Effect of moxibustion on expression profile of miRNAs in Tripterygium glycoside-induced decreased ovarian reserve

Abstract

OBJECTIVE:

To explore the possible regulatory mechanism of microRNA (miRNA) in moxibustion treatment for decreased ovarian reserve (DOR).

METHODS:

The DOR model was constructed by intragastrical Tripterygium glycoside suspension administration, and moxibustion therapy was simultaneously given. The morphological ovarian changes were observed by hematoxylin and eosin staining. The miRNA expression profile was detected by RNA sequencing, and bioinformatics analysis was performed. Cytoscape software 3.6.1 was used to establish a regulatory network and differentially expressed miRNAs were verified by reverse transcription quantitative polymerase chain reaction (RT-qPCR).

RESULTS:

Decreased number of mature follicles, increased atresia follicles, and abnormal granulosa cell morphology were observed in the model group compared with the control group. The moxibustion group demonstrated increased mature follicles, decreased atretic follicles, and significantly decreased abnormal morphology of granulosa cells compared with the model group. Additionally, RNA sequencing results manifested significantly up-regulated miRNA expressions (miR-92b-3p, miR-26-5p_R + 1_1ss10TC, miR-206-3p, miR-9993b-3p_1ss6GA, miR-7857-3p_R-1, miR-219a-2-3p_1ss10GC, miR-3968-p5_1ss10AT, and PC-5p-6478_1795) and down-regulated miR-664-2-5p_R + 1 in the model group, compared with the control group, and the moxibustion group reversed abnormal disorder levels of these miRNAs. Moreover, these differentially expressed miRNAs were mainly involved in the phosphatidylinositol-3-kinase / protein kinase B signaling pathway and nuclear factor erythropoietin-2-related factor 2 / heme oxygenase 1 signaling pathway. Finally, network and RT-qPCR verification revealed miR-9993b-3p_1ss6GA as the most critical miRNA.

CONCLUSION:

This experiment proved the effectiveness of moxibustion in improving the ovarian reserve of rats by regulating miRNA expression, especially miR-9993b-3p_1ss6GA.

1. INTRODUCTION

Decreased ovarian reserve (DOR) is characterized by decreased number or quality of healthy follicles and reproductive endocrine dysfunction.¹ The incidence of DOR is currently increasing, affecting younger females. Patients with DOR can develop premature ovarian failure within 6 years with no effective intervention.² Additionally, DOR has adverse effects on several body systems, including cardiovascular, skeletal function, and nervous systems.³

Moxibustion, which is a Traditional Chinese Medicine therapy, mainly uses Aiye (Folium Artemisiae Argyi) as moxibustion materials to stimulate acupoints to achieve the treatment goal.⁴ Clinical studies have proved that moxibustion helps regulate the menstrual cycle and accelerate ovarian blood circulation.⁵ Studies have reported that moxibustion improves ovarian function by regulating hormone levels in the body, such as estradiol, aromatase, and testosterone.⁶ Moxibustion is now being accepted by more and more people in Traditional Chinese Medicine as a complementary and replacement therapy with an active effect on ovarian function based on its positive effect on ovarian function.⁷ However, the molecular mechanism of moxibustion in DOR treatment remains unknown.

MicroRNAs (miRNAs), which is one of the non-coding RNAs, play an important role in ovarian development.⁸ Some studies compared miRNAs in atresia follicles with those in other follicles and revealed that miR-23-27-24, miR183-96-182, and miR-17-92 were correlated with follicular atresia.⁹ Wei et al ¹⁰ demonstrated that miR-221-3p decreases the apoptosis rate of ovarian granulosa cells in females with DOR by targeting forkhead box O1. Additionally, miRNA is a target for moxibustion treatment of diseases. For instance, moxibustion can improve inflammation in rats with immune thyroiditis by regulating the miRNA-346 and miRNA-331-5P expressions.¹¹ However, the association between moxibustion treatment for DOR and miRNA is unclear.

