Effects of Heat Clearing and Stasis Resolving Method on PTS, Inflammatory Factors and T-lymphocyte Subsets in Peripheral Blood of URM Patients with Suppressed Internal Heat

Fenqin ZHAO, Jieying LIU, Yan ZHAO, Yajiao GOU, Zhixia HU

Medical College of Integrated Chinese and Western Medicine, Gansu University of Chinese Medicine, Lanzhou 730000, China

Abstract [Objectives] To explore the effects of heat clearing and stasis resolving method on prethrombotic state, inflammatory factors and T-lymphocyte subsets in peripheral blood of unexplained recurrent miscarriage (URM) patients with suppressed internal heat. [Methods] Thirty cases of URM patients with suppressed internal heat and 30 normal women were collected, and characteristics of changes in peripheral serum D-dimer (D-D), fibrin degradation product (FDP), fibrinogen (FIB), IL-6, IL-10 and TNF-α, levels were detected. URM patients were treated with traditional Chinese medicine for clearing heat and resolving blood stasis for 3 menstrual cycles, and the changes of indicators before and after treatment were observed. [Results] Compared with normal women, the peripheral serum levels of D-D, IL-6, TNF-α and decreased after 3 menstrual cycles (P<0.05), while the contents of IL-10 and T lymphocytes increased significantly (P<0.05). [Conclusions] The heat clearing heat and stasis resolving method can effectively improve the prethrombotic state of URM, and the action mechanism may be related to the regulation of immune and peripheral blood inflammatory factors.

Key words Unexplained recurrent miscarriage (URM), Suppressed internal heat type, Prethrombotic state (PTS), Heat clearing and stasis resolving, Inflammatory factors, T-lymphocyte subsets

1 Introduction

Recurrent spontaneous abortion (RSA) fetal loss has become a main threat for women’s reproductive health. Repeated pregnancy and repeated abortions seriously affect the physical and mental health of patients. The causes for RSA are complex and diverse, including known genetic, anatomical, endocrine, infectious, immune factors, and prethrombotic state (PTS)[1]. Unexplained recurrent miscarriage (URM) is often believed to be related to immune tolerance disorders[2]. Concurrent autoimmune diseases lead to coagulation and immune dysfunction, which in turn leads to pregnancy failure, accounting for 15% of the causes of recurrent miscarriage[3]. PTS[4]is a dysfunction of the coagulation and anticoagulation system, and it leads to a persistent hypercoagulable state and increased uterine artery blood flow resistance[5-6]. Poor intimal receptivity[7]is a key factor in the occurrence of URM, and its occurrence process involves coagulation, platelet aggregation, and chronic inflammation[8]. PTS is treated with low-dose aspirin or low-molecular-weight heparin, but there are risks such as bleeding, osteoporosis, liver and kidney damage, and the efficacy is limited[9]. For immune dysfunction, it is able to take low-dose immunosuppressants (glucocorticoids) about 3 months before pregnancy, but the teratogenicity and liver and kidney damage caused by it, as well as the uncertainty of efficacy[3], have attracted much attention.

This disease belongs to the category of "habitual abortion" in the traditional Chinese medicine. Autoimmune antibodies are mostly yang pathogen, and enter the nutrient and blood, and damage the fluid and humor, leading to blood sticky and accordingly forming the blood stasis syndrome; veins on the uterus are connected with the kidney, the fetus resides in the mother’s body and feeds on the mother’s uterus veins, and the mother’s qi can carry the fetus, blood can nourish it, and thoroughfare and conception vessels secure it[10]. The blood can nourish the fetus, qi deficiency will lead to blood stasis, blood stasis is not only the pathogenic factor, but also the pathological product of URM[11]. The method of invigorating the kidney and activating blood has become a research hotspot in the treatment of this disease. Through regulating the function of the hypothalamus-pituitary-ovarian axis, regulating reproductive immunity, and promoting microcirculation, it can improve the blood supply of the placenta[12]. However, there are few studies about the treatment of URM with suppressed internal heat. In view of this, we explored the correlation between the changes in Th1 cells, Th2 cells, T lymphocytes, NK cells and coagulation function in the peripheral blood of URM with suppressed internal heat and the intervention effect of the heat clearing heat and stasis resolving method.

2 Data and methods

2.1 Source of casesWe selected 30 URM patients, aged 25-38 and with an average of (28.27±3.51) years old, treated at the Affiliated Hospital of Gansu University of Chinese Medicine and the Reproductive Center of the Second Hospital of Lanzhou University from March 2015 to March 2018, and the number of miscarriages was 2 to 4 times. At the same time, we collected 30 normal female patients, aged 20-40, with an average of (27.25±5.89) years old, ≥1 normal birth history, no history of miscarriage, normal menstrual cycle, and no history of thrombotic diseases.

2.2 Diagnostic criteria for recurrent miscarriageWith reference toObstetricsandGynecologychiefly edited by Xie Xing[13], more than two times of continuous miscarriage, but without chromosomes, infections, endocrine factors, abnormal uterine anatomy, antiphospholipid antibody syndrome and other autoimmune diseases.

