Application of BCAT2 recombinant protein in preeclampsia rat model treatment

Abstract

The invention discloses application of BCAT2 recombinant protein in preeclampsia rat model treatment, and belongs to the technical field of biomedicine, preeclampsia symptoms of a preeclampsia rat model can be improved or treated by taking an effective dose of BCAT2 recombinant protein as an injection medicine, including improvement of preeclampsia typical symptoms and signs such as blood pressure rise and proteinuria of the model, and the preeclampsia typical symptoms and signs of the model can be improved. The BCAT2 recombinant protein provided by the invention can be used for improving the preeclampsia phenotype, improving liver and kidney function impairment, and improving fetal rat intrauterine development limitation and other performances, so that the treatment or improvement effect of the BCAT2 recombinant protein on the preeclampsia phenotype can be proved, and an animal model can be provided for discussing pathogenesis, clinical diagnosis and treatment strategies and the like of severe preeclampsia. And a related research basis is also provided for preparing a medicine for improving or treating preeclampsia by using the BCAT2 recombinant protein.

Description

Technical Field

[0001] The present invention is an application of BCAT2 recombinant protein in the treatment of preeclampsia rat model, and specifically relates to a new application of BCAT2 recombinant protein in improving or treating preeclampsia rat model, and belongs to the field of biomedical technology. Background Art

[0002] Preeclampsia is a common pregnancy-related complication characterized by new-onset hypertension, and / or proteinuria and multiple organ dysfunction after 20 weeks of pregnancy. According to the progression of the disease, it is divided into preeclampsia and severe preeclampsia. To date, the etiology and pathogenesis of preeclampsia remain unclear, but since placental delivery is the only effective treatment, preeclampsia is considered to be a placental disease, with spiral artery remodeling disorders and reduced placental blood perfusion as its pathological basis. At present, the treatment of preeclampsia mainly focuses on relieving hypertension and preventing complications. Since its pathological mechanism has not been fully clarified, there is still a lack of specific therapeutic drugs.

[0003] Due to the medical ethics restrictions on human experiments, animal models have become an important tool for studying the occurrence and development of preeclampsia and exploring preventive and therapeutic measures. The classic rat model of preeclampsia is an Ad-sFlt-1 adenovirus constructed by rat tail vein injection, which simulates the clinical characteristics of human preeclampsia, such as increased blood pressure, proteinuria, placental dysfunction, and endothelial cell damage. In recent years, therapeutic research on this model has focused on improving placental angiogenesis, reducing systemic inflammatory responses, and alleviating vascular endothelial function damage.

[0004] At present, there are a variety of treatment strategies that have shown certain efficacy in preeclampsia-like rat models, including antioxidant therapy, anti-inflammatory therapy, and endothelial function protection drugs. For example, antioxidants such as vitamin C and vitamin E can effectively reduce oxidative stress and improve vascular function; anti-inflammatory drugs such as non-steroidal anti-inflammatory drugs and specific immunomodulators also reduce inflammatory responses to a certain extent and relieve vascular damage. Studies have reported that tail vein injection of BMP2 recombinant protein has a therapeutic effect on the model based on the construction of preeclampsia-like model with Ad sFlt-1. In addition, Japanese patent JP2012211131A also discloses that when treating or preventing pregnancy-related hypertension disorders in subjects, a compound that increases the level of growth factors with soluble endothelin binding ability can be administered to the subject, and the treatment or prevention of preeclampsia or eclampsia in the subject can be achieved under the control of appropriate dosage and time during the administration stage. The compound can be selected from TGF-β 1, TGF-3 β, activin A, BMP-2 and BMP-7.