The present study aimed to discover the molecular mechanism of moxibustion’s protective effect on Tripterygium glycoside (TGS) suspension-induced DOR mice. MiRNA expression profile of moxibustion-treated DOR rats was identified by RNA sequencing (RNA-seq) to explore the potential regulatory mechanism. Our study provides a reference to further explore the mechanism of moxibustion on DOR and a theoretical basis for clinical diagnosis and treatment of DOR.

2. MATERIALS AND METHODS

2.1. Experiment equipment and materials

Hunan Qianjin Co., Ltd. (Changsha, China) (Z43020138) provided TGS suspensions. Gosen Biological Co., Ltd. (Changsha, China) provided the moxa sticks. The hematoxylin and eosin (HE) staining kit was purchased from Abcam (Cambridge, UK) (ab245880). OLYMPUS (Kyoto, Japan) (U-ND6-2 Fluorescence Multifunctional Microscopy) provided the light microscope. Qiagen (Hilden, Germany) (217084) provided the miRNeasy Kit. GE Healthcare (Milwaukee, WI, USA) (NanoVue Plus) provided the Nanovue Spectrophotometer. Illumina (San Diego, CA, USA) provided the TruSeq Small RNA Sample Prep Kits (FC-121-4001/4002/4003) and Hiseq 2000 sequencing (HiSeq 2000). Agilent (Santa Clara, CA, USA) (2100) provided Bioanalyzer 2100. KAPA Library Quantification Kits were purchased from Roche (Basel, Switzerland) (7960255001). Tiangen (Beijing, China) (4992865) provided the miRcute miRNA Isolation Kit. Shanghai Bioengineering Technology Service Co., Ltd. (Shanghai, China) (B532451) provided the miRNA First Stand cDNA Synthesis. Yeasen Biotech (Shanghai, China) (11201ES30) provided the Hieff qPCR SYBR Green Master Mix.

2.2. Animals

Shanghai Xipuer-Bikai Lab Animal Co., Ltd. [license number: SCXK (Hu)-2018-0006] provided 35 specific pathogen free female Sprague-Dawley rats [(200 ± 10) g] with non-pregnant aged 8 weeks. Only 30 rats with normal estrous cycles were accepted after 3 consecutive estrous cycles. The rats were housed in an environment with ventilation, 12∶12 hours light-dark cycles, temperature of 24-26 ℃, and humidity of 50%-60%, and were free to eat and drink. This experiment passed the ethical review of animal experiments of our ethics committee (202106A002).

2.3. Experimental design

Thirty rats were divided into the control, model, and moxibustion groups (n = 10/per group). TGS suspension (50 mg·kg^-1·d^-1) was used in the DOR model and moxibustion groups through intragastric administration for 14 consecutive days, as reported by previous studies.¹² The control group received intragastric physiological saline (10 mL·kg^-1·d^-1). Rats in the moxibustion group had a mild moxibustion with moxa sticks (diameter: 5.3 mm; length: 85 mm) above bilateral 1 h after TGS administration. The specific operation was as follows: moxibustion group included two groups of acupoints: one is the ventral side: Guanyuan (CV4) and Zhongwan (CV12), and the other is the dorsal side: bilateral Shenshu (BL23). Supplementary Figure 1 shows the acupoint map of the rat. The two sets of acupoints were used in turn with one moxibustion each day. Moxa sticks were lit 1 cm above the acupoints for 10 min, once a day, until the end of modeling. The rats were euthanized on day 15, and then ovaries were resected.

2.4. HE staining

HE staining was strictly performed according to kit procedures. The ovaries from 5 rats in each group were fixed with paraffin blocks in 4% neutral buffer paraformaldehyde and sliced into 5 μm sections for histopathological study. The development of follicles, luteum, and granulosa cells in the ovary were observed under a light microscope. Follicles were classified and counted in reference to the previous procedure.^13,14 Briefly, six fields were randomly selected and cell counts were performed.