2.3 Criteria for judging the physique of suppressed internal heatWith reference to the "criteria for determination of suppressed internal heat physique" inClassificationandDeterminationofPhysiqueofTraditionalChineseMedicine[14]formulated by the China Association of Chinese Medicine in 2009: bitterness in the mouth and dry throat, vexation, dark red tongue, or with ecchymosis, yellow slimy tongue fur, thready and slippery pulse or sunken pulse.

2.4 Inclusion criteria(i) Women aged 20-40 who are married or have a sexual history; (ii) patients who meet the western medicine diagnostic criteria for URM; (iii) patients who meet the TCM diagnostic criteria for suppressed internal heat physique; (iv) human chorionic gonadotropin β (β-HCG)<5 IU/L and contraception during medication; (v) patients who have not received any treatment recently and signed informed consent to participate voluntarily and meet the requirements of medical ethics.

2.5 Exclusion criteria(i) Those who do not meet the criteria for inclusion; (ii) those with clear cause for recurrent miscarriage; (iii) those with mental illness, or liver and kidney insufficiency; (iv) those who have been taking other drug treatment before seeking medical advice; (v) those who cannot complete the treatment course.

2.6 Treatment methodsThe heat clearing and stasis resolving prescription. 15 g of Pinelliae Rhizoma Praeparatum, 12 g of wine processed Scutellariae Radix, 9 g of Coptidis Rhizoma, 12 g of Citri Reticulatae Pericarpium, 15 g of Angelicae Sinensis Radix, 18 g of Spatholobi Caulis, 30 g of Salviae Miltiorrhizae Radix Et Rhizoma, 10 g of Curcumae Rhizoma, 30 g of Astragali Radix, 15 g of Eupatorii Herba, 15 g of Cuscutae Semen, 15 g of salt processed Eucommiae Cortex, 15 g of Plantaginis Semen, 30 g of stir-fried Atractylodis Rhizoma, and 30 g of stir-fried Coicis Semen. One dose daily, decocted with water in the morning and evening. According to the symptoms, added 15 g of Ziziphi Spinosae Semen and 12 g of Poria Cum Radix Pini for insomnia; for those with fallopian tubes not smooth, added 15 g of Liquidambaris Fructus, 15 g of Gleditsiae Spina, and 6 g of Pangolin Scales (taking with water). All Chinese medicines were obtained from the Chinese Pharmacy of the Affiliated Hospital of Gansu University of Chinese Medicine, and the 3 months were used as a course of treatment.

2.7 Detection indicators and methods

2.7.1Comparison of TCM symptom scores. The clinical symptoms of the two groups of patients before and after treatment: primary symptoms (sore and weak waist and knees, phlegm in throat, depressed complexion, fat tongue, white and slimy tongue fur); according to no, mild, moderate, and severe symptoms, 0, 2, 4, and 6 points were scored separately; secondary symptoms (dizziness, lethargy, dull complexion, decreased libido, chills and cold limbs); according to no, mild, moderate, and severe symptoms, 0, 1, 2, and 3 points were scored separately.

2.7.2Serum coagulation function test. Before and after treatment, blood was taken on an empty stomach in the morning, and serum fibrinogen (FIB), D-dimer (D-D) and fibrin degradation products (FDP) were detected by turbidimetry.

2.7.3Detection of serum IL-6, IL-10 and TNF-α levels in peripheral blood. Blood was taken on an empty stomach in the morning, the blood samples of patients were placed in sterile test tubes and stored at 2-8 ℃ for 6 h. After coagulation at room temperature, centrifuged with a centrifugation radius of 16 cm at 3 000 r/min for 10 min, collected at least 300 UL of serum, and detected by chemiluminescence. Added IL-6 (concentrations of 1 000, 4 000 and 8 000 pg/mL, respectively), mixed with the sample at a ratio of 1∶10. The experiment was carried out with the program of Interleukin-6 of Siemens Immulite 2000 immunoassay system. The rest of the detection procedures were similar. Kits used in the experiment: interleukin-6 assay kit [Siemens Immulite 2 000 immunoassay system, batch No.:10381452 (L2K6P2)], interleukin-10 assay kit (Siemens Immulite 1 000 immunoassay system, batch No.:10381383 (LKXP1)), and TNF-α assay kit [Siemens Immulite 1 000 immunoassay system, batch No.:10381417 (LKNF1)].

3 Results and analysis

3.1 Comparison of TCM symptom scores of URM patients before and after treatmentCompared with the score before treatment (36.57±2.68) points, the score of TCM syndrome in the score (31.28±2.95) points after treatment was significantly decreased (P<0.05).

3.2 Comparison of FDP, D-D and FIB contents in URM patients before and after treatmentFrom Table 1, it can be seen that compared with the normal group, the D-D content of URM patients was significantly increased before treatment (P<0.05); compared with that before treatment in this group, the D-D content of URM patients was significantly decreased after treatment (P<0.05).

