Application of vimentin in the preparation of drugs for the treatment of angiostrongylus cantonensis

Abstract

The present invention discloses the application of vimentin in the preparation of a drug for the treatment of Guangzhou angiostrongylus disease. The present invention constructs a mouse infection model of Guangzhou angiostrongylus disease, and finds that the level of vimentin and its phosphorylation level in the brain tissue of mice in the late stage of infection are significantly increased, and the permeability of the blood-brain barrier is significantly increased. After knocking out vimentin using the Vimentin-knockout mouse model, it was found through magnetic resonance imaging and Evans blue experiments that the degree of blood-brain barrier damage in infected mice was effectively alleviated, the expression of AQP4, a molecule related to blood-brain barrier damage, was significantly reduced, and the expression of tight junction protein occludin was significantly increased. The research results of the present invention show that vimentin is a key regulatory factor involved in the destruction of the blood-brain barrier of Guangzhou angiostrongylus disease, and downregulating vimentin can play a role in the treatment of Guangzhou angiostrongylus disease. Vimentin can be used as a therapeutic target for Guangzhou angiostrongylus disease for the preparation or screening of drugs for the treatment of Guangzhou angiostrongylus disease.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and more specifically, to the use of vimentin in preparing a drug for treating angiostrongylus cantonensis. Background Art

[0002] Angiostrongyliasis cantonensis is a zoonotic infectious disease that seriously damages the central nervous system. It is caused by the larvae of Angiostrongyliasis cantonensis that penetrate the blood-brain barrier (BBB) ​​of the body and enter the central nervous system when humans ingest uncooked freshwater snails, terrestrial snails and other mollusks or contaminated vegetables. Angiostrongyliasis cantonensis is the most common cause of eosinophilic meningitis in Southeast Asia, the Pacific Islands, the Caribbean and South America, and is considered one of the major public health issues in the world. Angiostrongyliasis cantonensis can cause different pathological damage in suitable hosts and non-suitable hosts. The symptoms of suitable hosts are relatively mild, while non-suitable hosts often have severe symptoms of meningitis and even death. Humans are non-suitable hosts for Angiostrongyliasis cantonensis. After infecting non-suitable hosts, the larvae can cross the blood-brain barrier through the blood circulation system to reach the central nervous system and grow and develop in the brain tissue. However, after the worm develops into a V-stage larva in the brain of a non-suitable host, it cannot further migrate to other organs, thus causing severe eosinophilic meningoencephalitis or meningitis. In the later stages of infection, Angiostrongylus cantonensis can cause serious damage to the integrity of the blood-brain barrier, leading to increased intracranial pressure and a significant increase in cerebrospinal fluid in patients. The destruction of the integrity of the blood-brain barrier is an important reason for the further deterioration of brain damage. Therefore, it is urgent to conduct in-depth research on the specific mechanism by which Angiostrongylus cantonensis destroys the blood-brain barrier and to discover the key regulatory factors that regulate the destruction of the blood-brain barrier.

[0003] Vimentin is a member of the intermediate filament protein family. It is mainly found in mesenchymal cells such as endothelial cells, glial cells and some undifferentiated cells such as neural stem cells and neural progenitor cells. It is an important component of the cytoskeleton and plays an important role in maintaining the morphology of supporting cells, fixing intracellular subcellular structures, cell differentiation, and growth regulation. Recently, it has also been reported that Vimentin is involved in the body's immune response. However, the specific role of Vimentin in Guangzhou roundworm disease is still unknown. Summary of the invention

[0004] The purpose of the present invention is to overcome the above-mentioned defects and deficiencies in the prior art and provide the use of vimentin in the preparation of a drug for treating angiostrongylus cantonensis.

[0005] Another object of the present invention is to provide a drug for treating Angiostrongylus cantonensis.

[0006] The above-mentioned object of the present invention is achieved through the following technical solutions:

[0007] The present invention first constructs a mouse model of Guangzhou angiostrongylus disease, and detects changes in the blood-brain barrier permeability of mice and normal mice at different time points of infection by Evans blue permeability experiment, blood biochemical analysis experiment and enhanced magnetic resonance imaging, and finds that the permeability of the blood-brain barrier of infected mice is significantly increased. Next, the microstructural components of the blood-brain barrier are observed by transmission electron microscopy, and it is found that the end feet of astrocytes are significantly swollen. RT-qPCR and Western blot detections find that the expression of water channel protein AQP4 increases, and the water content of brain tissue increases significantly. Further RT-qPCR, Western blot, immunofluorescence and flow cytometry are used to find that it is significantly highly expressed in the brain tissue of infected mice. Then, by constructing Vimentin knockout mice, it is found that after knocking out Vimentin, the permeability of the blood-brain barrier is significantly reduced, the expression of AQP4 is reduced, the expression of tight junction protein occludin is significantly increased, and the water content of brain tissue is significantly reduced, suggesting that Vimentin is a potential drug target for the treatment of Guangzhou angiostrongylus disease. By down-regulating Vimentin, the expression of AQP4 can be inhibited, the water permeability can be reduced, and the permeability of the blood-brain barrier can be alleviated, thereby achieving the effect of treating the blood-brain barrier damage caused by Guangzhou roundworm disease. Therefore, the present invention first provides new applications of vimentin in the following aspects:

[0008] Application of vimentin as a therapeutic target in the preparation or screening of drugs for the treatment of Angiostrongylus cantonensis.

[0009] The use of vimentin as a therapeutic target in the preparation or screening of drugs for preventing and / or treating blood-brain barrier damage caused by Angiostrongylus cantonensis.

[0010] Application of a preparation for inhibiting or down-regulating the expression of vimentin in the preparation of a drug for treating angiostrongylus cantonensis.

[0011] Use of a preparation for inhibiting or downregulating the expression of vimentin in a drug for preventing and / or treating blood-brain barrier damage caused by Angiostrongylus cantonensis.

[0012] The methods of inhibiting or down-regulating the expression of vimentin include, but are not limited to, RNAi interference technology, CRISPR / Cas9 gene editing technology, etc. Any method that can achieve the inhibition or down-regulation of vimentin in the patient's body should be within the scope of protection of the present invention.

[0013] Preferably, the preparation is a reagent containing a vimentin RNA interference vector or a reagent containing a vimentin CRISPR / Cas9 gene editing vector.

[0014] The present invention also provides a drug for treating Angiostrongylus cantonensis, which comprises a preparation for inhibiting or down-regulating the expression of vimentin.

[0015] Preferably, the preparation is a reagent containing a vimentin RNA interference vector or a reagent containing a vimentin CRISPR / Cas9 gene editing vector.

[0016] Preferably, pharmaceutically addible excipients are also included.

[0017] Further preferably, the auxiliary material includes one or more of a diluent, an excipient, a filler, a binder, a wetting agent, a disintegrant, an absorption promoter, a surfactant, an adsorption carrier or a lubricant.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] The present invention provides a new use of vimentin in the preparation of a drug for the treatment of Guangzhou angiostrongylosis. The present invention constructs a mouse infection model of Guangzhou angiostrongylosis and finds that the level of vimentin and its phosphorylation level in the brain tissue of mice in the late stage of infection are significantly increased, and the permeability of the blood-brain barrier is significantly increased. After knocking out vimentin using the Vimentin-knockout mouse model, magnetic resonance imaging and Evans blue experiments found that the degree of blood-brain barrier damage in infected mice was effectively alleviated, the expression of blood-brain barrier-related molecule AQP4 was significantly reduced, and the expression of tight junction protein occludin was significantly increased. The research results of the present invention show that vimentin is a key regulatory factor involved in the destruction of the blood-brain barrier in Guangzhou angiostrongylosis, and downregulating vimentin can play a role in treating the destruction of the blood-brain barrier in Guangzhou angiostrongylosis. Vimentin can be used as a therapeutic target for Guangzhou angiostrongylosis for the preparation or screening of drugs for the treatment of Guangzhou angiostrongylosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The results of the Evans blue experiment on mice infected with Angiostrongylus cantonensis at different time points (0, 3, 7, 14, and 21 days). Figure A is the gross observation of the brain, Figure B is the quantitative result of Evans blue in brain tissue, Figure C is a representative fluorescence image of frozen sections of brain tissue, and Figure D is the relative quantitative result of fluorescence.

[0021] Figure 2 These are the enhanced magnetic resonance imaging results of mice infected with Angiostrongylus cantonensis at different time points (0, 3, 7, 14, and 21 days).

[0022] Figure 3 These are the transmission electron microscopy results of mice infected with Angiostrongylus cantonensis and mice in the blank control group.

[0023] Figure 4 These are the results of brain tissue water content in mice infected with Angiostrongylus cantonensis at different time points (0, 3, 7, 14, and 21 days).

[0024] Figure 5 Figure 1 shows the changes in mRNA and protein levels of Vimentin in the hippocampus of mice infected with Angiostrongylus cantonensis at different time points (0, 3, 7, 14, and 21 days). Figure A shows the mRNA level of Vimentin in the hippocampus, Figure B shows the representative Western blot bands, and Figure C shows the relative quantitative Western blot bands.

