Anti-phospholipid antibody (APA) is a family of autoimmune antibodies, mainly anticardiolipin antibody (ACA) and lupus anticoagulant (LA), which is one of the main immune factors for pregnancy loss. Studies have shown that Chinese medicine plays an important role in immune regulation. In this study, we used animal experiments to preliminarily observe the effect of kidney tonifying and blood invigorating formula-Danshou Tang on pregnancy loss in pregnant rats caused by APA. Wei Aiwu, Reproduction Center, The First Affiliated Hospital of Henan College of Traditional Chinese Medicine
Materials and methods
1. Animals One hundred and twenty SPF-grade healthy female and male SD rats, 10 weeks old, weighing 200g±10g for females and 250g±20g for males, were used, provided by Henan Provincial Experimental Animal Center (Certificate of Conformity No. 0009881). The female and male rats were caged together at 1:1, and those found with pubic plugs or spermatozoa on vaginal smear early the next morning were designated as day 0 of pregnancy, and a total of 100 rats were conceived as the enrolled experimental animals. All SD rats were fed in a clean environment with constant temperature and humidity of 26℃ and 70% humidity, and 12h of light/darkness per day.
2. Drugs The kidney tonic formula (|Silk Seeds 32 g, Chuanjian 16 g, Mulberry 16 g, Agaricus 16 g), blood invigorating formula (Salvia 20 g, Radix et Rhizoma 20 g), and Danshou Tang (the above two formulae were combined) were made into a concentrated decoction containing 1 g/ml, 0.67 g/ml, and 0.34 g/ml of raw herbs, respectively, according to the weight ratio of human rats. It was prepared by the preparation room of the First Affiliated Hospital of Henan College of Traditional Chinese Medicine.
3. Human serum collection and purification
3.1 Entry criteria for serum collection
Patients with recurrent miscarriage who attended the outpatient clinic and ward of the First Affiliated Hospital of Henan College of Traditional Chinese Medicine in March C September 2007, with positive blood ACA by ELISA and medium and high quantitative tests; patients with systemic lupus erythematosus (SLE) who were in the outpatient clinic and ward at the same time, with positive lupus anticoagulant activity test by monoclonal antibody; sera from healthy individuals.
3.2 Exclusion criteria for serum collection
(1) Patients with positive ACA should be excluded from other causes of miscarriage; (2) SLE patients, those with positive ACA and other positive antibodies should be excluded; (3) All the above three populations, and at the same time, those with various other diseases in combination and without recent drug treatment should be excluded, and serum should be retained with the consent of patients.
3.3 Purification and protein solution concentration determination After purification of the protein solution by secondary salting, the protein concentration was determined by UV spectrophotometry, and the OD value of the solution was measured at 280 nm, with reference to the formula: protein mass concentration (mg/ml) = 1.2×OD280 nm to find the protein mass concentration in the solution [1]. The protein solution was prepared at a mass concentration of 15 mg/ml protein solution (equivalent to 75 mg/(kg・d)) and stored at -20°C in portions.
4. Animal modeling, grouping and drug administration methods The model replication was based on the modeling method of Holers [2] and modified. The 100 pregnant rats were randomly divided into two groups of 50 rats each, and each group was divided into kidney tonic and blood activation group (A), kidney tonic group (B), blood activation group (C), model group (D), and blank pregnant rat group (E), and groups A, B, and C were given Danshou Tang, kidney tonic formula, and blood activation formula concentrated decoction from the date of discovery of Yin embolism, while groups D and E were given saline, 4 ml/d, in two gavages for 15 days, during which groups A, B, C, and Groups A, B, C and D were injected subcutaneously with ACA purified protein solution and LA purified protein solution 15mg/(ml・d) on the back of multiple parts on gestation days 8 and 12, respectively, while group E was injected subcutaneously with the same amount of normal human serum on the back of multiple parts on gestation days 8 and 12, respectively. All the animals were dead on the 15th day of gestation.
3. Observation indexes and examination methods
3.1 Determination of ACA level in rat blood 3ml of blood was collected from the abdominal aorta on day 15 of pregnancy, and the serum ACA level was measured by enzyme-linked immunoassay, and the OD value was measured by enzyme-labeled instrument. (ACA enzyme-linked immunoassay kit, produced by Shenzhen Xaar Biotechnology Co., Ltd, batch number 20070721).