Table 1 Comparison of coagulation indicators in each group

Table 2 Comparison of serum levels of IL-6, IL-10, TNF-α, and in each group n=30)

Fig.1 Correlation between TCM symptom scores and peripheral blood inflammatory factors in 30 URM patients

Table 3 Comparison of changes in serum T lymphocytes in URM group after treatment n)

4 Discussion

Angiogenesis and good blood supply to the embryo are key factors for early embryo implantation and successful pregnancy[15-16]. The occurrence of unexplained recurrent spontaneous abortion (URSA) is related to the insufficient progesterone concentration resulted from the imbalance of the ratio of Th1 cells (IL-2, IL-12, IFN-γ and TNF-β/α and other cytokines)/Th2 cells (IL-4, IL-6 and IL-10,etc.)[17]. In particular, the overexpression of Th1 cytokines will activate cytotoxic T cells and NK cells,etc., mediate the apoptotic damage of trophoblast cells and the generation of maternal hypercoagulability (thrombophilia), ultimately leading to the occurrence of abortion[18]. Some studies[19-20]have confirmed that the prethrombotic state markers Fbg, D-D, BPC, and PAg T in the plasma of URM patients were significantly higher than those of the normal group, the anticoagulation and fibrinolytic system functions are out of balance in the uteroplacental tissue[21]. The formation of tiny thrombus causes hypoperfusion of placental blood flow, ischemia and hypoxia of the embryo or fetus, and eventually leads to poor fetal development and miscarriage[1]. A commonly used and meaningful indicator for monitoring thrombophilia is the determination of D-D level (a specific and sensitive serum marker of hyperfibrinolysis system, which can reflect the hypercoagulable state of blood)[22-23].

According to the TCM theory[24], repeated abortion consumes much qi and damages the blood, and deficiency of qi leads to failure to transport blood, then leading to stasis of the uterus, disharmony of the thoroughfare and conception vessels. If the blood stasis is not resolved, new blood will not be generated, and fetal vitality will not be secure. Such cycle goes back and forth, and the disease belongs to deficiency in origin and excess in superficiality. Over time, there will become phlegm. Suppressed phlegm-dampness physique is generally accompanied with weak immune functions, and imbalance of cellular and humoral immunity. If the foreign body (antibody) cannot be removed in time, the antibody activates the endogenous coagulation system and produces a large amount of fibrinogen degradation products AFP and D-D[25], resulting in the formation of microthrombosis, that is, "stasis".

Banxia Xiexin Decoction is used to treat the stuffiness with cold-heat complex, spleen-stomach weakness, spleen ascending and liver descending, and disorder of rise and fall of yin and yang, suppressed heat-dampness, and not smooth movement of qi. Pinelliae Rhizoma Praeparatum has functions of normalizing stomach by checking the upward perverted qi flow; Coptidis Rhizoma has functions of clearing the upper body heat; the whole prescription has both functions of warming and clearing, supplementation and draining, dispelling the cold and clearing the heat, realizing upper and lower harmony, and restoring the ascending and descending. On this basis, it is able to add drugs of tonify the kidney, activating the blood, and removing the dampness. Angelicae Sinensis Radix, Salviae Miltiorrhizae Radix Et Rhizoma, Spatholobi Caulis, and Curcumae Rhizoma have functions of nourishing and activating blood, enter the liver meridian to regulate blood vessels. Modern pharmacological studies have found that Angelicae Sinensis Radix, Salviae Miltiorrhizae Radix Et Rhizoma and other blood-activating traditional Chinese medicines have functions of anticoagulation, inhibiting platelet aggregation, preventing platelet activation, inhibiting thrombosis and improving adverse pregnancy outcomes[26-28]. Eupatorii Herba, stir-fried Atractylodis Rhizoma, and stir-fried Coicis Semen have functions of invigorating the spleen and removing dampness; Astragali Radix can strengthen the middle and boost qi, and improve the body immunity; Cuscutae Semen, Eucommiae Cortex, and Plantaginis Semen have functions of tonifying the kidney, especially stir-fried Atractylodis Rhizoma can remove the dampness and invigorate the spleen. This prescription combines the functions of dispelling the dampness and activating the blood, and tonifying the kidney, and follows the basic treatment principle of reinforcing the healthy qi and eliminating the pathogenic factors, regulating the yin and yang, benefiting qi and tonifying the deficiency, so that it can improve and regulate the body immunity and realizing the objective of treating both principal and secondary aspect of disease, and it also takes into account the menstrual cycle of patients with reference to the TCM sequential therapy[29].

In summary, the causes of recurrent miscarriage are complex. The study results indicate that abnormal expression of TNF-α, IL-6 and IL-10 in peripheral blood may play an important role in the occurrence of URM. The method of clearing the heat and resolving the stasis may play an important role in regulating the immune balance of the body and improving the microcoagulation state of blood vessels.