Abstract

Graphical abstract

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1. Introduction

Premature ovarian failure (POF) refers to a gynecological endocrine disease, with symptoms that female's ovarian function declines ahead of time before 40 years old, followed by amenorrhea and sexual organatrophy, rising levels of follicle stimulating hormone (FSH) and luteinizing hormone (LH), and declining estrogen (E2).¹ Women with POF ovulate abnormally and are unable to conceive normally due to complex factors, including environmental influence, immunity and psychology. The incidence rate is about 1%~3%. As economy develops and life pace accelerates, women's childbearing age is gradually postponed, and the incidence rate of POF is rising year by year due to various influence from environment and diseases.² Only 5% of POF patients can naturally conceive and complete normal pregnancy.³ As POF develops, menopause will follow, and patients may lose normal endocrine function prematurely, resulting in premature depletion of primordial follicle pool.⁴ The treatment is difficult because of the fact that the etiology of POF is complex and the pathogenesis is unclear. Therefore, POF is recognized as one of the most tough gynecological diseases by the World Health Organization.

At present, the general treatment of POF using western medicine is hormone replacement therapy (HRT). Although HRT can alleviate partial symptoms, it doesn't work in restoring function and fertility of ovary, and may increase the risk of cancer on ovarian, breast and endometrial aspects, meningioma, thrombotic disease, etc.⁵ In comparison, traditional Chinese medicine has a long research history of effects on human body aging. It fundamentally regulates the endocrine function of hypothalamic-pituitary-ovarian reproductive axis and cures diseases from the root cause.⁶ Recent studies show that combination of apoptosis and oxidative stress forms a “vicious circle” in the process of POF. It involves various changes between molecules, among which oxidative stress may play an important role in the occurrence and development of POF. However, both compound chinese medicine and monomer of herbal medicine can significantly relieve the oxidative stress in the ovary, delay the apoptosis of ovarian granulosa cells, regulate the secretionfunction of ovarian reproductive hormone, and consequently improve POF situation.⁷ Ancient medical books such as “Compendium of Materia Medica” records that pearl has the effect of detoxification and muscle regeneration. The traditional Chinese medicine compatibility theory is used to analyze the compatibility law of pearl,⁸ and the network pharmacology method is used to study the pearl, sort out the interaction relationship of “medicine compound target gene disease”, explore the correlation of its component-target, component-disease and disease-cause, suggesting that the pearl has anti-oxidation, anti-inflammatory, endocrine regulation, astringent and myogenic effects, and has a good application prospect, It involves 192 diseases.⁹ Modern scholars also found that pearl extract has good effect on anti-oxidation and anti-aging in vitro and vivo, and can be used to treat various aging diseases.^10,11 The previous study by our group showed that pearl has great capacity to resist oxidation, alleviate oxidative stress and play an important role in the efficacy of “detoxification and muscle regeneration".^12,13

As recorded in the ancient books, “Baopuzi” told, pearl with size of one inchor larger, once taken, give people a better look and longer life span¹⁴; “Extrinsic Materia Medica” told, pearl removes facial spot; “Kaibao Materia Medica” told, pearl make face glossy with bright color".¹⁵ The “Pearl Pill”, stated in the “Taiping Shenghui recipe”, mainly consisting of 30g Pearl (grounded into extremely fine powder), are made to tonify primordial Qi, benefit essence, delight color, brighten ears and eyes, and can strengthen viscera function and calm mind. It is mainly used to treat “physical decline due to aging, cold limb, haggard face and uneasy mind".¹⁶ GuichengXia, a great master of national medicine in Nanjing University of traditional Chinese medicine, focused on application of pearl powder to treat early-onset ovarian insufficiency based on the theory of “heart-kidney-uterus axis"¹⁷; Professor ZhenyuTan's team, from the First Affiliated Hospital of Hunan University of traditional Chinese medicine, adopted “climacteric formula” which is composed of Chinese medicine calcined with mother of pearl, to treat menopausal syndrome (deficiency of kidney yin)¹⁸; Professor Lin Jiang in our research group is experienced in employing pearl compatibility to treat various syndromes caused by ovarian insufficiency in clinical practice, she found that pearl has a good effect on “abnormal uterine bleeding” in gynecology in addition to the above symptoms and signs, and has a good improvement on perimenopausal symptoms and osteoporosis.¹⁹ We wonder whether the above efficacy of pearl is related with its role in enhancing ovarian function. According to the literature review, there is no relevant experimental report. Therefore, we carried out the research on the effect of pearl on POF rats to fill the gap.

