Moxibustion ameliorates ovarian function in premature ovarian insufficiency rats by activating cAMP/PKA/CREB to promote steroidogenesis in ovarian granulosa cells

Journal of Steroid Biochemistry and Molecular Biology 242 (2024) 106547 Contents lists available at ScienceDirect Journal of Steroid Biochemistry and Molecular Biology journal homepage: www.elsevier.com/locate/jsbmb Moxibustion ameliorates ovarian function in premature ovarian insufficiency rats by activating cAMP/PKA/CREB to promote steroidogenesis in ovarian granulosa cells Rui Zhao a, 1, Lingxiang Ran a, d, 1, Hanyue Yao a, Yizhi He a, Xinru Lu a, Weina Zhu b, c, Yajie Zhang b, c, Tianyi Zhang a, Shijie Shi a, Zheng Luo a, Cairong Zhang a, * a Department of Acupuncture and Moxibustion, Nanjing Hospital of Chinese Medicine affiliated to Nanjing University of Chinese Medicine, Nanjing, Jiangsu 210022, China b Central Laboratory, Nanjing Hospital of Chinese Medicine affiliated to Nanjing University of Chinese Medicine, Nanjing, Jiangsu 210022, China c Department of Biobank, Nanjing Hospital of Chinese Medicine affiliated to Nanjing University of Chinese Medicine, Nanjing, Jiangsu 210022, China d Department of Urology, the Second Affiliated Hospital of Soochow University, Suzhou, Jiangsu 215004, China A R T I C L E I N F O A B S T R A C T Keywords: Premature ovarian insufficiency Moxibustion Granulosa cells Steroidogenesis Premature ovarian insufficiency (POI) presents a substantial challenge to women’s physiological and psychological well-being. Hormone replacement therapy, as the preferred therapeutic approach, involves solely exogenous supplementation of estrogen. Moxibustion, a traditional Chinese external treatment, has been investigated in our previous studies. It not only improves hormone levels and clinical symptoms in POI patients but also safeguards ovarian reserve. This study aims to explore the regulatory mechanisms by which moxibustion modulates hormone levels and restores ovarian function in POI. A POI rat model was established using cyclophosphamide, and moxibustion treatment was applied at acupoints "CV4" and "SP6" for a total of four courses. Subsequently, ovaries from each group were subjected to transcriptome sequencing (Bulk RNA-seq). Target pathways and key genes were selected through enrichment analysis and GSVA scoring, with validation using various techniques including electron microscopy, ELISA, Western blot, and immunohistochemistry. The results demonstrated that moxibustion restored the estrous cycle in POI rats, improved sex hormone levels, reduced the number of atretic follicles, and increased the count of dominant follicles (P<0.05). Bulk RNA-seq analysis revealed that moxibustion downregulated pathways associated with ovarian dysfunction, infertility, and immune responses, upregulated pathways related to follicular development and ovarian steroidogenesis. Furthermore, our data confirmed that moxibustion significantly increased the number of ovarian granulosa cells (GCs) and upregulated the expression of proteins related to steroidogenesis in GCs, including FSHR, P450 arom, cAMP, PKA, and CREB (P<0.05), with no significant effect observed on proteins related to steroidogenesis in theca cells. These outcomes aligned with the RNA-seq results. In conclusion, these findings propose that moxibustion enhances steroidogenesis in GCs through the activation of the cAMP/PKA/CREB pathway, consequently improving impaired ovarian function in POI rats. This study provides robust evidence supporting moxibustion as a targeted intervention for treating POI by specifically regulating steroidogenesis in GCs. Abbreviations: POI, premature ovarian insufficiency; CTX, cyclophosphamide; CAMP, cyclic adenosine monophosphate, PKA, protein kinase A; CREB, cAMP response element binding protein; GCs, granulosa cells; StAR, Steroidogenic Acute Regulatory Protein; CYP19A1, P450 arom, Aromatase P450; FSH, Follicle Stimulating Hormone; LH, Luteinizing Hormone; E2, 17β-Estradiol; AMH, anti-Mullerian hormone; T, Testosterone; CV4, Guanyuan; SP6, Sanyinjiao; HRT, Hormonal Replacement Therapy; PBS, phosphate buffer saline. * Corresponding author. E-mail address: fsyy00297@njucm.edu.cn (C. Zhang). 1 Rui Zhao and Lingxiang Ran contributed equally to this work https://doi.org/10.1016/j.jsbmb.2024.106547 Received 20 December 2023; Received in revised form 5 May 2024; Accepted 12 May 2024 Available online 14 May 2024 0960-0760/© 2024 Elsevier Ltd. All rights are reserved, including those for text and data mining, AI training, and similar technologies. R. Zhao et al. Journal of Steroid Biochemistry and Molecular Biology 242 (2024) 106547 1. Introduction them, standard food and distilled water were provided ad libitum. All animal experiments in this study were performed according to ARRIVE guidelines[12]. The experiment protocol was approved by the Experimental Animal Ethics Committee (JHVC-IACUC-2023-B019). Currently, there is a significant global burden of fertility-related issues. According to WHO estimates, approximately 518.6 million couples worldwide experience infertility, with female factors contributing to around 50%[1]. Premature ovarian insufficiency (POI) is a leading cause, and spontaneous pregnancy rates in affected individuals range from 5% to 10% [2]. POI, denoting ovarian dysfunction in women under 40, is characterized by abnormal menstruation (oligomenorrhea, amenorrhea, etc.), elevated gonadotropin and fluctuating estrogen (E2) levels, with follicular atresia being the main pathological feature [3]. E2, the most bioactive in vivo estrogen, retards follicular atresia development