3.2 Determination of lupus anticoagulant activity by murine blood monoclonal antibody 1.8 ml of blood was taken from the abdominal aorta on day 15 of pregnancy, and its activity was measured by the endpoint agglutination method to determine the activated partial thromboplastin time (APTT) by reference [3]. (Semi-automatic chronograph, Becton Dickinson, Franklin Lakes, NJ; Mouse monoclonal antibody kit MABTEST, Adiatec SA, Diagnositic }Biotechnologies, LA, France, item number 20070091)
3.3 Weighing of embryos After Ulatan anesthesia, mouse embryos were separated openly and weighed wet with a precision electronic balance.
3.4 Detection of embryo absorption rate The embryo absorption rate (embryo absorption rate = number of absorbed embryos/number of absorbed embryos + number of unabsorbed embryos × 100%) was calculated by referring to the literature [4-5].
3.5 Placental pathological detection The stripped part of placenta was fixed with 4% paraformaldehyde for 24-48h, routinely paraffin embedded, sectioned (thickness 4um), HE staining and SP immunohistochemical staining for ACA positive pregnancy loss model group, HE staining for LA positive pregnancy loss model group, and image acquisition by HMLAS-2000 high definition color medical graphic analysis system.
3.6 Statistical analysis SPSS13.0 software was used for analysis, and all data were subjected to chi-square test, and t-test was used for data between two groups.
Results
1. The comparison of embryo uptake rate, embryo weight, ACA and LA activity in different groups of ACA-positive and LA-positive pregnant rats is shown in Tables 1 and 2.
Compared with group D, (1) the embryo uptake rate, ACA and APTT levels were decreased in groups A, B and C (P<0.01), but the decrease was significant in group A among the three groups (P<0.05), and the difference was not statistically significant with group E. (2) There was a significant increase in embryo weight in group A and group C (P<0.01, P<0.05), and only the embryo weight in group A was not statistically significant from group E (P>0.05). It is suggested that the formula of tonifying kidney and activating blood is superior to the formula of tonifying kidney or activating blood.
2. Histopathological observation of rat placenta in each group (1) Immunohistochemical section Group E did not see brown ACA immune complex deposition; ACA pregnancy loss Group D rat placental tissue could be seen with different degrees of brown ACA immune complex deposition, and there were cell nuclei solidification and nuclear fragmentation, suggesting placental tissue degeneration and necrosis; Group B and Group C all had significant improvement compared with Group D; Group A did not see placental tissue degeneration and necrosis (2) HE stained pathological sections (2) HE stained pathological sections The placental tissue in group E had neat cell arrangement, clear structure, and abundant and uniform blood sinus; the placental tissue in LA and ACA pregnancy loss group D had different degrees of degeneration and necrosis, inflammatory cell infiltration was seen in the trophoblast layer, and different degrees of fibrin clotting and venous stasis in the placental vessels, suggesting impaired placental blood circulation; group B and group C both had significant improvement compared with the model group, while group A The placental pathology was basically normal in group A.