[0025] Figure 6 Figure 1 shows the changes in the mRNA and protein levels of Vimentin in the cortex of mice infected with Angiostrongylus cantonensis at different time points (0, 3, 7, 14, and 21 days). Figure A shows the mRNA level of Vimentin in the cortex, Figure B shows the representative Western blot protein bands, and Figure C shows the relative quantitative Western blot protein bands.

[0026] Figure 7 The immunofluorescence results of Vimentin expression in mice infected with Angiostrongylus cantonensis at different time points (0, 3, 7, 14, and 21 days). Figure A is a representative immunofluorescence image of Vimentin in the hippocampus and cortex, and Figure B is the relative quantitative result of fluorescence.

[0027] Figure 8 These are the flow cytometry results of Vimentin expression in mice infected with Angiostrongylus cantonensis and mice in the blank control group.

[0028] Fig. 9 The results of double immunofluorescence labeling of Vimentin and astrocyte marker (GFAP).

[0029] Fig.10 The results of double immunofluorescence labeling of Vimentin and neuronal marker (NeuN).

[0030] Fig.11 The results of double immunofluorescence labeling of Vimentin and microglial marker (Iba1).

[0031] Fig.12 These are the flow cytometry results of mice infected with Angiostrongylus cantonensis and the blank control group, detecting the ratio of Viemntin and GFAP, NeuN, and Iba1 double-positive cells.

[0032] Fig.13 These are the Evans blue results for the wild-type mouse blank control group (WT-NC), wild-type mouse infection group (WT-AC), knockout mouse blank control group (KO-NC), and knockout mouse infection group (KO-AC). A is the gross brain observation result, B is the representative fluorescence image of the brain tissue frozen section, and C is the fluorescence relative quantification result.

[0033] Fig.14 These are the enhanced MRI results of the wild-type mouse blank control group (WT-NC), wild-type mouse infection group (WT-AC), knockout mouse blank control group (KO-NC), and knockout mouse infection group (KO-AC).

[0034] Fig.15 These are the results of brain tissue water content in the wild-type mouse blank control group (WT-NC), wild-type mouse infection group (WT-AC), knockout mouse blank control group (KO-NC), and knockout mouse infection group (KO-AC).

[0035] Fig.16 The results of changes in the mRNA and protein levels of AQP4 and Occludin in the wild-type mouse blank control group (WT-NC), wild-type mouse infection group (WT-AC), knockout mouse blank control group (KO-NC), and knockout mouse infection group (KO-AC). Figure A is a representative image of Western blot protein bands in hippocampal tissue, Figure B is a representative image of Western blot protein bands in cortical tissue, Figure C is a relative quantitative image of Western blot protein bands in hippocampal tissue, and Figure D is a relative quantitative image of Western blot protein bands in cortical tissue. DETAILED DESCRIPTION

[0036] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0037] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0038] 7-8 week old SPF BALB / c mice were purchased from Guangdong Medical Experimental Animal Center, Vimentin knockout mice were donated by Professor Peng Hongjuan from Southern Medical University, and stage III larvae of Angiostrongylus cantonensis were from the smooth biumbilical snails preserved in our laboratory.

[0039] Example 1 Increased blood-brain barrier permeability in mice after infection with Angiostrongylus cantonensis

[0040] The specific method for constructing the mouse model of Guangzhou roundworm disease is as follows: crush the smooth biumbilical snails containing stage III larvae, remove the snail shells, clamp the snail meat into a centrifuge tube and cut it into pieces, add digestive fluid and incubate at 37° for 1 hour, terminate the digestion with PBS, count the worms under a dissecting microscope, and infect each mouse with 30 stage III larvae by gavage.

[0041] After successfully constructing the Guangzhou Angiostrongylus infection mouse model, Evans blue permeability experiments and enhanced magnetic resonance imaging experiments were performed on days 0, 3, 7, 14, and 21 of infection. The Evans blue experiment operation steps include preparing a 2% Evans blue solution, injecting it into the tail vein of mice at a dose of 200 μL / mouse, and after circulating for 2 hours, perfusion sampling and photography are performed. The results of the Evans blue experiment at different time points (0, 3, 7, 14, and 21 days) of Guangzhou Angiostrongylus infection mice are shown in the figure. Figure 1 The experimental procedure of enhanced magnetic resonance imaging is to inject the contrast agent GD-DTPA through the tail vein at a dose of 287 mg / kg, and then use the magnetic resonance imaging device to scan and image 20 minutes later. The enhanced magnetic resonance imaging results of mice infected with Guangzhou roundworms at different time points (0, 3, 7, 14, and 21 days) are shown in the figure. Figure 2 As shown, the above results showed that the blood-brain barrier permeability of infected mice increased significantly on the 21st day.