and promotes follicular growth, differentiation, and development. Hormonal Replacement Therapy (HRT) is the primary clinical choice for POI treatment. However, its hormone supplementation has limited effects on improving ovarian function and fertility. Moreover, significant toxic and side effects are associated with this treatment[4]. Consequently, there is an urgent need to explore new treatments aimed at elevating endogenous estrogen levels or restoring ovarian function. Moxibustion, a traditional Chinese medical therapy, garners widespread acceptance among patients due to its straightforward application, cost-effectiveness, efficacy, and minimal adverse effects. The distinctive benefit of moxibustion stems from its "warm" stimulation, luminous radiation effect and moxa combustion products. These external stimulation facilitates the regulation of the patient’s own function, achieving the effect of "stimulation outside and regulation inside"[5]. Studies demonstrate that moxibustion improves hormone levels and clinical symptoms in POI patients. Additionally, it effectively regulates ovarian artery blood flow supply, increases diastolic blood perfusion, and enhances ovulation and conception rates[6,7]. In comparison to HRT, moxibustion presents natural and substantial advantages in POI treatment. However, further clarification of the moxibustion mechanism in POI treatment is needed. Ovarian Granulosa Cells (GCs) constitute the principal functional cells within follicles, playing a crucial role in ovarian reserve, follicular development, maturation, and secretory function[8]. Approximately ninety percent of estrogen in women originates from the ovary, governed by ovarian steroidogenesis-related pathways[9]. This regulation primarily occurs in GCs and involves various proteins and enzymes[10]. While moxibustion is recognized for enhancing sex hormone levels in POI patients, the current understanding of its relationship with improving ovarian function, particularly steroidogenesis regulation in GCs, remains unclear. Bulk RNA-seq technology, a well-established method, enables the comprehensive and rapid assessment of transcriptional activity in various tissues or cells under different conditions. It is employed for analyzing the expression of differentially expressed genes and has found extensive applications in biological processes, clinical disease research, and drug innovation[11]. In a nutshell, this study aims to investigate the potential mechanism by which moxibustion improves ovarian function in POI rats using Bulk RNA-seq technology. It will experimentally demonstrate the regulatory relationship between moxibustion and GCs. The findings will offer robust evidence for moxibustion in the clinical treatment practice of POI and similar diseases. 2.2. Drugs and reagents Cyclophosphamide for injection (Jiangsu Hengrui Medicine Co., Ltd., China); Pure moxa stick (Jiangsu Kangmei Pharmaceutical Co., Ltd., China); Amobarbital Sodium for Injection(Shanghai New Asia Pharmaceutical Co., Ltd., China); Rat Follicle Stimulating Hormone (FSH) ELISA Kit (EK17665, Signalway Antibody LLC, America), Rat antiMullerian hormone (AMH) ELISA kit (CSB-E11162r, Wuhan Huamei BIOTECH Co., Ltd., China), Rat LH(Luteinizing Hormone) ELISA Kit, QuicKey Pro Rat E2(Estradiol) ELISA Kit, QuicKey Pro Rat T(Testosterone)ELISA Kit (E-EL-R0026c、E-OSEL-R0001、E-OSEL-R0003, Elabscience); RIPA Lysis Buffer(P0013B, Beyotime), Phosphatase Inhibitor Cocktail(B15001, Bimake), Protease Inhibitor Cocktail(B14001, Bimake), Enhanced BCA Protein Assay Kit(P0010, Beyotime), Antibodies: StAR antibody(# 8449 S, CST), cAMP antibody (ab76238, Abcam), PKA antibody(ab75991， Abcam)， CREB antibody(# 9197 S, CST), Aromatase antibody(# 14528 S, CST); Histone-H3 Polyclonal antibody(17168–1-AP, Proteintech), HRP-abeled Goat Anti-Rabbit laG (H+L)(A0208, Beyotime). Super-sensitive ECL chemiluminescent substrate(BL520B, Biosharp); FSHR Polyclonal antibody(22665–1-AP, Proteintech). 2.3. Grouping and treatment After all animals were arrived, we first adaptively fed them for 7 days, performed vaginal smears every morning from 9:00–11:00 to observe the estrous cycle of rats. Then 15 female rats with normal estrous cycle were randomly divided into three groups: CON group, CTX group, CTX+MOX group, with 5 rats in each group. CTX group and CTX + MOX group were injected intraperitoneally with cyclophosphamide at a dose of 50 mg/kg on day 8, decreasing to 8 mg/kg in later 14 days; CON group was injected intraperitoneally with the same amount of saline daily. Meanwhile, according to the results of previous studies, we made the following adjustments to the experimental protocol: CTX + MOX group was given moxibustion fumigation at "Guanyuan point" and "Sanyinjiao point (bilateral)" from 15:00–17:00 from the first day of administration，2 – 3 cm away from the acupoint skin, 15 min per day, 5 times a week, and the experiment was terminated after 2 weeks of maintenance treatment after the end of administration[13,14] (Fig. 1. A). During this period, rats were weighed daily and observed for vital signs, such as diet, water intake, hair and activity. In addition, vaginal smears were performed every day from 9:00–11:00, and immediately fixed with 95% alcohol, stained with Papanicolaou. Exfoliated cells from the vaginal epithelium of rats were observed microscopically to determine the estrous cycle (characteristics of vaginal epithelial cells in each estrous cycle: leukocytes and nucleated epithelial cells were predominant in diestrus, nucleated epithelial cells were predominant in proestrus, keratinized epithelial cells were predominant in estrus, and keratinized epithelial cells and leukocytes were predominant in metestrus[15]). 