Table 1 Changes in embryo resorption rate, embryo weight and ACA level in different groups of ACA-positive pregnant rats
Group
Number of pregnant rats
Embryo uptake rate
Embryo weight
OD value of ACA level
Group A
10
0.03±0.03＊＊
0.38±0.05＊＊
0.10±0.03＊*
Group B
10
0.12±0.08＊＊#
0.32±0.06△
0.19±0.05＊＊##△
Group C
10
0.10±0.04＊＊#
0.29±0.10＊△
0.22±0.04＊＊##△
Group D
10
0.21±0.09
0.23±0.04
0.32±0.05
Group E
10
0
0.39±0.06
0
Note: Compared with group D, *P<0.05, **P<0.01; compared with group E, △P<0.01
Compared with group A, #P<0.05, ##P<0.01
Table 2 Comparison of embryo uptake rate, embryo weight and LA activity in different groups of LA-positive pregnant rats
Group
Number of pregnant rats
Embryo uptake rate
Embryo weight
APTT(S)
Group A
10
0.01±0.03＊＊
0.42±0.03＊*
20.03±0.78＊*
Group B
10
0.08±0.07＊＊ #
0.34±0.06△
21.40±1.11＊＊ ## △
Group C
10
0.06±0.07＊＊ #
0.36±0.06＊△
21.31±1.18＊##△
Group D
10
0.19±0.06
0.32±0.04
24.34±1.31
Group E
10
0
0.43±0.04
19.86±0.95
Note: Compared with group D, *P<0.05, **P<0.01; compared with group E, △P<0.01
Compared with group A, #P<0.05, ##P<0.01
Discussion
Pregnancy loss is often manifested as recurrent miscarriage, stillbirth or intrauterine growth retardation, etc. Except for genetic, anatomical and endocrine factors, it is closely related to immune factors, of which APA is one of them. At present, the exact mechanism of pregnancy loss caused by APA is not well understood, but most tend to favor the hemagglutination theory. Animal experiments have confirmed that APA prevents prostacyclin synthesis, causing thromboxane/prostacyclin ratio imbalance, leading to blood hypercoagulation and thrombosis, especially in the placental circulation, which can cause the formation of placental thrombosis and placental infarction, resulting in placental hypoplasia, embryonic resorption, and intrauterine growth retardation [6-7]. The clinical treatment of these drugs mainly uses aspirin, heparin and prednisone, but the potential side effects of these drugs make patients feel panic.
In Chinese medicine, there are the names of “abortion”, “slippery fetus”, “fetal atrophy without growth”, and “child dying in the abdomen”, which are similar to In modern medicine, it is similar to the manifestation of pregnancy loss. According to TCM, the above diseases are caused by damage to the punching point. Therefore, kidney deficiency and weakness are the root of the disease. However, if the kidney Yang deficiency loss of warmth, or Yin deficiency heat, heat burning essence and blood, resulting in blood stagnation does not work, or repeatedly fall punch and Ren injury, blood stasis blocking the ligament, resulting in fetal displacement, the symptoms of the disease. The “Nei Jing”, “four oyster bones and a pill” opened the precedent of tonifying the kidney and invigorating the blood, and Zhang Xichen pointed out that “the kidney pill is a medicine for tonifying the kidney, but it is also a medicine for opening stasis”, and that the organic combination of tonifying the kidney and invigorating the blood has a mutually reinforcing effect. Based on this, he established the principle of tonifying the Kidney and invigorating Blood to calm the fetus, and formulated the formula of tonifying the Kidney and invigorating Blood – Dan Shou Tang, which is composed of Shou Fetus Pill with Salvia miltiorrhiza and Radix Rehmanniae. In this formula, |Serum seeds and Dan Shen are the rulers, combining tonicity and activation, complementing each other; the minister uses Fructus Parviflora and Radix et Rhizoma Polygonati to benefit the kidney and calm the fetus, increasing the function of consolidating and protecting the flushing and the Ren; the adjuvant uses Radix Aconiti to help Dan Shen invigorate the Blood and move the Blood, and Aconite to tonic the Blood and stop the Blood, nourishing the fetus with the Blood, and preventing the blood stasis from dispersing the Blood, so that one tonic and one line can treat both the symptoms and the root of the problem. Modern research also shows that |serum can promote the increase of ovarian luteinizing hormone, enhance humoral immune function, and have estrogen-like effects, can inhibit the production of immunoglobulins; mulberry and sequestra are rich in sterols and steroids, conducive to the development of the uterus and embryo and inhibit the role of uterine contraction; Agaricus contains a variety of trace elements, can improve the capital of macrophages to support the ΓΓ bad Wei Wei Dizi Senatorial file rabbit wish. It has been shown that it can improve macrophages’ capital and the development of embryo and inhibit uterine contraction. The results of this study showed that the formula for tonifying the kidney and invigorating the blood was more effective than the formula for tonifying the kidney and invigorating the blood alone in reducing APA titers, thereby improving placental microcirculation, reducing embryo resorption, increasing the weight of rat embryos, and improving the success rate of repeat pregnancies.
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