Traditional Chinese Medicine can significantly reduce the oxidative stress in the ovary and reduce the apoptosis of ovarian granulosa cells and oocytes, regulating the development of follicles, and reducing the incidence of POF.²⁰ Polycystic ovarian syndrome (PCOS) and POF have similar gynecological characteristics, and PCOS is a continuum of reproductive, endocrine and metabolic disorders and is characterized by suppressed granulosa cell proliferation, early antral follicle growth arrest, chronic anovulation, hyperandrogenemia, and insulin resistance.²¹ Increasing evidence has revealed that increased autophagy is associated with chronic inflammatory behavior in polycystic ovaries.²² Li et al. reported that 41 autophagy-related genes were differentially expressed in PCOS patients and animal models.²³ Therefore, Using the POF rat models induced by tripterygium glycosides, we compared the results of serum reproductive hormone, ovarian tissue structure, oxidative stress, and expression levels of apoptosis and autophagy protein before and after pearl intervention on POF rats, then preliminarily discussed the effect and mechanism of pearl on improving the ovarian function of rats with POF induced by tripterygium glycosides. It was found for the first time that pearl could improve POF situation of rats, which lay a foundation for further research.

2. Materials and methods

2.3. Grouping, POF modeling and administration of SD rats

68 female SD rats with regular estrous cycle were randomly distributed into normal (N) group (12 rats) and tripterygium glycosides group (56 rats). Referring to the methods of Fu et al.,²⁴ the rats in the tripterygium glycosides group were administered intragastrically with tripterygium glycosides at 40 mg kg⁻¹ per day for ten weeks, and the rats in the N group were given the same volume of normal saline simultaneously. After nine weeks of modeling, the cell changes of vaginal smears in the rats were observed. After ten weeks, the vagina mucosa cell smears showed that continuous disorder of estrous cycle occurred in 53 rats, characterized with prolongation and stagnation. The success rate of modeling was 94.6%; Finally, 48 POF rats with estrous cycle disorder were selected and divided into model (M) group, low, medium and high-dosepearlgroup (PL, PM and PH), with 12 rats in each group. The PL group, PM group and PH group were treated intragastrically with pearl powder at the doses of 185 mg kg⁻¹, 370 mg kg⁻¹ and 740 mg kg⁻¹ per day respectively for 30 consecutive days. The grouping, POF modeling and administration of SD rats is shown in Fig. 1. Each dose of pearl powder is given to rats by gavage with the same volume of normal saline. Among them, the medium dose of 370 mg kg⁻¹ was the equivalent dose for rats, which was calculated according to the clinical dosage (2.5 g d⁻¹ for adults) and the “conversion table of dosage between human and rat”. The N group and the M group were given the same volume of normal saline.

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Fig. 1

Thegrouping, POF modeling and administration of SD rats.

3. Results

3.1. Effect of pearl on estrous cycle of POF rats

Most of vagina mucosa cells proved to be oval nucleated epithelial cells in proestrus, lamellar anucleated keratinocytes in estrous stage, leukocyte in the estrous interval, and white blood cells, nucleated epithelial cells and lamellar keratinocytes in late estrus; see Fig. 2. After experiment, the recovery ratio of estrous cycle in the M group was 0, in comparison with 50% for PL group,58.33% for PM group, and 75% for PH group. It can be seen that pearl improved the estrous cycle of POF rats, and PH group behaved best in terms of the recovery effect. See Table 1.

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Fig. 2

Morphological changes of vagina mucosa cells at each stage of estrous cycle in rats ( × 200).

Table 1

Recovery ratio of estrous cycle of rats in each group.