2. Materials and methods 2.4. Samples collection and organ index calculation 2.1. Animals and ethics Next day after end of treatment, rats were anesthetized and samples were collected. 2% Pentobarbital sodium was used for anesthesia at a first dose of 0.5 mL/100 g and was increased according to the consciousness of rats (no more than 0.1 mL/time). After anesthesia, the rats were fixed on the operating table with their stomach upwards, and blood was collected and centrifuged (4 ◦ C, 3000 rpm, 20 min). The upper serum was loaded into new EP tubes, labeled and stored at −80 ◦ C 6-week-old specific pathogen free (SPF) grade female SpragueDawley (SD) rats, weighing 200–220 g, unmated, were purchased from Zhejiang Vital River Laboratory Animal Technology Co., Ltd. (Zhejiang, China), license No. SCXK(Zhejiang)2019–0001. All animals were kept in SPF animal room: temperature 20 – 25 ◦ C, humidity 40 – 60%, and 12 h: 12 h diurnal cycle. The bedding was changed daily for 2 R. Zhao et al. Journal of Steroid Biochemistry and Molecular Biology 242 (2024) 106547 Fig. 1. Moxibustion restores the weight gain rate in POI rats. A. The diagram of constructing POI rat model and moxibustion treatment. B. CTX decreases the rate of body weight gain in rats, moxibustion increases it in POI rats. *P<0.05, ***P＜0.001，**** P＜0.0001. pending subsequent testing. The uterus and ovaries were completely removed, the surrounding adipose tissue was removed, washed with PBS and dried before they were weighed on a balance and photographed for documentation. One ovary from each group was removed, approximately 1.0 mm*1.0 mm*1.0 mm of ovaries containing follicles were cut and immersed in 2.5% glutaraldehyde solution (precooled at 4 ◦ C), and the remaining portion was fixed in 4% paraformaldehyde solution. Remaining ovaries were stored at −80 ◦ C. Calculation formula of organ index: organ index = organ weight/ body weight*100%. 2.6. Transmission electron microscopy (TEM) of ovarian granulosa cells Ovaries of 1 mm3 was fixed in 2.5% glutaraldehyde, rinsed in PBS, fixed in 1% osmic acid, dehydrated in ethanol, embedded and polymerized in epoxy resin, sectioned in an ultramicrotome (RMC POWERTOME XL), stained with 2% uranyl acetate and 2.6% lead citrate solution to observe the ultrastructure of ovarian tissue under transmission electron microscopy (FEI Tecnai G2 Spirit Bio TWIN), and images were collected. 2.7. ELISA of serum sex hormone levels 2.5. HE staining of ovaries and follicles counting Serum FSH, LH, AMH, E2, and T levels were measured in strict accordance with the instructions of the ELISA kit. Absorbance values for each standard and sample were detected using a microplate reader (BioTek SynergyHI MFD) at 450 nm. Origin 9.0 was used to obtain the standard curve by four-parameter fitting method, and the absorbance values of the samples were substituted to calculate the hormone concentration. Ovaries were fixed in 4% paraformaldehyde solution for 24 h, embedded in paraffin and sectioned into 4 μm sections, then stained with hematoxylin-eosin (HE). The structure of ovaries was observed by microscope, so that histopathological changes of ovaries were evaluated according to the characteristics and number of follicles at each grade. Primordial follicles: oocytes are surrounded by a layer of flat granulosa cells, the boundary of granulosa cells is not obvious, and oval nuclei can only be seen; preantral follicles (primary follicles): the shape of granulosa cells changes from flat to cuboidal, from one layer to multiple layers, and radiates from the center to the periphery to form corona radiata; antral follicles (secondary follicles): multiple layers of columnar granulosa cells, and theca cells layer appears, and oocytes float in follicular fluid; atretic follicles: follicles collapse, irregular arrangement of granulosa cells, pyknosis or disappearance of oocyte cell nuclei, or only irregular zona pellucida can be seen[16]. 2.8. Bulk RNA-seq of ovaries Total RNA was isolated using the Trizol Reagent (Invitrogen Life Technologies), after which the concentration, quality and integrity were determined using a NanoDrop spectrophotometer (Thermo Scientific). Three micrograms of RNA were used as input material for the RNA sample preparations. Sequencing libraries were generated according to the following steps. Firstly, mRNA was purified from total RNA using 3 R. Zhao et al. Journal of Steroid Biochemistry and Molecular Biology 242 (2024) 106547 poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature in an Illumina proprietary fragmentation buffer. First strand cDNA was synthesized using random oligonucleotides and Super Script II. Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities and the enzymes were removed. After adenylation of the 3′ ends of the DNA fragments, Illumina PE adapter oligonucleotides were ligated to prepare for hybridization. To select cDNA fragments of the preferred 400–500 bp in length, the library fragments were purified using the AMPure XP system (Beckman Coulter, Beverly, CA, USA). DNA fragments with ligated adaptor molecules on both ends were selectively enriched using Illumina PCR Primer Cocktail in a 15 cycle PCR reaction. Products were purified (AMPure XP system) and quantified using the Agilent high sensitivity DNA assay on a Bioanalyzer 2100 system (Agilent). The sequencing library was then sequenced on NovaSeq 6000 platform (Illumina) Shanghai Personal Biotechnology Cp. Ltd. Ovaries samples are sequenced on the platform to get image