Study on the mechanism of Haizao Yuhu Decoction in inhibiting proliferation of thyroid papillary carcinoma cells

Feng-Nuan Zhang, Tian-Shu Gao

1. Liaoning University of Traditional Chinese Medicine, Shenyang 110847, China

2. Affiliated Hospital of Liaoning University of Traditional Chinese Medicine, Shenyang 110000, China

Keywords:Haizao Yuhu Decoction Papillary thyroid carcinoma Cell proliferation

ABSTRACT Objective: To clarify the inhibitory mechanism of Haizao Yuhu Decoction on BRAFV600E mutation-driven papillary thyroid carcinoma cells. Methods: Prepare seaweed Yuhu Decoction medicated serum, select human normal thyroid cells Nthy-ori3-1 as the normal control group,and PLX4032 as the positive control drug. The experiment was divided into normal control group, model control group, PLX4032 group, Haizao Yuhu Decoction group (referred to as HYD group), Haizao Yuhu Decoction group + PLX4032 (referred to as HYD+PLX4032 group), and high iodine water group, at 8h and 24h respectively. At 72h, the cell proliferation of each group was detected by MTT method; at 24h, the expression of ERK and p-ERK protein in each group was detected by Western blot. Results: The longer the time, the more obvious the inhibitory effect of Haizao Yuhu Decoction-containing serum on the proliferation activity of BCPAP cells: Compared with the normal control group, the BCPAP cell proliferation activity of the model control group was significantly enhanced at 24h and 72h (P ＜0.05); At 24h, the PLX4032 group, HYD group, and HYD+PLX4032 group all showed a tendency to inhibit the proliferation of BCPAP cells, but there was no statistical difference; at 72h, compared with the model control group, the PLX4032 group The cell proliferation activity of the HYD group and HYD+PLX4032 group was significantly inhibited (P ＜0.05), and there was no difference between the high iodine water group and the model control group (P＞0.05). In inhibiting the proliferation of BCPAP cells, the medicated serum of Shanghai Zaoyuhu Decoction has a synergistic effect with PLX4032 (F=10.87, P=0.005). Western blot results showed that there was no difference in the expression of ERK1/2 protein between the groups, but there were significant differences in the expression of p-ERK1/2 protein between the groups: Compared with the normal control group, the expression of p-ERK1/2 protein in the model group increased significantly (P ＜ 0.05); Compared with the model control group, the expression of p-ERK1/2 protein in the PLX4032 group, the HYD group, and the HYD+PLX4032 group decreased significantly (P ＜0.05), and there was no difference between the high iodine water group and the model control group (P＞ 0.05). Conclusion: The medicated serum of Haizao Yuhu Decoction has an inhibitory effect on the proliferation of BCPAP cells, and its mechanism may be inhibiting the proliferation of BCPAP cells by inhibiting ERK1/2 protein phosphorylation, a protein post-translational modification process; the medicated serum of Haizao Yuhu Decoction may have Help enhance the effect of PLX4032 curative effect.✉Corresponding author:GAO Tian-shu, M.D., Chief Physician.

1. Introduction

Papillary thyroid carcinoma (PTC) belongs to differentiated thyroid cancer (DTC) and is the most common histological type of thyroid cancer (TC), accounting for about 80-80% of all thyroid malignancies. 90% [1], most DTCs respond well to surgery, 131I residual nail ablation and TSH suppression therapy, and can survive for a long time, with a 5-year survival rate of 98% [2]. However,about 23% of DTC patients will develop distant metastasis, and about 1/3 of them will gradually show a decline in the ability to gather iodine in their natural history or during treatment, are

insensitive to radioactive 131I treatment, and develop into radioactive iodine-refractory differentiated type Thyroid cancer [3] (Radioiodine refractory differentiated thyroid cancer, RAIR-DTC) is a difficult clinical treatment, and the 10-year survival rate has dropped to 10%[4]. BRAFV600E gene mutation is the most common mutation site of PTC and is one of the important molecular basis for the formation of RAIR-DTC [5,6]. BRAF is one of the key activators of the mitogenactivated protein kinase (MAPK) pathway. The amino acid at position 600 on exon 15 of the BRAFV600E gene, valine (V), is mutated to glutamic acid ( E), leading to sustained activation of the MAPK pathway, stimulating PTC cell proliferation and RAIR-DTC formation [7].