[0042] Next, transmission electron microscopy was used to observe the changes in the microstructure of the blood-brain barrier in the blank control group and on the 21st day of infection. Figure 3 As shown, the end-feet of astrocytes in infected mice were found to be significantly swollen.

[0043] The changes in brain tissue water content at different infection time points were detected by weighing method. The results are as follows Figure 4 As shown, the water content of the brain tissue of infected mice was significantly increased, suggesting that the mice showed obvious pathological manifestations of cerebral edema.

[0044] Example 2 Increased expression of Vimentin in mouse brain tissue after infection with Angiostrongylus cantonensis

[0045] The hippocampus and cortex were isolated on days 0, 3, 7, 14, and 21 of mouse infection, and the expression of Vimentin was detected by RT-qPCR, Western blot, immunofluorescence, and flow cytometry. The RT-qPCR experimental steps include extracting tissue RNA, then reverse transcribing it into cDNA using a reverse transcription kit, then preparing the RT-qPCR system, and finally testing it on the machine. The Western blot operation steps include extracting tissue protein, quantifying it using BCA, then preparing protein denaturation samples for SDS-PAGE electrophoresis, transferring the membrane, blocking, incubating with primary antibodies, washing, incubating with secondary antibodies, and developing with a chemiluminescent instrument. The immunofluorescence operation steps include baking the slides, dewaxing and dehydrating, and then performing antigen retrieval, blocking, incubating with primary antibodies, washing, incubating with secondary antibodies, and photographing with a fluorescence microscope. The flow cytometry operation steps include taking fresh tissue, grinding it to make a single cell suspension, then incubating it with fluorescent antibodies, washing it, and then analyzing it on a flow cytometer. The results of RT-qPCR and Western blot are shown in the following figure. Figure 5 and Figure 6 The results of immunofluorescence and flow cytometry are shown in Figure 7 and Figure 8 As shown, it was found that Vimentin expression was significantly increased in both the hippocampus and cortex.

[0046] Example 3 Vimentin is mainly expressed in astrocytes

[0047] On days 0, 3, 7, 14, and 21 of mouse infection, immunofluorescence co-localization and flow cytometry were used to detect the main cell types expressing Vimentin. Immunofluorescence co-localization is based on immunofluorescence, incubating two primary antibodies from different sources, and then incubating two secondary antibodies from different sources. Fig. 9 It showed that Vimentin and GFAP were co-localized. Fig.10 Vimentin and NeuN were not co-localized. Fig.11 Vimentin and IBA1 were not co-localized. Fig.12 Flow cytometry analysis showed that the number of Vim+GFAP+ cells and Vim+IBA1+ cells increased significantly, while the number of Vim+NeuN+ cells did not change significantly. The above results indicate that Vimentin is expressed in astrocytes, but not in neurons, microglia and vascular endothelial cells.

[0048] Example 4 Knockout of Vimentin inhibits AQP4 expression and increases occludin expression, reducing blood-brain barrier permeability

[0049] In order to prove the role of Vimentin in the mouse model of Angiostrongylus cantonensis, a Vimentin knockout mouse model was used, and then the knockout mice were infected with Angiostrongylus cantonensis. A wild-type mouse blank control group, a wild-type mouse infection group, and a knockout mouse blank control group were set up. Evans blue experiments and enhanced magnetic resonance imaging experiments were performed. Fig.13 The enhanced magnetic resonance imaging results are shown in Fig.14 As shown, it was found that knocking out Vimentin could significantly alleviate the increase in blood-brain barrier permeability and reduce the degree of blood-brain barrier damage.

[0050] The changes in brain water content of mice in each group were detected by weighing method. The results are as follows: Fig.15 As shown, it was found that knocking out Vimentin can reduce brain water content and alleviate brain edema in mice.

[0051] Next, Western blot was used to detect the changes in the expression of AQP4 and occludin. The results are as follows Fig.16As shown, both in the hippocampus and cortex, compared with the WT-NC group, the expression of AQP4 in the WT-AC group was significantly increased, and the expression of occludin was significantly decreased. Compared with the WT-AC group, the expression of AQP4 in the KO-AC group was significantly decreased, and the expression of occludin was significantly increased, indicating that knocking out Vimentin can significantly reduce the upregulation of AQP4 expression and alleviate the degradation of occludin, suggesting that Vimentin participates in the destruction of the blood-brain barrier in mice infected with Guangzhou roundworm by mediating the changes in AQP4 / occludin. Vimentin can be used as a potential target for the treatment of Guangzhou roundworm disease.

Claims

1. Application of vimentin knockout reagent in the preparation of drugs for the treatment of Guangzhou roundworm disease.

2. Use of a vimentin knockout reagent in the preparation of a drug for preventing and / or treating blood-brain barrier damage caused by Angiostrongylus cantonensis.

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