2.5. RNA sequencing

The total RNA was obtained from granular cells of ovaries (n = 3 per group) by miRNeasy Kit (Qiagen, Hilden, Germany). RNA concentration and purity were measured by the Nanovue Spectrophotometer (GE Healthcare, Buckinghamshire, UK). MiRNA sequencing was performed at the Lianchuan-biotechnology (Hangzhou, China). TruSeq Small RNA Sample Prep Kits (Illumina, San Diego, CA, USA) Kits were used to prepare small RNA sequencing libraries. RNA was used for deep sequencing using the Hiseq 2000 sequencing platform of Illumina (San Diego, CA, USA). Briefly, small RNA fragments of 16-26 nt were purified from total RNA and enriched by denatured polyacrylamide gel electrophoresis. The RNA was reverse transcribed into cDNA after T4 ligase to connect the 3’ and 5’ ends of the adapter. Subsequently, polymerase chain reaction (PCR) amplification was performed using Illumina-compatible index primers (San Diego, CA, USA). Finally, library quality was assessed using Bioanalyzer 2100 (Agilent, Santa Clara, CA, USA), and reverse transcription quantitative PCR (RT-qPCR) was performed using KAPA Library Quantification Kits for Illumina (San Diego, CA, USA). The sequencing file is from the image file generated by Illumina Genome Analyzer (San Diego, CA, USA).

2.6. Identification of differentially expressed miRNAs

The Fast-QC (Illumina, San Diego, CA, USA) was performed to check the quality of raw reads. The number of read images of reference miRNAs obtained from the free miRNA database was correlated with the number of detected miRNAs to evaluate the miRNA detection rate. The differentially expressed miRNAs were explored by the DE-Seq 2.0 algorithm with |fold change| of > 1 and a P-value of < 0.05 as the inclusion criteria.

2.7. Target gene prediction and functional enrichment analysis

The miRanda and TargetScan software (La Jolla, San Diego, CA, USA) were performed to predict the target genes of miRNAs with significant differences. Target genes with a context score percentile of < 50 were removed in the TargetScan algorithm, and target genes with maximum free Energy of > －10 were removed in the miRanda algorithm. Finally, the intersection of the two algorithms was selected as the final target gene of differentially expressed miRNAs. Gene Ontology (GO) enrichment analysis was used to label target genes of the differential miRNA from the aspects of molecular function, cellular component, and biological process. Kyoto Encyclopedia of Genes and Genomes was used for pathway analysis of target genes of differentially expressed miRNAs.

2.8. Construction of the miRNA-target genes-pathway network

A miRNA-target genes-pathway network was constructed to explore the molecular regulation mechanism of moxibustion therapy DOR. The target gene overlapped with the differentially expressed miRNAs, and the negative interaction pairs between target genes and differentially expressed miRNAs (based on their expression level) were used to construct the miRNA-mRNA network using Cytoscape software version 3.6.1 (Cytoscape Consortium, San Diego, CA, USA).

2.9. RT-PCR of miRNAs

The total RNA was extracted using miRcute miRNA Isolation Kit (Tiangen, Beijing, China), according to the manufacturer’s protocol. The miRNA First Stand cDNA Synthesis (Tailing Reaction) (Sangon Biotech, Shanghai, China) was employed for synthetic cDNA, and the Hieff qPCR SYBR Green Master Mix (Low Rox Plus) (Yeasen, Shanghai, China) was used for RT-PCR. The PCR program was set to incubate at 95 ℃ for 15 min, followed by 40 cycles at 94 ℃ for 15 s, 60 ℃ for 30 s, and 70 ℃ for 30 s. MiRNA was normalized using U6. The 2^∆∆Ct method was used to calculate relative miRNA expressions.

2.10. Statistical analysis

Data are shown as the mean ± standard deviation. The difference between the two groups was measured by Student’s t-test. GraphPad Prism 8.0 (La Jolla, San Diego, CA, USA) was performed to analyze data. P-values of < 0.05 indicate a significant difference.