Thyroid cancer belongs to the category of "stone gall" in traditional Chinese medicine. In "Three Causes, Extremes and One Disease Syndrome", "the hard and immovable person is called stone gall.""Surgical Authenticity" records that "the disease of the wife's gall tumor is not a swelling of yin and yang. It is caused by blood stasis,turbid qi, and phlegm stagnation in the five internal organs." The"Golden Mirror of Medicine" in the Qing Dynasty said that "Shiying Seaweed Yuhu Decoction is the main one." ", more than 400 years ago, is a famous prescription for the treatment of gall tumors.Seaweed Yuhu Soup is composed of seaweed, Fritillaria, tangerine peel, kelp, green peel, Chuanxiong, Angelica, Pinellia, Forsythia,Licorice, Duhuo, and kelp.

Studies have shown that the addition of Haizao Yuhu Decoction on the basis of TSH inhibitory therapy after PTC can effectively improve thyroid function and reduce Tg[8]; Haizao Yuhu Decoction can significantly inhibit the chemokine receptors of experimental TC tumor-bearing mice CXCR4 gene and protein expression slowed tumor progression [9]. Haizao Yuhu Decoction can also inhibit the proliferation of experimental liver cancer by affecting the cell cycle and inducing apoptosis [10], and by reducing whole blood viscosity and erythrocyte aggregation in thymic lymphoma mice, alleviating the signs of phlegm coagulation and blood stasis, and inhibiting tumor progression [11]. The above studies all suggest that the seaweed Yuhu decoction may play an anti-cancer effect from many aspects. The previous research of our team showed that Haizao Yuhu Decoction and its disassembled recipes can reduce oxidative stress in thyroid tissue of experimental low-iodine goiter rats[12, 13], promote NIS protein expression[14], and iodine-rich traditional Chinese medicine and inorganic iodine There are differences [15]. However,there are few researches on the application of seaweed Yuhu decoction in PTC at home and abroad, so this study was carried out.In this study, the most common oncogene in PTC, the PTC cell line BCPAP cells driven by BRAFV600E mutation, was selected as the research object.

2. Materials and methods

2.1 Cell lines and experimental animals

2.1.1 Cell line

Human papillary thyroid carcinoma cells BCPAP and human normal thyroid cells Nthy-ori3-1 were purchased from Shanghai Cell Bank, Chinese Academy of Sciences. Culture in 1640 medium containing 10% fetal bovine serum, 37℃, 5% CO2 incubator.

2.1.2 Experimental animals

Forty healthy female SPF SD rats, 6-8 weeks old, weighing 190-220 g, animal certificate number: SCXK (Liao) 2015-0001, were purchased from Liaoning Changsheng Biotechnology Co., Ltd.

2.2 Drugs and Reagents

Preparation of Haizao Yuhu Decoction: seaweed 15g, kelp 15g,kelp 7.5g, Zhe Fritillaria 15g, Pinellia 15g, Qingpi 15g, tangerine peel 15g, Chuanxiong 15g, Angelica 15g, Forsythia 15g, Licorice 15g, Duhuo 15g. It was purchased from the Chinese Pharmacy of the Affiliated Hospital of Liaoning University of Traditional Chinese Medicine. The above medicines were soaked in water for 2 hours,decocted for 3 times, each decocted for 40 minutes, combined with the three decocting liquids, concentrated to 2g/ml containing crude drug, and stored in a refrigerator at 4℃ for later use.

1640 Medium (Cat. No. 31800-014), purchased from Gibco,USA; Fetal Bovine Serum (Cat. No.: 04-001-1ACS), purchased from BI, USA; BRAFV600E Inhibitor PLX4032 (Cat. No.: HY-12057), purchased from America MCE Company; KIO3 (Cat. No.:P116291), purchased from Aladdin, China; Extracellular regulated protein kinase (ERK, Cat. No.: WL02195), purchased from Wanleibio, China; Phosphorylated extracellular regulated protein kinase (p-ERK, Cat. No.: WLP1512 ), purchased from wanleibio,China; BCA protein concentration assay kit (item: WLA004),purchased from wanleibio; ECL luminescent solution (item:WLA003), purchased from wanleibio; PVDF membrane (item:IPVH00010), purchased from the United States Millipore Company;MTT (Item No.: KGA311), purchased from KGI, USA.