Group	Total number of rats (n)	Normal cycle (n)	Disordered cycle (n)	Disordered ratio (%)	Recovery ratio (%)
N	12	12	0	0	0
M	12	0	12	100	0
PL	12	6	6	50	50
PM	12	7	5	41.67	58.33
PH	12	9	3	25	75

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3.2. Effect of pearl on tissue structure of ovary in POF rats

In the N group, growing follicles at all levels could be seen in the ovarian tissue with clear outline, and there were many layers of granulosa cells which were arranged densely around the periphery of follicles; Structural destruction, decreased follicles at all levels, increased atretic follicles, and no dominant follicles were shown in ovarian tissue of M group, which signified degenerative development. Compared with M group, the reduced ovarian tissue lesions in PL, PM and PH groups with increased follicles at all levels and decreased atretic follicles could be seen. The layers of granulosa cells increased and the structure was arranged regularly, and the development degree of follicle rised in a pearl dose-dependent manner. See Fig. 3 and Table 2.

Table 2

Number of follicles of ovary in each group.

Group	Total follicles(n)	Primary follicles(n)	Mature follicles
N	17.35 ± 3.07	4.97 ± 1.34	4.82 ± 0.69
M	5.17 ± 1.44	1.05 ± 0.45	0.71 ± 0.12
PL	11.08 ± 2.25#	3.01 ± 0.66#	2.15 ± 0.33#
PM	13.82 ± 2.97#	3.26 ± 1.02#	2.62 ± 0.45#
PH	16.91 ± 3.13#	4.30 ± 1.13#	3.21 ± 0.82#

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Note: Compared with the N group, P < 0.05; compared with the M group, ^#p < 0.05.

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Fig. 3

Effect of pearl on ovarian tissue structure of POF rats (HE, × 40), scale bar = 20 μm. Compared with M group, the ovarian tissue lesions in PL, PM and PH groups were reduced with increased follicles at all levels and decreased atretic follicles can be seen, and the development degree of follicle rised in a pearl dose-dependent manner.

3.3. Effect of pearl on reproductive hormone level in POF rats

Compared with N group, the contents of E2 and AMH in M group decreased significantly (P < 0.05). Compared with M group, the content of E2 in PH group increased significantly (P < 0.05), and the content of AMH in PM and PH groups increased significantly (P < 0.05). At the same time, there was no significant difference in the contents of E2 and AMH between PH and N group. See Fig. 4.

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Fig. 4

Effect of pearl on the contents of E2(A), AMH(B), FSH(C) and LH(D) in POF rats. Compared with M group, the content of E2 and AMH in PH group increased significantly (P < 0.05), and the content of LH and FSH in PH group decreased significantly (P < 0.05). Compared with the N group, P < 0.05; compared with the M group, ^#P < 0.05.

Compared with N group, the contents of FSH and LH in M group were significantly higher (P < 0.05). While compared with M group, the content of LH in PH group decreased significantly (P < 0.05), and the content of FSH in PM and PH groups decreased significantly (P < 0.05). At the same time, there was no significant difference in the contents of FSH and LH between PH group and N group. See Fig. 4.

3.4. Effect of pearl on oxidative stress in POF rats

Compared with N group, the contents of ROS and MDA in M group were significantly higher (P < 0.05). While compared with M group, the content of ROS in PL, PM and PH groups decreased significantly (P < 0.05), and the content of MDA in the PM and PH groups decreased significantly (P < 0.05). At the same time, there was no significant difference in the content of MDA between PH group and N group. See Fig. 5.

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Fig. 5

Effect of pearl on the contents of ROS(A), MDA(B), GSH(C) and the activities of SOD(D), GSH-Px(E) and CAT(F) in POF rats. Compared with M group, the content of ROS, MDA in the PM and PH groups decreased significantly (P < 0.05), the content of GSH and the activities of SOD, GSH-PX and CAT in PM and PH groups were significantly increased (P < 0.05). Compared with N group, P < 0.05; Compared with M group ^#p < 0.05.

Compared with N group, the content of GSH and the activities of SOD, GSH-PX and CAT in M group decreased significantly (P < 0.05). While compared with M group, the content of GSH and the activities of SOD, GSH-PX and CAT in PM and PH groups were significantly increased (P < 0.05). At the same time, there was no significant difference in the activities of GSH-PX and CAT between PH and N group. See Fig. 5.

3.5. Effect of pearl on the apoptotic protein expression of cleaved-caspase 3 and Bax in POF rats

Compared with N group, the apoptotic protein expressions of cleaved-caspase3 and Bax in M group increased significantly (P < 0.05). While compared with M group, the apoptotic protein expressions of cleaved-caspase3 and Bax in PL, PM and PH groups decreased significantly (P < 0.05). At the same time, there was no significant difference in the apoptotic protein expression of Bax between PH and N group. See Fig. 6 (A, B, C).