3. RESULTS

3.1. Moxibustion improves the ovarian damage of TGS-induced DOR rats

Abundant follicles at primary and secondary levels in the ovary, more and larger corpus luteum tissue, and fewer atretic follicles were observed in the control group, as shown in Figures 1A-1C. Additionally, a large amount of connective tissue can be seen in the ovaries in the model group, with fewer mature follicles, more atretic follicles, and a significant reduction in the volume of the corpus luteum. Concurrently, the granulosa cells around the follicles in the model group were disordered with fuzzy edges and cell shrinkage. However, the number of normal follicles and corpus luteum significantly increased, while the number of atretic follicles was reduced in the moxibustion group. Subsequently, we counted the follicles of all stages in each group (Figure 1D). The model group had lower numbers of follicles at primordial and mature stages and more atretic follicles than those in the control group. However, the number of follicles at all levels in the moxibustion group was greater than that in the model group, except for atretic follicles, which is significant. These results indicated that moxibustion can improve follicular development and increase the number of healthy follicles.

Figure 1. Moxibustion has a protective effect on ovarian damage in TGS-induced DOR rats.

A-C: hematoxylin and eosin staining was used to detect the morphological changes in ovarian tissue. A1-C1: ×40. A2-C2: Enlarged part (×100). A-C: control, model, and moxibustion. Control group: rats treated with intragastric physiological saline (10 mL·kg^-1·d^-1) for 14 d; Model group: rats treated with TGS suspension (50 mg·kg^-1·d^-1) for 14 d; Moxibustion group: rats treated with TGS suspension (50 mg·kg^-1·d^-1) and moxibustion for 14 d. PF: primary follicle; SF: secondary follicle; MF: mature follicle; AF: atretic follicle; circles represent corpus luteum. D: number of follicles in each ovarian stage. The difference between the two groups was measured by Student’s t-test. Data are shown as the mean ± standard deviation (n = 6). ^aP < 0.01, compared with the control group; ^bP < 0.05 and ^cP < 0.05, compared with the model group. DOR: decreased ovarian reserve; TGS: Tripterygium glycoside.

3.2. Moxibustion affects miRNA expression profile in the ovary of DOR rats

RNA-seq was performed on the control, model, and moxibustion groups to analyze miRNA levels between different groups. Supplementary figure 2 shows the quality control information. The comparison between the model and control groups identified 22 differentially expressed miRNAs, including 14 up-regulated miRNAs and 8 down-regulated miRNAs (Figure 2A). Additionally, 30 up-regulated miRNAs and 13 down-regulated miRNAs were observed in the moxibustion group compared with the model group (Figure 2B). We analyzed the up-regulated miRNAs in the model group versus the control group and the down-regulated miRNAs in the moxibustion group versus the model group to confirm which miRNAs are involved in DOR in moxibustion treatment. The results revealed that 8 miRNAs (miR-92b-3p, miR-206-3p, miR-9993b-3p_ 1ss6GA, miR-7857-3p_R-1, miR-219a-2-3p_1ss10GC, miR-3968-p5_1ss10AT, miR-26-5p_R + 1_1ss10TC, and PC-5p-6478_1795) were abnormally up-regulated in the model, while the expression levels were significantly decreased after moxibustion stimulation (Figure 2C). Additionally, one common miRNA (miR-664-2-5p_R + 1) was determined in the model group versus the control group (down) and the moxibustion group versus the model group (up) (Figure 2D). Therefore, we speculated that moxibustion may lessen miR-206-3p, miR-9993b-3p_1ss6GA, miR-7857-3p_R-1, miR-219a-2-3p_1ss10GC, miR-3968-p5_1ss10AT, miR-26-5p_R + 1_1ss10TC, and PC-5p-6478_1795, and enhance miR-664-2-5p_R + 1 to protect DOR.

Figure 2. Effects of moxibustion on DOR on a transcriptome-wide scale.

A: volcano map of miRNA of the model versus the control groups; B: volcano map of miRNA of the moxibustion versus the model groups; C: venn diagram of the model versus the control (up) and the moxibustion versus the model (down) groups; D: venn diagram of the model versus the control (down) and the moxibustion versus the model (up) groups. Control group: rats treated with intragastric physiological saline (10 mL·kg^-1·d^-1) for 14 d; Model group: rats treated with TGS suspension (50 mg·kg^-1·d^-1) for 14 d; Moxibustion group: rats treated with TGS suspension (50 mg·kg^-1·d^-1) and moxibustion for 14 d. Mod: model; Con: control; Mox: moxibustion; DOR: decreased ovarian reserve; TGS: Tripterygium glycoside. The difference between the two groups was measured by Student’s t-test. Data are shown as the mean ± standard deviation (n = 3).