files, which are transformed by the software of the sequencing platform, and the original data in FASTQ format (Raw Data) is generated. Sequencing data contains a number of connectors, low-quality Reads, so we use Cutadapt (v1.15) software to filter the sequencing data to get high quality sequence (Clean Data) for further analysis. All bioinformatics analyses were performed in the R (Version 4.1.3) environment, and the “DESeq package” was used for differential expression analysis with a data screening threshold of " |log2FoldChange | > 1& P-value < 0.05". Filtered data were used for subsequent enrichment analysis. next day, after washing three times with TBST buffer, membranes were incubated with secondary antibody horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (1:1000) for 1 h at room temperature. Immunoreactive bands were detected by exposing the membranes to Clarity Western ECL chemiluminescent substrate and imaged using the chemiluminescent gel imaging analysis system (Bio-Rad ChemiDoc XRS +). Finally, Image J software was used to calculate the gray values of each protein, and the target protein/Histone h3 ratio was defined as the relative expression of the target protein. 2.12. Immunohistochemistry (IHC) staining of ovaries Paraffin-embedded ovaries, sectioned at 4 μm thickness, were boiled for 5 min in Tris-EDTA buffer (10 mM Tris base 1 mM disodium EDTA dihydrate, pH 8.0) for antigen retrieval. Sections were washed three times with PBS, blocked with goat serum and incubated with specific antibodies against FSHR (1:600). Next, the sections were incubated with Goat Anti-Rabbit IgG H&L HRP for 1 h. The images were captured using an upright fluorescent microscope (Olympus BX43). 2.13. Statistical analysis All data were analyzed and all graphics were plotted using Graphpad Prism 8.4 (GraphPad Software, Inc, La Jolla, CA, USA) and are presented as mean ± standard error (SEM). If data met normal distribution and homogeneity of variance, One-way analysis of variance (ANOVA) and Bonferroni tests for multiple comparisons were used for comparison among multiple groups. P < 0.05 was considered statistically significant. 3. Results 2.9. GO, KEGG and GSEA enrichment analysis 3.1. Moxibustion restores the weight gain rate in POI rats Differentially expressed genes (DEGs) were used to complete enrichment analysis, and GO, KEGG and GSEA enrichment analysis were mainly used in this study: The Gene Ontology (GO) system provides structured, computable information regarding the functions of genes and their products[17].The Kyoto Encyclopedia of Genes and Genomes (KEGG) is a widely used database for the systematic pathway investigation of DEGs [18]; Gene set enrichment analysis (GSEA) analysis independent of DEGs, including all genes in the analysis to assess relevant pathways and molecular mechanisms [19]. GO, KEGG and GSEA enrichment analysis conducted based on the “ClusterProfiler” package [20], threshold criteria is as follows: “|log2FoldChange| > 1 & P-value < 0.05”. As shown in Fig. 1A, we used cyclophosphamide to construct POI rat model and performed moxibustion treatment (subsequent experiments were grouped on this basis). The results showed that the initial body weights of rats in the three groups were similar, and then the body weights of CON group steadily increased rapidly; compared with CON group, the body weight gain rate in CTX group was the slowest and fluctuating, while the body weight gain rate in CTX + MOX group was between the two groups (Fig. 1B). 3.2. Moxibustion improves estrous cycle and reproductive system in POI rats 2.10. GSVA pathway scoring To verify the effect of moxibustion on the reproductive system of POI rats, we first performed vaginal smears and analyzed the estrous cycle, and then collected samples to complete the weighing of the ovary and uterus, especially focused on ovarian HE staining. The results demonstrated that the estrous cycle in CTX group was apparently disturbed compared with CON group, while it in CTX + MOX group was similar to CON group and basically normal (Fig. 2.A, B). Compared with CON group, the volume and organ index of the ovary and uterus became smaller in CTX group, with slight inflammation and significant congestion, while the above parameters were better in CTX + MOX group than in CTX group (Fig. 2.C, D). HE staining results showed that follicles were abundant in CON group, follicles at each grade were visible, and atretic follicles were not observed in the visual field; compared with CON group, the number of normal follicles was decreased and the number of atretic follicles was increased in CTX group; however, the number of dominant follicles was significantly more in CTX + MOX group than in CTX group (Fig. 2.E, F). According to the results of DEGs, designated pathways were selected for GSVA pathway scoring, and the pathway genesets were downloaded from GSEA official website (https://www.gsea-msigdb.org/gsea/index. jsp). GSVA pathway scoring were completed by using the “GSVA package” to show the trend of target pathway scores in the experimental data. 2.11. Western Blot Ovaries were lysed in RIPA buffer containing protease inhibitor and phosphatase inhibitor cocktails， supernatants of lysates were collected by centrifugation (12,000 rpm, 4 C, 15 min), with protein concentrations determined by BCA assay. 