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Fig. 6

Effect of pearl on the expression of cleaved-caspase3 (A, B) and Bax (A, C) proteins in POF rat. Compared with M group, the apoptotic protein expressions of cleaved-caspase3 and Bax in PL, PM and PH groups decreased significantly (P < 0.05). Compared with N group, P < 0.05; Compared with M group ^#p < 0.05.

3.6. Effect of pearl on the phosphorylation level of MAPK signaling pathway in POF rats

Compared with N group, relative expression of p-ERK1/2,p-p38and p-JNK proteins in the ovary of rats in M group increased significantly (P < 0.05). While compared with M group, relative expression of p-ERK1/2, p-p38 and p-JNKproteins in the ovary of rats in PL, PM and PHgroups decreased significantly (P < 0.05). See Fig. 7 (A, B, C, D).

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Fig. 7

Effect of pearl on the relative expression of p-JNK (A, B), p-p38 (A, C), and p-ERK1/2 (A, D) proteins in POF rats. Compared with M group, relative expression of p-ERK1/2, p-p38, and p-JNK proteins in the ovary of rats in PL, PM and PH groups decreased significantly (p < 0.05). Compared with N group, P < 0.05; Compared with M group ^#p < 0.05.

3.7. Effect of pearl on the autophagy protein expression of LC3II, beclin-1 and p62 in POF rats

Compared with N group, the autophagy protein expressions of LC3II + I, LC3II, beclin-1 and p62 in M group decreased significantly (P < 0.05). Compared with M group, the autophagy protein expressions of LC3II + I, LC3II and beclin-1 in PM and PH groups were significantly increased (P < 0.05), the autophagy protein expression of p62 in PL, PM and PH groups were significantly increased (P < 0.05). See Fig. 8 (A, B, C, D, E, F).

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Fig. 8

Effect of pearl on the expression of beclin-1 (A, B), LC3II + I (A, C), LC3II (A, C) and p62 proteins in POF rats. Compared with M group, the autophagy protein expressions of LC3II + I, LC3II, beclin-1 and p62 in PM and PH groups were significantly increased (P < 0.05). Compared with N group, P < 0.05; Compared with M group ^#p < 0.05.

4. Discussion

Tripterygium glycoside is widely used clinically, since it has effects on anti-inflammatory, antibacterial, immunosuppressive, antitumor and other aspects, however it brings reproductive toxicity which lead to POF.²⁵ Synergistic effect of LH with FSH facilitates E2 secretion.²⁶ As the contents of FSH and LH increase, number of follicles and content of E2 will decrease, and the growth of follicles and ova will be restrained.²⁷ AMH is involved in the regulation of follicular development and serves as the best predictor of ovarian reserve function.²⁸ Reduced expression of AMH was also detected during normally premenopausal and aging processes in mice.^29,30 Fu et al. established rat POF models by administering rats intragastrically with quantitative tripterygium glycosides, and concluded the pathogenic pathway of rat POF was the same as that of humans.²⁴ In this experiment, POF rat models were replicated in the same way as above using tripterygium glycosides. The results showed disordered estrous cycle in the rat models, and the contents of FSH and LH increased, the contents of E2 and AMH decreased, follicles at all levels decreased, atretic follicles increased, and layers of granular cells decreased, suggesting that the modeling of POF rats was successful. The estrous cycle of rats in PL, PM and PH groups recovered in a high proportion, with growing follicles increased, and PH group behaved best in terms of the recovery effect. In PH group, the contents of serum E2 and AMH increased significantly, while the contents of LH and FSH decreased significantly, and the levels of above reproductive hormone tended to be normal. It can be seen that pearl can improve the estrous cycle and serum reproductive hormone level of POF rats, promote follicular development and effectively improve ovarian function.