3.3. Functional enrichment analysis of differentially expressed miRNAs

We performed function prediction of the above miRNAs for functional analysis to further understand the possible function and mechanism of moxibustion in DOR treatment. GO analysis demonstrated some main biological processes for differentially expressed miRNA enrichment, such as “intracellular membrane-bounded organelle,” “protein homodimerization activity,” “molecular function,” and “extracellular vesicular exosome” (Figure 3A). Moreover, several KEGG pathways were significantly enriched in the differentially expressed miRNAs, which mainly were the “phosphatidylinositol-3-kinase protein kinase B (PI3K-Akt) signaling pathway,” “MAPK signaling pathway,” “AMPK signaling pathway,” and “heme oxygenase 1 (Nrf2/HO-1) signaling pathway” (Figure 3B).

Figure 3. Enrichment analysis of differential miRNA.

A: GO terms analysis of differential miRNA; B: pathway analysis of differential miRNA. GO: Gene Ontology; PI3K-Akt: phosphatidylinositol-3-kinase-protein kinase B; MAPK: mitogen-activated protein kinase; Nrf2/HO-1: nuclear factor erythropoietin-2-related factor 2/heme oxygenase 1; AMPK: adenosine 5‘-monophosphate (AMP)-activated protein kinase; GPI: glycosylphosphatidylinositol, mTOR: mammalian target of rapamycin; FDR: false discovery rate.

3.4. Construction of the miRNA-target gene-pathway

The literature reports that moxibustion may regulate the estrus cycle of DOR rats through the PI3K-Akt and Nrf2/HO-1 signaling pathways.^15,16 Thus, we constructed a regulatory network of miRNA-target gene-pathway (Figure 4A). The miR-7857-3p_R-1 regulated Shox2 and Klf4 to affect the PI3K-Akt and Nrf2/HO-1 signaling pathways, respectively. The miR-206-3p and miR-7857-3p_R-1 regulated Edn1 together to affect the PI3K-Akt signaling pathway. Moxibustion may improve DOR rats by mediating PI3K-Akt and Nrf2/HO-1 signaling pathways through the above regulatory network.

Figure 4. Construction of the miRNA-target gene-pathway and validation of miRNAs.

A: the network of miRNAs-target genes-pathway. The square represents the pathway; the diamond represents the mRNA; the octagon represents the miRNA. B: the differentially expressed miRNA was verified by RT-PCR. RT-PCR: reverse transcription polymerase chain reaction; Nrf2/ARE: nuclear factor erythropoietin-2-related factor 2/AU-rich element; PI3K/Akt: phosphatidylinositol-3-kinase/protein kinase B; Cdx2: caudal type homeobox 2; E2f1: E2 promoter binding factor 1; Atp5c1: adenosine triphosphate synthase subunit gamma; Sall1: spalt like transcription factor 1; Klf5: krüppel-like factor 5; Edn1: endothelin 1; Zfp382: zinc finger protein 382; Hmg20a: high mobility Group 20a; Bach2: BTB domain and CNC homolog 2; Bcl6b: B-cell Lymphoma 6b; Rnf2: ring finger protein 2; Cbx2: chromobox 2; Esr1: estrogen receptor 1; Shox2: short Stature homobox 2; Cav1: caveolin-1; Pias1: protein inhibitor of activated signal transducer and activators of transcription 1; Dcaf1: DDB1 and CUL4 associated factor 1. The difference between the two groups was measured by Student’s t-test. Data are shown as the mean ± standard deviation (n = 5). ^aP < 0.05, compared with the control group. ^bP < 0.01 and ^cP < 0.05, compared with the model group.