[0005] Branched-chain amino acid transaminase 2 (BCAT2) is an enzyme that plays an important role in amino acid metabolism, especially in the decomposition of branched-chain amino acids. BCAT2 has an important influence on energy production, nitrogen balance, and metabolic processes under various physiological and pathological conditions. Studies have reported that BCAT2 overexpression is associated with tumor proliferation, invasion, and migration, but there are currently no studies reporting the effects of BCAT2 on the biological behavior of trophoblasts and its relationship with preeclampsia. Summary of the invention

[0006] The purpose of the present invention is to provide an application of BCAT2 recombinant protein in the treatment of a preeclampsia rat model. By administering BCAT2 recombinant protein to the preeclampsia rat model, the characteristic symptoms and sign changes of the model preeclampsia can be improved or treated, and the operation is controllable and stable.

[0007] The present invention is achieved through the following technical scheme: application of BCAT2 recombinant protein in the preparation of a drug for improving or treating preeclampsia; an effective dose of BCAT2 recombinant protein as an injectable drug can improve or treat the preeclampsia symptoms of a preeclampsia rat model.

[0008] On the 9th day of pregnancy, 2×10 9 PFU Ad sFlt-1 was used to construct a rat model of preeclampsia.

[0009] The SD pregnant mice were SPF grade and 8 weeks old.

[0010] 100 μg / kg BCAT2 recombinant protein was injected into SD pregnant mice via the tail vein on gestational days 10 to 19.

[0011] Symptoms of preeclampsia include blood pressure, 24-hour urine protein, puppies' condition, placenta condition, liver condition and kidney condition.

[0012] The preeclampsia refers to severe preeclampsia.

[0013] Compared with the prior art, the present invention has the following advantages and beneficial effects: (1) The present invention constructs a rat model of preeclampsia and administers BCAT2 recombinant protein for treatment, which can improve the model's typical symptoms and signs of preeclampsia, such as increased blood pressure and proteinuria, improve liver and kidney function damage, and improve manifestations such as intrauterine growth restriction in fetal mice. This can prove the therapeutic or improvement effect of BCAT2 recombinant protein on preeclampsia-like phenotypes.

[0014] (2) The present invention provides a standardized, safe, efficient, stable and reliable treatment method for preeclampsia rat model, which provides a relevant animal model for studying the pathogenesis, clinical diagnosis and treatment and disposal strategies of severe preeclampsia, and also provides a relevant research basis for the preparation of BCAT2 recombinant protein for the improvement or treatment of preeclampsia. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 Non-invasive blood pressure changes of pregnant SD mice.

[0016] Figure 2 Invasive blood pressure changes in pregnant SD mice.

[0017] Figure 3 The figure shows the changes of urine protein measurement values ​​in SD pregnant mice.

[0018] Figure 4 is the serum sFlt-1 / PlGF ratio of SD pregnant mice.

[0019] Figure 5 This is the intrauterine development of fetal mice.

[0020] Figure 6 This is the fluorescence expression of BCAT2 in the placenta of SD pregnant mice.

[0021] Figure 7 The expression changes of BCAT2 in the placenta of pregnant SD mice.

[0022] Figure 8 Pathological changes in the placenta, kidney and liver of pregnant SD mice. DETAILED DESCRIPTION

[0023] The invention objectives, technical solutions and beneficial effects of the present invention are further described in detail below.

[0024] It should be noted that the following detailed descriptions are exemplary and are intended to provide further explanation of the claimed invention, and unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs.

[0025] The present invention studies the changes of characteristic symptoms and signs of a rat model of preeclampsia caused by BCAT2 recombinant protein, and obtains relevant evidence of BCAT2 recombinant protein in improving or treating the rat model of preeclampsia. In particular, in a pregnant mouse model constructed by Ad sFlt-1, injection of BCAT2 recombinant protein through the tail vein can improve the phenotype of preeclampsia, indicating that BCAT2 recombinant protein has certain therapeutic potential, which is of great value for exploring the clinical diagnosis and treatment of severe preeclampsia and the selection of disposal strategies.

[0026] Several typical embodiments are listed below to illustrate the specific implementation of the present invention. Of course, the protection scope of the present invention is not limited to the following embodiments.