20 μg of protein of each sample was mixed with loading buffer and loaded onto 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)，then electrophoresed. Subsequently, proteins were transferred to polyvinylidene difluoride （PVDF） membranes, washed in TBST and blocked with 5% skim milk for 1 h and incubated overnight at 4 ◦ C with primary antibodies (1:1000). Histone h3 antibody was used as the internal reference. The 3.3. Moxibustion improves sex hormone levels in POI rats To assess the effect of moxibustion on ovarian function in POI rats, 4 R. Zhao et al. Journal of Steroid Biochemistry and Molecular Biology 242 (2024) 106547 Fig. 2. Moxibustion improves estrous cycle and reproductive system in POI rats. A-B. CTX disarranges the estrous cycle of rats, moxibustion restores the its regularity in POI rats. C-D. CTX damages the uterine and ovarian, moxibustion ameliorates the damage in POI rats. E-F. CTX promotes follicular atresia and hinders follicular development, moxibustion slows down follicular atresia and increases the number of dominant follicles. *P＜0.05，** P＜0.01，***P＜0.001，**** P ＜0.0001. 5 R. Zhao et al. Journal of Steroid Biochemistry and Molecular Biology 242 (2024) 106547 we examined sex hormone levels of serum. Compared with CON group, FSH levels increased, LH, AMH, E2 and T levels decreased, and FSH/LH ratio increased in CTX group; compared with CTX group, FSH levels and FSH/LH ratio decreased, LH and T levels did not change significantly, and AMH and E2 levels increased relatively in CTX + MOX group (Fig. 3A-F). MOX/CON comparison were mainly: “inflammatory response”，“acute inflammatory response”，“apoptotic process”; upregulated terms in CTX + MOX/CON comparison were mainly “reproductive process”，“female gamete generation”，“regulation of hormone levels” (Fig. 5A-C). KEGG pathway enrichment results showed that: the downregulated genes in CTX/CON comparison mainly enriched “Th1 and Th2 cell differentiation”，“Th17 cell differentiation”，“T cell receptor signaling pathway”; the pathways upregulated in CTX/CON comparison and downregulated in CTX + MOX/CTX comparison were mainly “Tight junction”，“Arginine and proline metabolism”; there were 3 KEGG pathways commonly significantly enriched in the upregulated DEGs between the comparisons of CTX + MOX/CTX and CTX + MOX/CON, which were of “cAMP signaling pathway”，“Calcium signaling pathway，“Ovarian steroidogenesis”; and 2 KEGG pathways commonly significantly enriched in the downregulated DEGs in CTX + MOX/CON comparison which were mainly involved in “HIF−1 signaling pathway”，“IL−17 signaling pathway” (Fig. 6A-C). GSEA enrichment results showed that: the pathways upregulated in CTX/CON comparison and downregulated in CTX + MOX/CTX comparison were mainly “E2F targets”，“G2M checkpoint”，“MYC targets”; the pathways downregulated in CTX/CON comparison and upregulated in CTX + MOX/CTX comparison were mainly "“Estrogen response early”，“Interferon gamma response”; the pathways downregulated in CTX + MOX/CON comparison were mainly “TNFA signaling via NFKB”，“Hypoxia”; the pathways upregulated in CTX + MOX/CON comparison were mainly “Interferon gamma response”，“Interferon alpha response” (Fig. 7A-C). The above results suggest that moxibustion ameliorates POI mainly 3.4. Bulk RNA-seq results of ovaries from rats in each group To investigate the potential mechanism of moxibustion making impact, bulk RNA-seq was completed on the ovaries of rats in each group. The PCA plot and clustering heatmap showed that the sequencing samples meet the quality standards and the gene expression patterns within group were similar and comparable (Fig. 4A, B). When the threshold was set as “P < 0.05”, there were a huge number of DEGs in each pairwise comparison (Fig. 4C). 3.5. GO, KEGG, GSEA enrichment analysis results of Bulk RNA-seq After that, we performed GO, KEGG and GSEA enrichment analysis of the DEGs. GO functional enrichment results showed that: the downregulated terms in CTX/CON comparison were mainly “T cell activation”，“T cell differentiation”，“immune system process”; the upregulated terms in CTX/CON comparison were not significantly specific; the downregulated terms in CTX+MOX/CTX comparison were mainly “inflammatory response”，“acute inflammatory response”，“cell death”; the upregulated terms in CTX + MOX/CTX comparison were mainly “lipid metabolic process”，“regulation of reproductive process”，“response to steroid hormone”; the downregulated terms in CTX + Fig. 3. Moxibustion improves sex hormone levels in POI rats. A-F. Moxibustion reduces the FSH level (A), FSH/LH ratio (C) of POI rats, increases the concentration of AMH (D), E2 (E), but has no significant effect on LH (B) and T (F) concentration. *P<0.05，**P<0.01，***P<0.01，ns indicates no significance. 6 R. Zhao et al. Journal of Steroid Biochemistry and Molecular Biology 242 (2024) 106547 Fig. 4. Overview of bulk RNA-seq sequence alignment and data dispersion. A-B. PCA and clustering analysis show clear separation in three groups. C. Volcano plot shows up-regulated (red color) and down-regulated (green color) significant genes in pairwise comparisons between groups. related to cAMP signaling pathway and is largely inseparable from steroidogenesis of GCs. ovarian GCs in POI rats, but not theca cells. To test this, we performed IHC experiments of FSHR (the maker of GCs[21]) on ovary sections. The results showed that the number of FSHR-positive cells decreased in CTX group compared with CON group, while they increased significantly in CTX + MOX group compared with CTX group. At the same time, the transmission electron microscopic results showed that there were intact and clear membranes, uniform nuclear chromatin, abundant lipid droplets and mitochondria in the GCs in CON group; while in CTX group, there were less uniform nuclear chromatin, less intracellular lipid droplets, and swollen mitochondria; in CTX + MOX group, there were intact membranes, uniform nuclear chromatin, abundant lipid droplets in the cells, and insignificant mitochondrial swelling (Fig. 9 E). In order to further inspect and verify the effect of moxibustion on the function of GCs, we used western blot to detect the expression of steroidogenic key proteins in each group, and the results showed that the relative expression of FSHR and P450 arom in CTX group decreased compared with CON group, while their relative expression were similar in CTX + MOX group and CON group. However, the relative expression of StAR was not significantly different between CTX and CTX + MOX groups (Fig. 9.F, G). 