Application of calcium ions in inhibition of survival and proliferation of echinococcus multilocularis protoscolex

Abstract

The invention discloses application of calcium ions in inhibition of survival and proliferation of echinococcus multilocularis protoscolex, and belongs to the technical field of biological medicines. The invention provides application of calcium ions in inhibition of survival and proliferation of protoscolex multilocularis of echinococcosis multilocularis. The invention also discloses a medicine for inhibiting the survival and proliferation of the protoscolex of the echinococcus multilocularis, and the medicine comprises the calcium ion. The specific action and mechanism of calcium ions in the survival and proliferation of the echinococcus multilocularis protoscolex are researched, the mechanism for inhibiting the survival and proliferation of the echinococcus multilocularis protoscolex by regulating and controlling the calcium ion pathway or oxidative stress of the organism is clarified, and a new theoretical basis and an intervention strategy are provided for prevention and treatment of parasitic diseases.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and specifically relates to the application of calcium ions in inhibiting the survival and proliferation of protoscolex of Echinococcus multilocularis. Background Art

[0002] Echinococcus multilocularis is a parasite that poses a serious threat to human health. Its larvae, Echinococcus multilocularis, mainly parasitize in the liver of mammals, forming invasive vesicles, posing a serious threat to human health. Alveolar echinococcosis (AE) is a chronic endemic parasitic disease that mainly parasitizes in the liver and grows in an infiltrative manner. The mortality rate of untreated patients within 10-15 years is as high as 90%. The protoscolex of Echinococcus multilocularis is its important life stage and is the main form of transmission of host infection.

[0003] Studies have shown that calcareous bodies are present in the protoscoleces of Echinococcus multilocularis, indicating that a calcium source is required for cyst development, and the calcium levels in the cysts of diseased animals have been shown to be higher than those in the host serum or plasma. Therefore, calcium ions play an important role in the growth and development of protoscoleces. However, the specific mechanism of action of calcium ions on the protoscoleces of Echinococcus multilocularis through oxidative stress has not yet been clarified. At present, the treatment of Echinococcus multilocularis disease mainly relies on surgery and drug therapy, but commonly used drugs such as albendazole and mebendazole can only inhibit the growth of parasites rather than effectively kill them, and some patients will have serious adverse reactions, such as hepatotoxicity. Therefore, the development of new treatment strategies and drugs is a hot topic in current research. Summary of the invention

[0004] The present invention aims to provide an application of calcium ions in inhibiting the survival and proliferation of protoscolex of Echinococcus multilocularis, thereby inhibiting the survival and proliferation of protoscolex of Echinococcus multilocularis by regulating the body's calcium ion pathway or oxidative stress, and thus treating parasitic diseases.

[0005] In order to solve the above technical problems, the technical solution adopted by the present invention is as follows:

[0006] Application of calcium ions in inhibiting the survival and proliferation of protoscolex of Echinococcus multilocularis.

[0007] The present invention also provides a medicine for inhibiting the survival and proliferation of the protoscolex of Echinococcus multilocularis, wherein the medicine contains calcium ions.

[0008] The present invention also provides the use of calcium ions in preparing medicines for preventing, alleviating and / or treating parasitic diseases.

[0009] Preferably, the parasitic disease is Echinococcosis.

[0010] Preferably, the drug for preventing, alleviating and / or treating parasitic diseases inhibits the survival and proliferation of Echinococcus multilocularis protoscolex by regulating the body's calcium ion pathways or oxidative stress, thereby treating the parasitic diseases.

[0011] The present invention also provides a medicine for preventing, alleviating and / or treating parasitic diseases, comprising the calcium ions.

[0012] Compared with the prior art, the present invention has the following advantages and technical effects:

[0013] The present invention studies the specific role and mechanism of calcium ions in the survival and proliferation of Echinococcus multilocularis protoscolex, clarifies the mechanism of inhibiting the survival and proliferation of Echinococcus multilocularis protoscolex by regulating the body's calcium ion pathway or oxidative stress, and provides a new theoretical basis and intervention strategy for the prevention and treatment of parasitic diseases.

[0014] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The morphology and structure of the protoscolecus of Echinococcus multilocularis in different concentrations of calcium ion culture medium provided in Example 1;

[0016] Figure 2 The results of the detection of ROS levels in the protoscoleces of Echinococcus multilocularis in different concentrations of calcium ion culture medium provided in Example 1, wherein: Figure 2 A in the figure is the ROS fluorescence image after different concentrations of calcium ions acted on Echinococcus multilocularis for 72 hours (40×). Figure 2 B in the figure is the ROS fluorescence intensity after different concentrations of calcium ions acted on Echinococcus multilocularis for 72 hours;

[0017] Figure 3 The results of SOD activity in the protoscolex of Echinococcus multilocularis in different concentrations of calcium ion culture medium provided in Example 1. DETAILED DESCRIPTION

[0018] The technical solution of the present invention is further described below through the accompanying drawings and embodiments.