2.3 Preparation of medicated serum

40 healthy female SD rats were randomly divided into traditional Chinese medicine group and blank control group, 20 rats in each group. The traditional Chinese medicine group was given the dosage converted according to the body surface area ratio, the crude drug content was 15.5g/Kg, twice a day, and the blank control group was given the same volume of deionized water. Consecutive gavage for 1 week, 1 hour after the last administration, routine anesthesia, blood collection from the abdominal aorta, serum separation at 3000 r/min, mixed with the same group of samples, sterilized by filtration through a 0.22 μm filter, and inactivated at 56℃ for 30-40 min , -80℃ storage.

2.4 Main instruments and equipment

Inverted phase contrast microscope (AE31); microplate reader(ELX-800); double vertical protein electrophoresis instrument(DYCZ-24DN); transfer tank (DYCZ-40D); gel imaging system(WD-9413B).

2.5 Experimental grouping and interventions

In vitro experiments were divided into 6 groups, A: normal control group (human normal thyroid cells Nthy-ori3-1 + blank serum); B model control group (BCPAP cells + blank serum); C: PLX4032 group (BCPAP cells + blank serum + 0.1 μM PLX4032[16]); D:HYD group (BCPAP cells + 20% HYD-containing serum); E:HYD+PLX4032 group (BCPAP cells + 20% HYD-containing serum+ 0.1 μM PLX4032); F: high iodine Water group (BCPAP cells +blank serum + 13.4 μg/mL KIO3).

2.6 MTT assay to detect cell proliferation ability

Nthy-ori3-1 and BCPAP cells in logarithmic growth phase were taken, and after trypsin digestion, the cell density was adjusted to 2×106cells/ml, inoculated in 96-well plates, and treated with drugs according to the experimental groups. Each group was designed with 5 replicates. After inoculation, place the cells in a 37℃, 5% CO2 incubator for 8h, 24h, and 72 hours, add 50 μl 1×MTT, incubate for 4h in a 37℃ incubator, remove the supernatant, add 150 μl The purple crystals formed by lysing the cells with DMSO were measured on a microplate reader at OD at 570 nm.

2.7 Western blot detection of ERK and pERK

Total protein was extracted from the lysate, protein quantification was performed by BCA method, 20 μl of protein sample was added to SDS-PAGE gel for electrophoresis separation, the protein was transferred to PDVF membrane, blocked with 5% nonfat milk powder for 1 h, primary antibody, 4℃ overnight. Rinse 4 times in TBST, add secondary antibody and incubate for 45 min at room temperature, after rinsing 6 times in TBST, add ECL developer dropwise, and analyze the optical density value of the target band with a gel image processing system.

2.8 Statistical methods

The obtained data were expressed as±s, and were analyzed using SPSS 26.0 statistical software. The comparison between groups was performed by one-way one-way ANOVA, and P＜0.05 was regarded as statistically significant.

3. Results

3.1 Effect of Haizao Yuhu Decoction-containing serum on the proliferation activity of BCPAP cells

The results of MTT assay showed that the inhibitory effect of Haizao Yuhu Decoction-containing serum on the proliferation of BCPAP cells gradually appeared over time. There was no difference in proliferation activity among the groups at 8h; compared with the normal control group, the proliferation activity of BCPAP cells in the model control group was significantly enhanced at 24h and 72h (P ＜ 0.05); at 24h, the PLX4032 group, 20%HYD group, HYD+PLX4032 Compared with the model control group,the cell proliferation activity of the PLX4032 group, 20%HYD group and HYD+PLX4032 group was significantly inhibited at 72 h (P ＜ Compared with the high iodine water group, the cell proliferation activity of the PLX4032 group, 20%HYD group, and PLX4032+20%HYD group was significantly inhibited (P ＜0.05). ),see Table 1, and the trend of proliferation activity of different groups over time is shown in Figure 1