3.6. GSVA pathway scoring results of Bulk RNA-seq To further clarify the phenotype and narrow down research scope, we continued the analysis using GSVA pathway scoring. The results showed that the scores of “cellular response to estrogen stimulus pathway” and “ovulation from ovarian follicle pathway” appeared a trend of "high-low-high" in CON group, CTX group and CTX+MOX group, while the scores of “peroxiredoxin 2 induced ovarian failure pathway” and “ovarian infertility pathway” showed the opposite trend (Fig. 8A-D). 3.7. Moxibustion improves the number and function of ovarian GCs in POI rats The GSVA scoring results showed that the scores of "response to follicle stimulating hormone pathway" and "regulation of steroid metabolic process pathway" appeared a trend of "high-low-high" in CON, CTX and CTX + MOX groups, while the scores of "response to luteinizing hormone pathway" showed the opposite trend (Fig. 9 A-C), which indicated that moxibustion may promote the number and function of 7 R. Zhao et al. Journal of Steroid Biochemistry and Molecular Biology 242 (2024) 106547 Fig. 5. GO functional enrichment results of Bulk RNA-seq. A-C. The biological process (BP), cellular component (CC), and molecular function (MF) enriched for DEGs in pairwise comparisons between groups revealed by GO analyses. Upper are pathways enriched for upregulated genes and below are pathways enriched for downregulated genes. Notably, E2, through a negative feedback mechanism, can inhibit the synthesis of FSH and LH, slowing the development of follicular atresia in POI pathogenesis and improving the elevated gonadotropin state in POI [23]. This forms the theoretical basis for HRT in POI and underscores why our study focused on granulosa cells (GCs). As one of the numerous somatic cells in the ovary, GCs are inseparable from follicular development and ovarian endocrine function: 1. GCs are essential for the normal development of follicles, and it is mainly localized in follicles, surrounding oocytes, can synthesize a variety of hormones and necessary growth factors, and express related receptors [24]. Under the guidance of pituitary FSH, GCs begin to proliferate, migrate, and express FSH receptor, E2 receptor, etc., so that primordial follicles develop into primary follicles that respond to hormones; then, under the stimulation of FSH and E2, they successively enter the stage of secondary follicle development, at which time GCs show explosive proliferation, produce a large amount of E2 and induce more FSH receptor production, promote the synthesis of estrogen, so that follicles grow rapidly and induce ovulation[25]. 2. GCs dominate ovarian endocrine function. Estrogen production is mainly divided into two processes: (1) Steroidogenic Acute Regulatory protein (STAR) acts as the initial carrier of the whole process, brings cholesterol into theca cells, and they converts cholesterol into Androstenedione (A2) and Testosterone (T); (2) The products diffuse through the basal membrane into GCs and accumulate continuously, while Aromatase P450 (P450 arom, CYP19A1) in GCs acts as the rate-limiting and critical enzyme in the catalytic step, converting A2 and T into 17β-Estradiol (E2) [26]. FSHR activates cAMP signaling to enhance CYP19A1 expression in GCs and promote estrogen production, while estrogen also enhances FSH action 3.8. Moxibustion regulates steroidogenesis by activating cAMP/PKA/ CREB signaling pathway in ovaries of POI rats To further explore the mechanism of moxibustion on POI, we continued to use GSVA pathway scoring to narrow down research scope, and the results showed that the scores of " Protein Kinase A signaling pathway" and "CREB pathway" showed a trend of "high-low-high" in CON, CTX, and CTX + MOX groups (Fig. 10A, B), and in combination with the results of KEGG pathway enrichment analysis in Fig. 6, we carried out western blot experiment of cAMP, PKA, and CREB in ovarian protein samples. The results showed that the relative expression of the above proteins was lower in CTX group compared with CON group, while they were significantly increased in CTX + MOX group compared with CTX group (Fig. 10C, D). 4. Discussion Currently, global concern regarding fertility issues has increased, with a growing trend of delayed conception. Reports indicate that from 1981 to 2019, the percentage of women over 30 giving birth to their first child rose from 15% to 51%[22]. This not only raises the risk of POI but also significantly increases fertility difficulty. Additionally, ovarian dysfunction can expedite the aging process of various organs in women, leading to hectic fever and night sweat, insomnia and dreaminess, anxiety and depression, or other symptoms similar to perimenopausal syndrome. This contradicts the contemporary pursuit of a high-quality life for women. Consequently, POI has emerged as a critical factor jeopardizing the physical and mental health of women at present. 