[0019] Unless otherwise defined, technical or scientific terms used in the present invention shall have the common meanings understood by one having ordinary skills in the field to which the present invention belongs.

[0020] Calcium chloride solution was purchased from Shanghai Yuanye Biotechnology Co., Ltd., BCA protein quantification kit, reactive oxygen species (ROS) detection kit, and 100× penicillin / streptomycin were purchased from Beijing Solebow Technology Co., Ltd., total superoxide dismutase (T-SOD) assay kit was purchased from Nanjing Jiancheng Bioengineering Institute, eosin stain was purchased from Shanghai Biyuntian Biotechnology Co., Ltd., fetal bovine serum was purchased from Gibco, USA, and RPMI1640 culture medium (with / without calcium ions) was purchased from Wuhan Boster Biological Co., Ltd.

[0021] In the present invention, unless otherwise specified, other test materials and instruments and equipment are conventional test materials in the art and can be purchased through commercial channels.

[0022] Example 1

[0023] Preparation and in vitro culture of protoscolices of Echinococcus multilocularis: Protoscolices of Echinococcus multilocularis were provided by mature gerbils that were long-term stocked in the Key Laboratory of Central Asian High Incidence Prevention and Control of the National Health Commission. Gerbils that were stocked for 5-6 months were selected, euthanized, and placed in 75% alcohol for disinfection for 30 minutes. The cysts in the gerbils were removed under sterile conditions, cut into pieces, and ground in a homogenizer. The homogenate was processed by filtering and washing steps. The harvested protoscolices of Echinococcus multilocularis were washed 5 times with PBS solution containing 1% penicillin-streptomycin to ensure that the activity reached more than 95%. The protoscolices of Echinococcus multilocularis were transferred to RPMI 1640 medium containing 1% penicillin-streptomycin and 10% fetal bovine serum and incubated in an incubator at 37°C and 5% CO2.

[0024] Eosin staining experiment: The experiment used a 6-well tissue culture plate, inoculated 5,000 protoscoleces in each well, and added 3 mL of complete culture medium (containing 1% penicillin-streptomycin and 10% fetal bovine serum). The final concentration of calcium ions in the culture medium was set to 0, 60, 90, and 120 μg / mL, which was prepared by calcium chloride solution and calcium-free RPMI 1640 culture medium. All samples were incubated in a 37°C, 5% CO2 incubator. During the culture period, the mortality rate of the protoscoleces was assessed using eosin staining and observed under an inverted optical microscope at 100× magnification. The culture lasted for 7 days, and the culture medium was changed every 3 days. 5 μL of the protoscolecene suspension was taken every day to observe its morphology and structural integrity to calculate the survival rate. Each group of experiments was repeated 3 times. The calculation formula is as follows:

[0025]

[0026] The results are shown in Table 1 and Figure 1 .

[0027] Table 1 Survival rate of protoscolecus

[0028]

[0029]

[0030] As shown in Table 1, different concentrations of calcium ions significantly affected the survival rate of protoscolex. The survival rate of the calcium-free (0 μg / ml) group remained at a high level. Although it decreased slightly, the decrease was small, from 98.44±0.79 on the first day to 82.57±0.50 on the seventh day. The inhibitory effect of low concentration (60 μg / mL) calcium ions on the survival rate was the most obvious, which rapidly decreased from 83.94±1.53 on the first day to 6.07±0.16 on the seventh day. The survival rates of the control (90 μg / mL) group and the high concentration (120 μg / mL) group on the first day were 96.01±1.04 and 95.27±0.61, respectively. By the seventh day, the survival rates of the two groups decreased to 71.56±0.51 and 70.63±0.87, respectively. The inhibitory effect was low and the difference was small.

[0031] Depend on Figure 1 It can be seen that the dead protoscolex showed typical morphological characteristics, including significant reduction in size, red staining, loss of hooks, microcracks and shedding, and obvious deformation of the sucker structure. On the seventh day, the proportion of protoscolex that stained red in the low calcium group increased significantly.