Table 1 Effects of Haizao Yuhu Decoction-containing serum on the proliferation activity of BCPAP cells (n=5,±s)

Table 1 Effects of Haizao Yuhu Decoction-containing serum on the proliferation activity of BCPAP cells (n=5,±s)

Note: Compared with normal control group: *P ＜0.05; compared with model control group: △ P ＜0.05; compared with high iodine water group: #P ＜0.05

group Cell proliferation activity (OD value)8h 24h 72h normal control group 0.186±0.031 0.252±0.032 0.435±0.080 model control group 0.216±0.031 0.333±0.031* 0.638±0.078*PLX4032 group 0.206±0.023 0.292±0.036 0.407±0.067△#20% HYD group 0.207±0.021 0.288±0.033 0.387±0.045△#HYD+PLX4032 group 0.195±0.022 0.265±0.030 0.328±0.032△#High iodine water group 0.211±0.036 0.315±0.037 0.599±0.046*F 0.793 4.118 20.87 P 0.565 0.008 0.000

Figure 1 The effect of Haizao Yuhu Decoction-containing serum on the proliferation activity of BCPAP cells

Table 2 BCPAP cell proliferation factorial design analysis of variance (±s)

Table 2 BCPAP cell proliferation factorial design analysis of variance (±s)

Intervening factors Proliferative activity F P HYD 0.387±0.045 40.01 0.000 PLX4032 0.407±0.067 30.90 0.000 HYD*PLX4032 0.328±0.032 10.87 0.005

3.2 Effects of Haizao Yuhu Decoction-containing serum on the expression of ERK1/2 and p-ERK1/2 proteins in BCPAP cells

Western Blot results showed that there was no difference in the expression of ERK1/2 protein among the groups, but there were significant differences in the expression of p-ERK1/2 protein among the groups: compared with the normal control group, the expression of p-ERK1/2 protein in the model group was significantly increased(P ＜ Compared with the model control group, the expression of p-ERK1/2 protein in the PLX4032 group, 20%HYD group, and HYD+PLX4032 group was significantly decreased (P ＜ 0.05), and there was no difference between the high iodine water group and the model control group (P ＞ 0.05). ), Figure 2(1) is the Western Blot band, Figure 2(2) is the histogram of the grayscale analysis of the bands, and Table 3 is the corresponding statistical table.

Figure 2 Effects of Haizao Yuhu Decoction-containing serum on the expression of ERK and p-ERK proteins in BCPAP cells

4. Discussion

Haizao Yuhu Decoction is a famous prescription for treating gall tumors, and it has been shown to inhibit cancer cell proliferation and delay tumor progression in PTC and various cancers [7-10]. In this study, the PTC cell line BCPAP cells with the BRAFV600E mutation genetic background were selected as the research objects,and the drug-containing serum was used for in vitro intervention.The traditional Chinese medicine compound is added to the cells in the form of drug-containing serum, which cleverly utilizes the metabolism and filtration of the complex components of the traditional Chinese medicine by the organism, which can reflect the real situation of the medicinal effect of the traditional Chinese medicine through digestion and absorption in the body [17, 18].20% Haizao Yuhu Decoction containing serum was selected, and the inhibition study of Haizao Yuhu Decoction on BCPAP cell proliferation was carried out at 8h, 24h and 72h as observation time points. PLX4032 was selected as the positive control drug.PLX4032, also known as Vemurafenib, is a B-Raf enzyme inhibitor.Haizao Yuhu Decoction is a compound of iodine-rich traditional Chinese medicine, and seaweed, kelp, and kelp are iodine-rich traditional Chinese medicines[19, 20]. Therefore, in this study, a KIO3 water group with the same iodine content as Haizao Yuhu Decoction was set as a control for interpretation. The difference between the effect of Haizao Yuhu Decoction compound and simple iodine.