[0027] Main reagents and materials: SPF-grade SD rats (Chengdu Dashuo Experimental Animal Co., Ltd.), recombinant adenovirus vector (Shanghai Hanheng Biotechnology Co., Ltd.), BCAT2 recombinant protein (Wuhan Pujian Biotechnology Co., Ltd.), BCA kit (Beijing Solebold Technology Co., Ltd.), rat PlGF and sFlt-1 ELISA kit (Wuhan Huamei Bioengineering Co., Ltd.).

[0028] Main instruments: rat blood pressure measurement system (Beijing Ruanlong Biotechnology Co., Ltd.), rat metabolic cage (Shanghai Yuyan Scientific Instrument Co., Ltd.), scanning electron microscope (Olympus Corporation, Japan).

[0029] Example 1: Establishment of preeclampsia rat model and BCAT2 recombinant protein treatment 8-week-old SD rats were selected and caged at a ratio of 2:1 between male and female. After the pregnancy was confirmed, the female rats were randomly divided into 4 groups, with 6 female rats in each group. The specific groups were as follows: (1) Ad sFlt-1 + BCAT2 group: 2×10 9 PFU of Ad sFlt-1, and 100 μg / kg BCAT2 recombinant protein was injected into the tail vein on gestational days 10-19; (2) Ad sFlt-1 + normal saline (NS) group: 2×10 9 PFU of Ad sFlt-1 was injected into the tail vein of NS on gestational days 10-19; (3) Ad Fc + NS group: 2×10 9 PFU of Ad Fc, NS was injected into the tail vein on gestational days 10-19; (4) NS group: An equal volume of NS was injected via the tail vein on gestational day 9, and an equal volume of NS was injected via the tail vein on gestational days 10-19.

[0030] Example 2: Preeclampsia Symptom Monitoring The 4 groups of SD pregnant mice in Example 1 (Ad sFlt-1+BCAT2 group, Ad sFlt-1+normal saline (NS) group, AdFc+NS group, NS group) were monitored for preeclampsia symptoms, as follows: 1. Blood pressure and 24-hour urine protein monitoring Non-invasive blood pressure was measured on the 6th, 9th, 12th, 15th, and 17th days of pregnancy, and urine was collected for 24-hour urine protein test. Invasive blood pressure was measured on the 19th day of pregnancy, and urine was collected for 24-hour urine protein test. The results are shown in Figures 1 to 3 shown.

[0031] Among them, the tail-cuff method was used to measure non-invasive blood pressure; the common carotid artery catheterization was used to measure invasive blood pressure; and the BCA method was used to detect 24-hour urine protein.

[0032] Figure 1 The non-invasive blood pressure changes of SD pregnant mice, A is the systolic pressure, B is the diastolic pressure. Figure 1 It can be seen that compared with the Ad Fc+NS group, the systolic blood pressure of pregnant mice in the Ad sFlt-1+NS group was increased on gestational days 12, 15, and 17, and the diastolic blood pressure was increased on gestational days 15 and 17; compared with the AdsFlt-1+NS group, the systolic blood pressure of pregnant mice in the Ad sFlt-1+BCAT2 group was decreased on gestational days 15 and 17, and the diastolic blood pressure was decreased on gestational day 17. This shows that an effective dose of BCAT2 recombinant protein as an injectable drug can improve or treat the symptoms of hypertension in the severe preeclampsia rat model.

[0033] Figure 1 In the figure, ***: P < 0.001, compared with Ad sFlt-1+NS group and Ad Fc+NS group; ##: P < 0.01, compared with Ad sFlt-1+BCAT2 group and Ad sFlt-1+NS group.

[0034] Figure 2 The invasive blood pressure changes of SD pregnant mice. Figure 2 It can be seen that compared with the Ad Fc+NS group, the invasive blood pressure of pregnant mice in the Ad sFlt-1+NS group on the 19th day of pregnancy was increased; compared with the Ad sFlt-1+NS group, the invasive blood pressure of pregnant mice in the Ad sFlt-1+BCAT2 group on the 19th day of pregnancy was decreased, which shows that an effective dose of BCAT2 recombinant protein as an injectable drug can improve or treat the symptoms of hypertension in the severe preeclampsia rat model.