8 R. Zhao et al. Journal of Steroid Biochemistry and Molecular Biology 242 (2024) 106547 Fig. 6. KEGG pathway enrichment results of Bulk RNA-seq. A-C. Top pathways involving DEGs in pairwise comparisons between groups revealed by KEGG analyses. On the left are pathways enriched for upregulated genes, on the right are pathways enriched for downregulated genes. 9 R. Zhao et al. Journal of Steroid Biochemistry and Molecular Biology 242 (2024) 106547 Fig. 7. GSEA pathway enrichment results of Bulk RNA-seq. A-C. Significant pathways enrichment in GSEA of DEGs in pairwise comparisons between groups. [27]. Up until now, moxibustion has extremely significant advantages as a treatment for POI when HRT is accompanied by numerous side effects. Compared with HRT, moxibustion can not only improve the hypoestrogenic state of patients, but also ameliorate the clinical symptoms of them as a whole and restore ovarian secretion and reproductive function [28]. The mild heat of moxibustion fire, light radiation effects, and volatile moxa components are the stimulating characteristic; warming yang and tonifying qi, promoting qi and activating blood circulation, regulating blood vessels are important effects of moxibustion; the local site of moxibustion is the direct object of moxibustion action; acupoints, skin, and meridians are the basis for the effect of moxibustion. Guanyuan and Sanyinjiao are the main acupoints selected, both of which are important acupoints for the treatment of gynecological diseases. Modern 10 R. Zhao et al. Journal of Steroid Biochemistry and Molecular Biology 242 (2024) 106547 Fig. 8. Moxibustion improves female reproductive related pathways in POI rats. A-D. GSVA scoring results show promoted effect on “response to estrogen stimulus” (A) and “ovulation” (B) pathways, antagonized effect on “ovarian failure” (C) and “infertility” (D) pathways in moxibustion on POI rats. medical research has also confirmed that the compatibility of Sanyinjiao with Guanyuan can effectively regulate the hypothalamic-pituitary-ovarian axis[13], but the related mechanism remains to be completed. Our team has been committed to the clinical practice and related research of moxibustion in the treatment of POI for a long time, so we have a lot of clinical experience and research basis. First, based on our previous work and other studies[14,29,30], we used the cytotoxic agent cyclophosphamide (CTX) to construct the POI rat model. Compared with CON group, rats treated with CTX showed slower weight gain rate (Fig. 1B), significantly disturbed estrous cycle (Fig. 2.A, B), smaller organ index of reproductive organs (Fig. 2.C, D), increased atretic follicle counts(Fig. 2.E, F), and reduced ovarian hormone secretion (Fig. 3 A-F); response estrogen stimulus and ovulation pathways were downregulated and ovarian failure and ovarian infertility pathways were upregulated in GSVA scoring results (Fig. 8 A-D), which indicated that our POI model was successfully constructed. Therefore, combined with the method previously established by our team[13,14], we treated POI model rats with moxibustion (hereafter referred to as CTX + MOX group). As shown in the results, the above phenotypic parameters were effectively improved in CTX + MOX group, and HE staining results showed that the dominant follicles increased, indicating that moxibustion could effectively ameliorate ovarian secretion and reproductive function in POI rats (Fig. 2.E, F). To further explore the potential mechanism of moxibustion action, we performed bulk RNA sequencing of ovaries from each group. The results of PCA and clustering analysis showed that the intra-group differences of samples were small and the comparability between groups was high (Fig. 4.A, B); volcano plot results showed that the expression patterns were significantly different between the groups at P < 0.05 (Fig. 4.C). Then, we used enrichment analysis to continue to explore, mainly using traditional GO and KEGG enrichment, as well as GSEA pathway enrichment and GSVA pathway scoring method which are more reliable. The results showed that the immune response ability, reproductive system function, T cell proliferation in CTX group were significantly inhibited (Figs. 5, 6, 7 A-C), which was largely related to the characteristics of cyclophosphamide-induced POI model[31], and also similar to the pathogenesis of POI [3], which more indicated the accuracy of our POI rat model. It is worth noting that moxibustion can effectively improve immune system function and reduce cell death (Figs. 5, 6, 7. A-C), which is also consistent with the mechanism of moxibustion action in other fields [32,33]; at the same time, the enrichment analysis results also suggest that moxibustion can also significantly improve ovarian hormone response (Figs. 5, 6, 7.A-C), come back to the neatly arranged and orderly GCs in MOX + CTX group compared with CTX group in Fig. 2B, and the ovarian hormone levels that are significantly improved in Fig. 3, it is reasonable to speculate that moxibustion may have a more significant promoting effect on ovarian GCs; more importantly, we noticed that cAMP signaling pathway was significantly enriched in CTX + MOX group (Fig. 6.A-C), and this pathway was inseparable from the steroidogenic function of GCs[34]. Therefore, combined with the known results, we hypothesized that moxibustion may improve steroidogenic function in GCs by affecting cAMP signaling pathway to achieve the goal of recovering ovarian function in POI rats. On the basis of the above results, to confirm our hypothesis, we first completed GSVA scoring for FSH, LH, and steroid-related pathways, the results showed that the target cells of moxibustion action were