understood that the above-mentioned embodiments are merely exemplary embodiments of the present invention, and are not intended to limit the scope of the present invention, and any modifications, equivalent substitutions, improvements and the like made within the spirit and principle of the present invention should be included in the scope of the present invention.
Claims (10)
1. Application of mycophenolic acid in inhibiting growth of liver infusorian is provided.
2. Application of mycophenolic acid in preparing medicine for inhibiting liver echinococcosis is provided.
3. Use according to claim 1 or 2, wherein said use is the killing of echinococcus granulosus and/or echinococcus multocida.
4. The use according to claim 3, wherein said use is for blocking the synthesis of purine nucleotides by inhibiting the action of inosine 5' -monophosphate dehydrogenase in echinococcus granulosus and/or echinococcus multilocularis, resulting in apoptosis of echinococcus granulosus and/or echinococcus multilocularis.
5. The medicine for inhibiting the echinococcosis hepatica is characterized in that mycophenolic acid is contained in a formula of the medicine.
6. The drug for inhibiting echinococcosis hepatica according to claim 5, further comprising a pharmaceutically acceptable excipient.
7. The drug for inhibiting echinococcosis hepatica according to claim 5, wherein the drug blocks the synthesis of purine nucleotides by inhibiting the action of inosine 5' -monophosphate dehydrogenase in echinococcus granulosus and/or echinococcus multilocularis, resulting in apoptosis of echinococcus granulosus and/or echinococcus multilocularis.
8. The medicine for inhibiting the growth of the liver infusorian is characterized in that mycophenolic acid is contained in the formula of the medicine.
9. The medicament for inhibiting the growth of hydatid according to claim 8, further comprising a pharmaceutically acceptable excipient.
10. The drug for inhibiting the growth of echinococcus hepatica according to claim 8, wherein the drug blocks the synthesis of purine nucleotides by inhibiting the action of inosine 5' -monophosphate dehydrogenase in echinococcus granulosus and/or echinococcus multilocularis, resulting in apoptosis of echinococcus granulosus and/or echinococcus multilocularis.
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| CN111973600A (en) * | 2020-08-20 | 2020-11-24 | 华中农业大学 | Application of mycophenolic acid or derivative thereof in preparation of canine distemper virus inhibitor |
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| CN111973600A (en) * | 2020-08-20 | 2020-11-24 | 华中农业大学 | Application of mycophenolic acid or derivative thereof in preparation of canine distemper virus inhibitor |
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| Title |
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| 王帅: "牛带绦虫和亚洲带绦虫全基因组测序、比较基因组及胰岛素信号通路研究", 《中国博士学位论文全文数据库 农业科技辑》, no. 1, pages 050 - 82 * |
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