3.5. Validation of miRNAs

We further performed RT-PCR detection on miRNA in the network diagram (Figure 4B). The results revealed that miR-92b-3p, miR-9993b-3p_1ss6GA, and miR-7857-3p_R-1 were significantly up-regulated in the model group compared with the control group. Moxibustion reduced the expression of miR-26-5p_R + 1_1ss10TC, miR-9993b-3p_1ss6GA, and miR-3968-p5_1ss10AT compared with the model group. However, only the miR-9993b-3p_1ss6GA expression was consistent with miRNAs transcriptome. Therefore, miR-9993b-3p_1ss6GA is a key target for DOR pathogenesis, as well as a therapeutic target for moxibustion protection of DOR rats.

4. DISCUSSION

DOR is a disease common in the Department of Gynecology, whose pathogenesis and mechanism remain unclear. DOR is not only associated with infertility in females but also develop into premature ovarian failure if left untreated.¹⁷ Thus, understanding the pathogenesis of DOR and effective therapeutic targets is crucial to improve ovarian reserve in females who want to become pregnant. Moxibustion, which is widely used in the clinical treatment of reproductive diseases, is beneficial to improve the sexual hormone levels of patients with DOR through the meridian system.¹⁸ This study investigated the protective effect of moxibustion on TGS-induced DOR to understand the molecular target of moxibustion for DOR treatment. This study revealed that moxibustion significantly improved follicular development of TGS-induced DOR and increased the number of healthy follicles. Moreover, moxibustion could reverse miRNAs that were abnormally expressed in DOR. In particular, miR-9993b-3p_1ss6GA may be a key target of DOR pathogenesis and moxibustion therapy.

Our study used moxibustion for two groups of acupoints [one group is bilateral Shenshu (BL23), and another group is Guanyuan (CV4) and Zhongwan (CV12)] and alternated with moxibustion on alternate days. The Traditional Chinese Medicine theory states that kidney essence deficiency and thoroughfare (Chong) and conception (Ren) vessel dysfunction is the main cause of primary ovarian insufficiency (POI). Bibliometric studies have shown that Guanyuan (CV4) and Shenshu (BL23) are the two most commonly used acupoints for clinical POI treatment.¹⁹ Guanyuan (CV4), which is an acupoint of the conception vessel, converges with the three Yin meridians of the foot. Stimulating Guanyuan (CV4) can regulate Qi and blood in the thoroughfare and conception vessels. Recent studies have indicated that stimulating Guanyuan (CV4) may adjust the hypothalamic-pituitary-gonadal axis, regulate sex hormone secretion,^20,21 and protect ovarian uterine function.²² Shenshu (BL23) belongs to the Taiyang bladder meridian and is located at the back of the waist. It collects the kidney essence and marrow and is the origin of the congenital constitution. Shenshu (BL23) functions to reinforce and nourish the kidney and fill the essence.²³ Meanwhile, this study used Zhongwan (CV12), which was a point on the conception vessel. It was the gastric Front-Mu point, which has the effect of replenishing Qi, promoting blood, and invigorating the spleen and stomach. Altogether, we selected the above acupoints to explore the therapeutic effect of moxibustion on DOR. Additionally, this study alternately used the acupoints on the abdomen and back, which is mainly the continuation of the previous animal experimental results of the research group,^24,25 and it is also a commonly used moxibustion method in clinical practice.²⁶ In turn, moxibustion is applied on the waist and back, which conforms to the basic treatment principle of Traditional Chinese Medicine, “guiding Yang from Yin and Yin from Yang.” The chest and abdomen belong to Yin, while the back is Yang. A state of Yin and Yang being too strong and too weak or both Yin and Yang being deficient occurs if the dynamic balance of Yin and Yang is broken. Moxibustion is applied at acupoints on the chest, abdomen, and back. Moxibustion regulates the function of Zang-Fu organs, dredges Qi and blood of meridians, promotes the growth of Yin and Yang, achieves the purpose of Yin, Yang, and constipation, and restores the balance of Yin and Yang in the body through the unique effect of warming and tonifying. Therefore, this study used two groups of acupoints for moxibustion, and the acupoints on the chest, abdomen, and back were alternately used.