Extracellular signal-regulated kinases (ERKs) are divided into ERK1 and ERK2, which are the final effectors of the MAPK signaling pathway and are mainly involved in cell growth and differentiation. The classical ERK activation pathway is composed of receptor tyrosine kinases bound to the cell membrane by different stimulatory ligands outside the cell, activating GTP protein,sequentially activating RAF and MEK, and finally phosphorylating ERK protein to promote its activation to p-ERK, which in turn facilitates the activation of p-ERK. It is located in the nucleus,phosphorylates transcription factors in the nucleus, and promotes cell growth and differentiation. BRAFV600E mutation, sustained activation of Raf-MEK-ERK signaling pathway, stimulates cell proliferation and metastasis, and plays a key role in cancer development [21].

This study showed that the proliferation activity of BCPAP cells was significantly higher than that of normal thyroid cells. After the intervention of Haizao Yuhu Decoction-containing serum, the proliferation activity of BCPAP cells was significantly inhibited.Analysis of ERK protein expression on the MAPK pathway showed that there was no difference in ERK protein expression between BCPAP cells and normal thyroid cells, while p-ERK protein expression increased significantly. But it is still higher than the normal thyroid cell group, and its efficacy is comparable to that of PLX4032. It is suggested that the effect of Haizao Yuhu Decoction on inhibiting the proliferation of BCPAP cells may be achieved by inhibiting the phosphorylation of ERK protein. Protein phosphorylation is the most basic and important mechanism for regulating protein activity and function, and plays a crucial role in cell signal transduction [22]. Studies have shown that quercetin from seaweed and forsythia, tangerine peel from green peel and tangerine peel, and baicalein from Pinellia are the important active components of seaweed Yuhu decoction, and have strong correlation with PTC core targets. The binding force may have a direct therapeutic effect on PTC [23]. Quercetin can increase the phosphorylation levels of p-MAPK and p-ERK1/2 in H9C2 cells [24],and Raf and MEK protein kinases are the direct molecular targets of quercetin's chemopreventive effect [25]. Baicalein can significantly reduce the p-ERK/ERK ratio of anaplastic thyroid cancer cells [26].The combination of Chuantheretin and cisplatin can effectively reduce the viability of anaplastic thyroid cancer cells [27], and has a strong synergistic effect with sorafenib [28]. The combination of Haizao Yuhu Decoction and PLX4032 has a synergistic effect.Whether Haizao Yuhu Decoction can alleviate the drug resistance of PLX4032 is worth further study.

Haizao Yuhu Decoction is an iodine-rich traditional Chinese medicine compound [19, 20]. The effect of iodine on thyroid cancer has been controversial for a long time. Most studies have shown that iodine deficiency is an initiating and promoting factor of thyroid tumors [29], and correction of iodine deficiency may shift thyroid cancer subtypes in a direction of less malignancy [30, 31], but does not affect thyroid cancer overall. risk [32]. There is insufficient evidence for iodine excess as a risk factor for thyroid cancer [33], limited case studies suggest that increased iodine intake is a protective factor for thyroid cancer [34], and iodine-rich foods may have a protective effect on the risk of thyroid cancer [32] , 35]. The results of this study showed that the high iodine water group with the same iodine content as Haizao Yuhu Decoction did not inhibit BCPAP cell proliferation and ERK phosphorylation, suggesting that the inhibition of BCPAP cell proliferation by Haizao Yuhu Decoction-containing serum may be related to the enrichment of BCPAP cells. Iodine was irrelevant; the high-iodine water group showed a similar proliferation state to the model group, and no excessive proliferation occurred, which to a certain extent supported that the dose of iodine contained in Haizao Yuhu Decoction was not a risk factor for thyroid cancer.

5. Conclusion

The medicated serum of Haizao Yuhu Decoction has an inhibitory effect on the proliferation of BCPAP cells, and the mechanism may be to inhibit the proliferation of BCPAP cells by inhibiting the post-translational modification process of ERK1/2 protein phosphorylation; the medicated serum of Haizao Yuhu Decoction may help enhance the The role of PLX4032 efficacy.

Author’s contribution

The experimental design is the first author and the corresponding author, the experimental implementation and writing are the first author, and the corresponding author is the reviewer.