[0035] Figure 2 In the table, *: P < 0.05, ***: P < 0.001.

[0036] Figure 3 The following is the change of 24h urine protein in SD pregnant mice. Figure 3It can be seen that compared with the Ad Fc+NS group, the 24-hour urine protein of pregnant mice in the Ad sFlt-1+NS group was increased on gestational days 12, 15, 17, and 19; compared with the Ad sFlt-1+NS group, the 24-hour urine protein of pregnant mice in the Ad sFlt-1+BCAT2 group was decreased on gestational days 17 and 19. This shows that an effective dose of BCAT2 recombinant protein as an injectable drug can improve or treat the symptoms of proteinuria in the severe preeclampsia rat model.

[0037] Figure 3 In the figure, ***: P < 0.001, compared with Ad sFlt-1 + NS group and Ad Fc + NS group; #: P < 0.05, ##: P < 0.01, compared with Ad sFlt-1 + BCAT2 group and Ad sFlt-1 + NS group.

[0038] (II) Serum PlGF and sFlt-1 detection The serum of SD pregnant mice on the 19th day of pregnancy was collected and tested for PlGF and sFlt-1 by ELISA. The results are shown in Figure 4 shown.

[0039] Depend on Figure 4 It can be seen that compared with the Ad Fc+NS group, the serum sFlt-1 / PlGF ratio of pregnant mice in the Ad sFlt-1+NS group was increased; compared with the Ad sFlt-1+NS group, the serum sFlt-1 / PlGF ratio of pregnant mice in the Ad sFlt-1+BCAT2 group was decreased. This shows that an effective dose of BCAT2 recombinant protein as an injectable drug can improve or treat the symptoms of high serum sFlt-1 and low PlGF in the severe preeclampsia rat model.

[0040] Figure 4 Middle, ***: P < 0.001.

[0041] 3. Fetal rats and placenta Rats were anesthetized with isoflurane and killed by cervical dislocation. The abdomen was cut open and the fetuses and placentas of SD pregnant mice on the 19th day of pregnancy were obtained. The fetuses and placentas were observed and weighed. Figure 5 shown.

[0042] Figure 5 In the figure, A is the size comparison of fetus and placenta, B is the weight comparison of fetus, and C is the weight comparison of placenta. Figure 5It can be seen that compared with the Ad Fc+NS group, the fetal weight of pregnant mice in the Ad sFlt-1+NS group was decreased; compared with the Ad sFlt-1+NS group, the fetal weight of pregnant mice in the Ad sFlt-1+BCAT2 group was increased; compared with the Ad Fc+NS group, the placental weight of pregnant mice in the AdsFlt-1+NS group was decreased; compared with the Ad sFlt-1+NS group, the placental weight of pregnant mice in the Ad sFlt-1 + BCAT2 group was increased. This shows that an effective dose of BCAT2 recombinant protein as an injectable drug can improve or treat the symptoms of intrauterine growth restriction in rat pups of severe preeclampsia rat model.

[0043] Figure 5 In the table, *: P < 0.05, ***: P < 0.001.

[0044] 4. Placental immunofluorescence and Western blot detection Placentas of SD pregnant mice were collected for immunofluorescence and Western blot detection. The results are shown in Figure 6 and Figure 7 shown.

[0045] Among them, the immunofluorescence detection method includes paraffin section dewaxing, rehydration, antigen retrieval, inhibition of endogenous peroxidase, blocking, 6×His primary antibody incubation, secondary antibody incubation, counterstaining with DAPI, sealing and image acquisition; the Western blot detection method includes total protein extraction, BCA determination of protein concentration, electrophoresis, membrane transfer, blocking, BCAT2 primary antibody incubation, secondary antibody incubation and development.