[0032] Detection of ROS levels in the protoscolex: The level of reactive oxygen species (ROS) was detected by LDCFH-DA probe in the protoscolex of Echinococcus multilocularis after intervention with different concentrations of calcium ions for 72 hours. The protoscolex was washed three times with PBS, the supernatant was discarded, and a 1:1000 diluted LDCFH-DA probe solution was added and incubated at 37°C in the dark for 1 hour. After incubation, the cells were washed three times with serum-free medium to remove unbound probes. The fluorescence signal was measured by a microplate reader at an excitation wavelength of 488 nm and an emission wavelength of 525 nm. At the same time, the fluorescence signal of the protoscolex was observed under a fluorescence microscope to evaluate the generation and distribution of ROS and to quantify the effect of different calcium ion concentrations on the oxidative stress level of the protoscolex. The results are shown in Tables 2 and Figure 2 .

[0033] Table 2 ROS levels in protoscolex

[0034]

[0035] From Table 2 and Figure 2It can be seen that different concentrations of calcium ions significantly affect the ROS of the protoscolecus of Echinococcus multilocularis. The fluorescence intensity increases with the increase of ROS, and the fluorescence intensity decreases with the decrease of ROS. The fluorescence intensity of the calcium-free group was 106.92±9.01, which was the lowest value among all groups; the fluorescence intensity of the low calcium group increased significantly to 188.07±0.43, which was the highest value in the experiment, indicating that low concentrations of calcium ions can significantly induce an increase in ROS levels, which may be related to the enhancement of intracellular calcium signals and oxidative stress response; the fluorescence intensities of the normal group and the high calcium group were 142.49±2.09 and 146.13±30.00, respectively, which were significantly higher than the control group, but lower than the 60μg / mL group, indicating that the induction effect of medium and high concentrations of calcium ions on ROS levels was weakened, which may be related to the activation of antioxidant mechanisms or the restoration of calcium homeostasis.

[0036] The low calcium group (60 μg / mL) significantly increased the ROS level of protoscolex, while the ROS levels induced by the normal group and high calcium group (90 μg / mL and 120 μg / mL) were relatively low, which may be related to the cellular oxidative stress tolerance or regulation mechanism.

[0037] Detection of SOD levels in protoscoleces: In the protoscoleces of Echinococcus multilocularis after intervention with different concentrations of calcium ions for 72 hours, the protoscoleces were washed three times with PBS, the supernatant was discarded and the protein sample was extracted, and the sample was diluted 10 times with PBS. The protein concentration was determined using the BCA protein quantification kit, and the superoxide dismutase (SOD) activity of the sample was detected according to the instructions of the SOD activity detection kit. GraphPad software was used to perform statistical analysis on the test results and generate visual charts to evaluate the effect of different calcium ion concentrations on SOD activity.

[0038]

[0039] Wherein: A1 is the absorbance value of the control group, A2 is the absorbance value of the control blank group, A3 is the absorbance value of the assay group, and A4 is the absorbance value of the assay blank group.

[0040]

[0041] The results are shown in Table 3 and Figure 3 .

[0042] Table 3 SOD activity

[0043]

[0044] From Table 3 and Figure 3It can be seen that different calcium ion concentrations significantly affect the superoxide dismutase (SOD) activity of protoscolex, and the SOD activity shows a concentration-dependent increase trend, indicating that with the increase of calcium ion concentration, the SOD activity of protoscolex gradually increases, indicating that calcium ions can significantly regulate the antioxidant capacity of protoscolex. High concentrations of calcium ions (120 μg / mL) may reduce the damage of oxidative stress to cells by promoting the expression or activity of antioxidant enzymes.

[0045] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solution of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solution to deviate from the spirit and scope of the technical solution of the present invention.

Claims

1. Application of calcium ions in inhibiting the survival and proliferation of Echinococcus multilocularis protoscolex.

2. A drug for inhibiting the survival and proliferation of Echinococcus multilocularis protoscoleces, characterized in that: The drug comprises the calcium ions as claimed in claim 1.

3. Use of calcium ions in the preparation of drugs for preventing, alleviating and / or treating parasitic diseases.

4. The use according to claim 3, characterized in that: The parasitic disease is Echinococcosis multilocularis.

5. The use according to claim 3, characterized in that: The drug for preventing, alleviating and / or treating parasitic diseases inhibits the survival and proliferation of Echinococcus multilocularis protoscolex by regulating the body's calcium ion pathway or oxidative stress, thereby treating the parasitic disease.

6. A drug for preventing, alleviating and / or treating parasitic diseases, characterized in that: Including the calcium ion as claimed in claim 3.

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