Inhibition of JAK3/STAT3 Activation in Rats Uterine Leiomyoma by formula of Taohong Siwu Decoction: A Randomized Controlled Trial

18 Background: Traditional Chinese medicine (TCM) formula of Taohong Siwu decoction (THSW) is 19 often used in traditional Chinese medicine for the treatment of uterine leiomyoma (UL). The 20 effectiveness of THSW for the treatment of UL has been confirmed in previous studies. At present, 21 there are few relevant mechanism studies. The purpose of this study is to explore the efficacy and 22 mechanism of THSW in the treatment of uterine leiomyoma. 23 Methods: A Sprague Dawley (SD) rat model of UL was established via estrogen and progesterone load 24 combined with external stimulation for 5 weeks. We randomly assigned adult female non-pregnant rats 25 into six groups: a control group, a UL model group, and a positive drug group,THSW(18g/ml) group, 26 THSW(9g/ml) group, THSW(4.5g/ml) group. THSW group and positive drug group were treated with 27 THSW medicinal solutions and gongliuxiao capsule medicinal solutions respectively after daily 28 modeling for 3 weeks. Histological analyses, Polymerase Chain Reaction and western blotting were 29 performed to evaluate the effect of THSW on UL and elucidate its mechanism of action. 30 Results: model group, the pathological changes of UL were relieved in the THSW group. Our data showed that 33 the treatment of rats with THSW significantly reduced the expression of cytotoxic T lymphocyte 34 associated antign 4 (CTLA4) and indoleamine 2, 3-dioxygenase 1 (INDO). The phosphorylation of 35 JAK3 and STAT3 which are the main signal transduction molecule of cytokines and growth 36 factorswere were also inhibited. 37 Conclusions: Our results suggest that THSW is effective in the prevention and treatment of UL in rats, 38 and THSW may exert its functions by inhibiting the activation of Tumor-related signal transduction 39 pathway (JAK3/STAT3), immune escape, proliferation of tumor cell and improving apoptosis.


42
Uterine leiomyoma, also known as uterine fibroma, is the most common benign tumors in women of shown that women with UL are usually more likely than normal women to feel depressed and restless, 49 and have a higher risk of depression [5], which seriously affects women's physical and mental health.

50
The global economic expenditure caused by UL is about $5.9-34.4 billion annually [6]. UL have 51 become a problem related to health and socio-economic all over the world. Due to the lack of 52 understanding of the underlying pathobiology, there is currently no fine medical treatment [7-9].The 53 current medical treatment of UL is mainly divided into surgical resection and drug treatment. Currently, 54 the mainstream theory believes that UL is a hormone-dependent tumor. Therefore, the medical 55 treatment of UL mainly is inhibiting steroid hormones at present, which has certain effect, but once the 56 use is stopped, there is a high tendency of recurrence. About three quarters of UL are treated with 57 hysterectomy [10]. Studies have shown [11] that there is~28% risk of surgical complications. In some 58 studies [12], the recurrence rate of surgery was more than 62%.In the United States, the lifetime risk of 59 myomectomy is 45% higher [13]. Therefore, the conventional Western medicine has the characteristics 60 of high recurrence rate, a lot of side effects and many complications at present. In fact, only 10% to 61 20% of UL patients need surgery. Therefore, it is important to find an effective and safe treatment for 3 [16] It is a key mediator in regulating cells of metabolism and development and immune response 74 [17,18]. In addition, JAK3 can affect the occurrence and development of diseases through various ways.

75
The overexpression of JAK3 can induce vascular smooth muscle proliferation [19]. Sustained 76 activation of JAK/STAT signal leads to increased expression of MMP [20]. These pathways are closely 77 related to the formation of UL. 78 STAT3 is an important part of signal transduction and transcriptional activation, which plays an 79 important role in the process of apoptosis and angiogenesis. It can mediate the interactive dialogue 80 between tumor cells and immune cells, and is related to tumor invasion, metastasis, and immune escape.

81
STAT3 plays a key role in tumor, including inhibition of apoptosis, alteration of cell cycle, and 82 mediating angiogenesis and immunosuppression. Studies have shown that STAT3 can inhibit 83 anti-tumor immunity by up-regulating apoptosis suppressor genes. STAT3 can also inhibit the 84 pro-inflammatory cytokines produced by tumor cells, contribute to immune escape and promote the 85 occurrence of tumor [21]. Continuous activation of STAT3 can promote tumor neovascularization [4,22]

121
After the mixture was mixed and filtered, the solution was concentrated to 18g/ml,9g/ml,4.5g/ml 122 separately and stored at 4℃.The medicine in gongliuxiao capsule (Shandong step size Shenzhou 123 Pharmaceutical Co., Ltd, z20055635) was poured into the mortar, ground thoroughly, dissolved with 124 appropriate amount of ultrapure water, and mixed evenly to obtain 0.09g/ml medicine liquid, which 125 was put in the refrigerator at 4 ℃ for standby. At the end of the experiment, the rats were sacrificed after an overnight fast, the rats were anesthetized 158 by 2% sodium pentobarbital (0.25 mL /100 g). We stripped the uterus, removed the surface adipose 159 tissue, removed the blood with normal saline, and then measured. We measure the transverse diameter 160 and diameter with vernier calipers. After measurement, we divided the uterus into three parts and put 161 them into the corresponding fixative.     Changes in the size of the uterus. Data are reported as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001 compared with control group; #P < 0.05, ##P < 0.01, ###P < 0.001 compared with model group (one-way ANOVA and LSD test).

Supplementary Files
This is a list of supplementary les associated with this preprint. Click to download.