Follicles are the material basis for maintaining ovarian function, so the development of follicles directly affects ovarian function. Under normal conditions, ROS oxidation in ovarian cells is in dynamic balance with endogenous antioxidant system, i.e., sufficient antioxidant GSH, and antioxidant enzymes including SOD, CAT and GSH-PX, etc. can remove most ROS.³¹ Under abnormal conditions, oxidative stress is induced in ovarian cells, and brings excessive ROS, which can attack liposomes in ovarian cells and produce MDA. Then MDA causes macromolecular cross-linking reaction and damages in ovarian tissue.³² Miyamoto reported that oxidative stress resulted in a significant reduction in the number of ovarian follicles and ovulating oocytes during ovulation.³³ This study found that the ovary of POF rats produced excessive ROS, and the content of GSH and the activities of antioxidant enzymes such as SOD, CAT and GSH-PX decreased, resulting in excessive accumulation of ROS and MDA, which further enhance oxidative stress and seriously damage the tissue structure. Medium and high doses of pearl can increase the content of GSH and the activities of SOD, CAT and GSH-PX in the ovary of POF rats, scavenge excessive ROS and MDA, alleviate oxidative stress and improve the situation of ovary structure. It turned out that the level of MDA in PH group tended to be normal. It can be seen that pearl can promote follicular development by alleviating oxidative stress after scavenging excessive ROS in the ovary of POF rats.

According to specific research of protein and micro element composition in pearl by Han et al.¹³ a total of 178 proteins were identified. These proteins included many antioxidant enzyme-related proteins, such as sigma classglutathione S-transferase, glutathione peroxidase, glutathi-one S-transferase A, superoxide dismutase, glutathione synthase, catalase, GST class pi, GST 2, peroxiredoxin 6, GST1, glutathione transferase, and superoxide dismutase [CuZn]. There were also some metal link-related proteins, such as metal binding protein, metal response element binding transcription factor-1, and metal response transcription factor 2, which may link metal elements in pearl to form metalloproteins. It is speculated that these proteins play a certain role in alleviating oxidative stress.

Under abnormal conditions, oxidative stress damages mitochondrial DNA, inhibits mitochondrial synthesis of ATP, restrains cell growth and accelerates cell apoptosis. In addition, oxidative stress facilitates mitochondria to release cytochrome C into the cytoplasm, activates caspase-3 and induces apoptosis.^34,35 Ovarian granulosa cell is one of the earliest cells during development of follicles. They surround the oocytes through gap junctions and play a decisive role in the development, maturation and atresia of follicles.³⁶ Previous studies show that ovarian granulosa cell is most sensitive to oxidative stress and prone to apoptosis, which is the root cause of follicular atresia. When the ratio of granulosa cells reaches 10%, it indicates that the follicle comes into atresia state, which will lead to POF.^{37, 38, 39} Caspase family protein activation is a typical biological feature of apoptosis. Cleaved-capase3, which can activate the transmission of apoptosis signal and destroy cell function, is the key protein to promote apoptosis.⁴⁰ Bax promotes the release of apoptosis inducing factors and cascades with caspase protein to induce apoptosis.⁴¹ This study found that the expression of apoptosis protein cleaved-caspase 3 and Bax in the ovary of POF rats increased significantly, which might promote the apoptosis of granulosa cells. Low, medium and high doses of pearl can significantly reduce the expression of apoptotic protein cleaved-caspase 3 and Bax in the ovary of POF rats, and high dose of pearl behaved best. It can be seen that pearl can promote follicular development by inhibiting apoptosis of granulosa cells after alleviating oxidative stress in the ovary ofPOF rats.

ROS is reported to play a key role in cell signaling, mainly in pathways of cell proliferation and apoptosis.⁴² Activation of mitogen-activated protein kinases (MAPKs) is known to play a major role in ROS-induced signaling pathways. It has been reported that increased ROS production in ovarian granulosa cells leads to the activation of MAPKs and cell apoptosis.⁴³ Three major MAPKs have been identified, including the c-Jun N-terminal kinase (JNK), the p38, and the extracellular signal-regulated kinases 1/2 (ERK1/2).⁴⁴ The results presented here show that POF could significantly upregulate expression of apoptosis protein cleaved-caspase 3 and Bax in the ovary of rats via activation of the MAPK signaling pathway, including ERK1/2, JNK and p38. Similar to our results, expression of apoptotic protein cleaved-caspase 3 and Bax increased through activation of AMPK signaling pathways in POF rats induced by -radiation in a study.⁴⁵ Nevertheless, treating POF rats with low, medium and high doses of pearl significantly decreased the phosphorylation of ERK1/2, JNK and p38, which resulted in the expression attenuation of apoptosis protein cleaved-caspase 3 and Bax in the ovary of rats,⁴³ and high dose of pearl behaved best. According to the results, we inferred that pearl could inhibit apoptosis of granulosa cells via suppression of MAPK signaling pathway after scavenging excess ROS in the ovary of POF rats.