[0046] Figure 6 BCAT2 fluorescence expression in the placenta of SD pregnant mice Figure 6 It can be seen that only the Ad sFlt-1+BCAT2 group had positive fluorescence expression, which indicates that the BCAT2 recombinant protein was successfully injected through the tail vein and widely distributed in the placenta.

[0047] Figure 7 The expression of BCAT2 in the placenta of SD pregnant mice. Figure 7 A in the middle is a representative image of Western blot. Figure 7 B is a Western blot statistical chart, Figure 7 It can be seen that compared with the Ad Fc+NS group, the expression of BCAT2 in the placenta of the Ad sFlt-1+NS group was decreased; compared with the Ad sFlt-1+NS group, the expression of BCAT2 in the placenta of the Ad sFlt-1+BCAT2 group was increased. This shows that an effective dose of BCAT2 recombinant protein as an injectable drug can increase the expression of BCAT2 in the placenta of the severe preeclampsia rat model.

[0048] 5. Pathological conditions of the placenta, liver, and kidneys Rats were anesthetized with isoflurane and killed by cervical dislocation. The abdomen was cut open and the liver and kidney of SD pregnant mice on the 19th day of pregnancy were obtained. The placenta, liver and kidney were collected for morphological staining observation. The results are shown in Figure 8 shown.

[0049] Morphological staining included HE staining of liver, PAS staining of kidney, Masson and HE staining of placenta.

[0050] Figure 8 Pathological changes in the placenta, kidney and liver of pregnant SD mice. Figure 8 Middle A is HE staining of the liver of SD pregnant mice (100×), showing that the hepatic cords of the liver of pregnant mice in the Ad sFlt-1+NS group were disordered and the interlobular veins were necrotic (black arrows); Figure 8 Middle B is PAS staining of the kidney of SD pregnant mice (400×), showing that the glomerular mesangial proliferation and glomerular atrophy (black arrow) of pregnant mice in the Ad sFlt-1+NS group; Figure 8 Middle C is Masson trichrome staining of the placenta of SD pregnant mice (200×), showing the presence of collagen deposition in the placental villous trophoblasts of the Ad sFlt-1 + NS group (black arrows); Figure 8 D in the middle is HE staining of the placenta of SD pregnant mice. It can be seen that compared with the Ad Fc+NS group, the area of ​​the labyrinth layer / junctional layer of the placenta of pregnant mice in the Ad sFlt-1+NS group decreased, and compared with the Ad sFlt-1+NS group, the area of ​​the labyrinth layer / junctional layer of the placenta of pregnant mice in the Ad sFlt-1+BCAT2 group increased. This shows that an effective dose of BCAT2 recombinant protein as an injectable drug can improve or treat the symptoms of organ dysfunction in the severe preeclampsia rat model.

[0051] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Any simple modification or equivalent change made to the above embodiment based on the technical essence of the present invention shall fall within the protection scope of the present invention.

Claims

1. Use of BCAT2 recombinant protein in the preparation of a drug for improving or treating preeclampsia, characterized in that: An effective dose of BCAT2 recombinant protein as an injectable drug can improve or treat the symptoms of preeclampsia in a rat model of preeclampsia.

2. The use according to claim 1, characterized in that: On the 9th day of pregnancy, 2×10 9 PFU Ad sFlt-1 was used to construct a rat model of preeclampsia.

3. The use according to claim 2, characterized in that: The SD pregnant mice were SPF grade and 8 weeks old.

4. The use according to claim 2, characterized in that: 100 μg / kg BCAT2 recombinant protein was injected into SD pregnant mice via the tail vein on gestational days 10 to 19.

5. The use according to claim 1, characterized in that: Symptoms of preeclampsia include blood pressure, 24-hour urine protein, puppies' condition, placenta condition, liver condition and kidney condition.

6. The use according to claim 1, characterized in that: The preeclampsia refers to severe preeclampsia.

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