ovarian GCs rather than theca cells (Fig. 9 A-C). Indeed, IHC staining of FSHR (the marker of GCs) and its quantitative analysis of mean optical density also verified this opinion, and the number of FSHR-positive cells was significantly increased after moxibustion treatment (Fig. 9 D). The results of TEM also fully demonstrated the rescue effect of moxibustion on the GCs in POI model (Fig. 9 E). Finally, western blot results of FSHR and P450 arom (mainly present within GCs) and StAR (mainly present within theca cells) also suggested that the function of GCs in POI model was improved after moxibustion treatment, but not theca cells (Fig. 9. F, G). Thus, we have enough evidence that moxibustion restores ovarian function in POI by improving steroidogenic function in GCs. Finally, based on the target cells identified in the previous data, to further explore the mechanism of moxibustion, that is, to clarify the changes of cAMP signaling pathway before and after moxibustion treatment, we performed GSVA pathway scoring of cAMP signaling and steroidogenesis pathway. In ovarian GCs, PKA and CREB are essential for their steroidogenic function: CREB, the full name cAMP Response Element Bound protein, whose main function is initiating aromatase gene transcription and is essential for the activity of aromatase such as P450 arom in GCs[34]; PKA, the full name of Protein Kinase A, directly phosphorylates CREB, and this process is irreplaceable by other protein kinases[35]. Because both are downstream of the cAMP signaling pathway[34], and the cAMP signaling pathway has a clear trend in KEGG enrichment results, we only assessed CREB and PKA signaling pathways. The results conformed to our hypothesis, suggesting that moxibustion may affect cAMP signaling pathway dominated by cAMP, PKA, and CREB to act (Fig. 10. A, B). To finally confirm this opinion, we performed western blot experiment on cAMP, PKA, and CREB proteins, and the results demonstrated that moxibustion improved steroidogenic function in POI rats GCs by activating cAMP/PKA/CREB signaling pathway (Fig. 10. C, D). In conclusion, our data demonstrate that moxibustion regulates steroidogenesis in ovarian GCs via activation of cAMP/PKA/CREB to upgrade P450 arom, thereby ameliorate ovarian function impairment in POI rats. This will provide strong evidence to support the promotion and use of moxibustion in the treatment of POI in clinical practice. Funding information This work was supported by the Natural Science Foundation of Jiangsu Province (BE2020624); the Natural Science Foundation of Nanjing University of Traditional Chinese Medicine (XZR2021051). 11 R. Zhao et al. Journal of Steroid Biochemistry and Molecular Biology 242 (2024) 106547 Fig. 9. The promotional effect of moxibustion on the number, structure and function of ovarian GCs in POI rats. A-C. GSVA scoring results show promoted effect on “follicle stimulating hormone” (A) and “regulation of steroid metabolic process” (B) pathways, antagonized effect on “response to luteinizing hormone” pathway (C) in moxibustion on POI rats. D-E. IHC results show moxibustion increase FSHR positive cell. F. Transmission electron microscopy results show moxibustion repair organelles of ovarian GCs in POI rats. G-H. Western blot results show moxibustion promoted FSHR and P450 arom levels, with no significant effect on StAR. *P<0.05，**P<0.01，***P＜0.001，**** P＜0.0001，ns indicates no significance. 12 R. Zhao et al. Journal of Steroid Biochemistry and Molecular Biology 242 (2024) 106547 Fig. 10. Moxibustion regulates steroidogenesis by activating cAMP/PKA/CREB signaling pathway in ovaries of POI rats. A-B. GSVA scoring results show promoted effect on “Protein Kinase A” (A) and “CREB” (B) pathways in moxibustion on POI rats. C-D. Western blot results show moxibustion promoted cAMP, PKA and CREB levels. *P<0.05，**P<0.01，***P<0.001. References CRediT authorship contribution statement [1] G. T, K. H, R. S, van R. F, Breaking the silence around infertility: a scoping review of interventions addressing infertility-related gendered stigmatisation in low- and middle-income countries, Sex. Reprod. Health Matters 31 (2023), https://doi.org/ 10.1080/26410397.2022.2134629. [2] X. Jiao, H. Zhang, H. Ke, J. Zhang, L. Cheng, Y. Liu, Y. Qin, Z.-J. Chen, Premature ovarian insufficiency: phenotypic characterization within different etiologies, J. Clin. Endocrinol. Metab. 102 (2017) 2281–2290, https://doi.org/ 20170706140016. [3] European Society for Human Reproduction and Embryology (ESHRE) Guideline Group on POI, L. Webber, M. Davies, R. Anderson, J. Bartlett, D. Braat, B. Cartwright, R. 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Sun, Exposure to Di-(2-Ethylhexyl) phthalate drives ovarian dysfunction by inducing granulosa cell pyroptosis via the Rui Zhao: Conceptualization, Data curation, Formal Analysis, Investigation, Methodology, Software, Validation, Visualization, Writing – original draft, Writing – review & editing; Lingxiang Ran: Conceptualization, Formal Analysis, Methodology, Software, Visualization, Writing – original draft, Writing – review & editing; Hanyue Yao: Investigation, Methodology; Yizhi He: Investigation, Methodology; Xinru Lu: Investigation, Methodology; Weina Zhu: Resources; Yajie Zhang: Resources; Tianyi Zhang: Investigation; Shijie Shi: Investigation; Zheng Luo: Investigation; Cairong Zhang: Funding acquisition, Project administration, Supervision. Declaration of Competing Interest All authors declare that they have no conflict of interest. Data availability Data will be made available on request. 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