Autophagy, as a double-edged sword, has the dual functions of “promoting survival” and “promoting death” of cells.⁴⁶ In most cases, oxidative stress activates autophagy as a negative feedback mechanism for cell protection against oxidative damage. However, high-intensity oxidative stress also inhibits autophagy, or excessive autophagy promotes the production of oxidative stress, both of which can lead to cell damage or death.⁴⁷ Microtubule-associated protein 3 (LC3) is one of the homologous genes of autophagy-related gene 8, which is mainly expressed in follicular granulosa cells and can reflect the expression level of follicular granulosa cell autophagy. Therefore, it is a marker protein of follicular granulosa cell autophagy.⁴⁸ Beclin-1 gene participates in the formation of early autophagosomes and promotes cell survival by maintaining autophagy level. It plays an important regulatory role in maintaining the life span of human luteal cells and ovarian estrogen secreting cells.⁴⁹ As a major autophagy receptor, p62 participates in the formation of autophagosomes and lysosomes to degrade harmful or unnecessary protein components in cells, and maintain the homeostasis of intracellular proteins.⁵⁰ Granulosa cell autophagy and apoptosis are involved in regulating the development and atresia of ovarian follicles. POF patients with poor autophagy and high apoptosis rate have lower pregnancy possibility and worse embryo quality.^51,52 This study found that the expression of autophagy protein LC3-II, beclin-1and p62 decreased significantly in the ovary of POF rats. Medium and high doses of pearl can significantly improve expression of autophagy protein LC3-II, beclin-1and p62. It is speculated that when the intensity and duration of oxidative stress in the ovary of POF rats reach the cellular limit, the adaptive mechanism of autophagy will be suppressed and apoptosis will be promoted; pearl can improve autophagy of granulosa cells, inhibit apoptosis of granulosa cells and promote follicular development by alleviating the intensity and duration of oxidative stress.^53,54

In conclusion, pearl can significantly improve the estrous cycle and serum reproductive hormone level of POF rats, promote follicular development and effectively enhance ovarian function. The optimal concentration was found tobe 740 mg kg⁻¹. The mechanism may be related with the enhanced follicular development through improving granulosa cell autophagy and inhibiting granulosa cell apoptosis by inhibition of MAPK signaling pathway after scavenging excessive ROS. This study explains the possible regulatory mechanism of pearl on POF from the aspects of anti-oxidation, autophagy and apoptosis of ovarian cells, and provides the basis for rational clinical compatibility and application, and in-depth development of Chinese patent medicine using pearl. However, in clinical practice, diseases with single symptom are far less than diseases with multiple symptoms. Therefore, it is necessary to use more traditional Chinese medicine to mix with pearls, which may be more effective than single component of pearl. In the future research, pearl combined with other traditional Chinese medicine to enhance the effect of POF treatment needs to be studied for further demonstration.

Data availability

The data used to support the findings of this study are included within the article. The supporting data is available upon reasonable request from the corresponding author.

Authors’ contributions

Siyin Han, and Jiang Lin conceived and designed the experiments; Siyin Han, HongxuanLi, RuiLu, Jiaxin Feng, KaiTang, SihuiLi performed the experiments; Siyin Han, HongxuanLi, RuiLu, and Jiang Lin analyzed the data; Siyin Han, and Jiang Lin wrote the paper.

Funding acknowledgments

This study was supported by grants from the Guangxi University of TCM Introduction of Doctor Scientific Research Startup Fund (Grant no.2018BS013), 2021 Undergraduate Innovation and Entrepreneurship Training Program (S20211060007), National marine economy innovation and development demonstration city industrial chain coordination and innovation project (Bhsfs002), Guangxi University of traditional Chinese medicine 2019–2021 Guangxi first-class discipline construction open project (Grant no. 2019xk058), Guangxi First-class Discipline: Traditional Chinese Medicine (Scientific Research of Guangxi Education Department (2022) No. 1), Guangxi famous traditional Chinese medicine Linjiang inheritance studio (Guangxi Traditional Chinese medicine science and education development [2021] No. 6).

Declaration of competing interest

The authors declare that there are no conflicts of interest associated with the manuscript.

Footnotes

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