Reviewer pointed out that it is a great scientific question. From the point of animal ethics and experimental outcome indicators, moxibustion operations in this experiment were performed under anesthesia in rats to avoid scald caused by a struggle in the process of moxibustion and the influence of severe struggle stress on serum sex hormone levels caused by grasping or restraining rats. Poulakos et al ²⁷ revealed that the test solution quickly passed through the pylorus into the intestine and emptied > 50% in the stomach at 60 min after gavage of the radioactive test solution to rats with a normal diet, and the distribution of radioactive substances in the intestine at 60 and 120 min demonstrated no significant difference. Moxibustion was given 1 h after the gavage of TGS suspension to prevent the reflux of gastric contents and asphyxia induced by direct anesthesia after gavage in rats. A series of previous studies adopted the same methods for modeling and intervention^{24,⇓,⇓,⇓-28} and confirmed its safety and effectiveness.

Numerous evidences proved that moxibustion may be an important method to restore ovarian function.²⁹ The theory of Traditional Chinese Medicine indicated kidney Qi deficiency as the most important cause of DOR. The kidney Qi can be injected into the back through Shenshu (BL23). This study used moxibustion Guanyuan (CV4) and Shenshu (BL23), which are similar to the autonomic nerves of the ovary and uterus, to supplement ovarian function.³⁰ Consistent with other research,⁴ moxibustion intervention on DOR rats can significantly promote the number of healthy follicles and reduce the occurrence of atretic follicles. Additionally, granulosa cells provided a large amount of energy and nutritional support for follicular maturation during ovarian development.³¹ Our study revealed a reduced corpus luteum volume in the ovary of the model group and deformed granulosa cells, while moxibustion could significantly improve this adverse situation. Altogether, moxibustion significantly improved ovarian injury.

MiRNA has attracted more and more attention because it may play a role in the regulation of nearly every cellular process. Some studies revealed that miRNA expression is different in females with different ovarian reserves.³² Relevant experiments have proved that moxibustion therapy can target miR-144-3p to effectively inhibit collagen-induced arthritis.³³ Hence, RNA-seq was performed on three groups of rats to study the molecular mechanism of moxibustion in DOR treatment. We found that TGS-induced DOR caused abnormal miRNA expression compared with normal mice, with 14 up-regulated miRNAs and 8 down-regulated miRNAs. However, moxibustion treatment in DOR mice can effectively alleviate abnormal miRNA expression, suggesting that moxibustion may treat DOR by regulating miRNA expression. Li et al ¹⁵ revealed that moxibustion alleviated DOR rats by restoring the PI3K-Akt signaling pathway. Furthermore, TGS-induced DOR mice lead to inappropriate NLRP3 inflammatory body activation, which affects ovarian development, while moxibustion activates the Nrf2/HO-1 pathway, which down-regulates NLRP3 and improves inflammation.³ Therefore, we performed functional enrichment analysis on differential miRNAs and revealed that most of these miRNAs were involved in PI3K-Akt, AMPK, and Nrf2/HO-1 signaling pathways. Then, the miRNA-target gene-pathway network was constructed to provide a reference for elucidating DOR pathogenesis. For example, miR-9993b-3p_1ss6GA targeted CBX2 to modulate the PI3K-Akt signaling pathway. CBX2 is a gene related to ovarian function which is mainly expressed in granulosa cells.³⁴ Briefly, miRNAs may be responsible for moxibustion treating DOR via targeting mRNAs related to ovarian development to regulate signaling pathways.

Finally, we performed PCR detection to determine whether moxibustion regulates DOR through miRNA and revealed that miR-9993b-3p_1ss6GA expression was consistent with the trend of the transcriptome. Thus, we speculated that moxibustion targeted down-regulation of miR-9993b-3p_1ss6GA, thereby affecting signaling pathways related to ovarian development, and thus alleviating ovarian damage in DOR rats.

Taken together, moxibustion improved TGS-induced DOR, promoted healthy follicle development, and reduced the number of atresia follicles, thereby decreasing the degree of ovarian dysfunction. Furthermore, moxi-bustion participated in the signaling pathway related to ovarian function to improve follicular development by regulating miRNA expression, among which miR-9993b-3p_1ss6GA is a potential therapeutic target.

5. SUPPORTING INFORMATION

Supporting data to this article can be found online at http://journaltcm.cn.