The Anti-Senescence Effect and Mechanism of 17β-estradiol on Pelvic Organ Prolapse Derived Fibroblasts

DOI: https://doi.org/10.21203/rs.3.rs-2469297/v1

Abstract

Background:

Recently, low estrogen and the age at menopause as independent risk factors for Pelvic Organ Prolapse (POP) were attracting high attention. In clinical practice, pre-/post-operative Local Estrogen Therapy (LET) shown effectiveness in alleviating POP symptoms. However, there is lack of scientific evidence to support the validity of these claims. Therefore, this study aimed to investigate the anti-senescence effect and mechanism of 17β-estradiol on POP derived fibroblasts.

Methods:

The primary fibroblasts cells were isolated and cultured form surgical POP samples (n = 8, age from 50–75), the passage-0 cells confluence at 80% takes about 15 days and the passage 3–5 cells were used for further test. Immunocytochemistry was used to characterize the primary fibroblasts, CCK8 assay was used to test the cell proliferative capacity and the Senescence-Associated β-Galactosidase (SA-β Gal) Staining was tested to calculate the senescence rate of fibroblasts. Moreover, western blotting was used to detect the expression of COL-I, COL-III, p16INK4A, p21, p-53, SIRT-1 and LC3-I/II protein. In addition, Transmission Electron Microscope (TEM) was used to observe the ultrastructure of fibroblasts.

Results:

The results showed that 17β-estradiol (E2) significantly promoted the POP derived-fibroblasts proliferation and reduced the staining rate of senescence-associated-β-galactosidase (SA-β-Gal), markedly enhanced the extracellular matrix protein COL-I and COL-III accompanied by the inhibition of senescent protein P16INK4a, as well as improved the cells autophagy and metabolic activity. In addition, E2 significantly up-regulated the anti-aging protein SIRT1 and markedly down-regulated p53 and p21, indicating the anti-senescence mechanism of E2 through mediated the Sirt1/p53/p21 axis pathway.

Conclusion:

We provide preliminary evidence that anti-aging effect and mechanism of estrogen on POP fibroblasts, hoping to provide a theoretical basis for estrogen against POP senescence, guide the clinical application and local administration of estrogen on POP treatment, thereby improve long-term maintenance and rejuvenation of the pelvic floor connective tissue.

Introduction

Pelvic Organ Prolapse (POP) is one of the common diseases of middle-age and elderly women, mainly manifested as uterine prolapse, anterior and posterior vaginal wall uterine prolapse, urinary retention, sexual dysfunction, et al. which seriously affected the life quality of women.(1) According to the epidemiological studies, the prevalence of POP in routine vaginal examinations is higher than 50%,(2) It is estimated that 12.6% of women by the age of 80 will require surgical correction due to POP, and 9.2 million women will be affected by POP By 2050 in the United States.(3, 4) At present, the causes of POP are not entirely understood, but may be multifactorial, including increased age, vaginal delivery, parity, decreased estrogen levels, high body mass index (BMI), increased intra-abdominal pressure and genetic factors, et al (5, 6)

At present, E2 have been widely used in clinical local estrogen therapy (LET) before and after POP surgery, patients are usually treated with E2 in the form of estrogen cream (0.5 or 1.0 g each time, E2 content is about 10%) or tablet (10 µg each time) for 4–6 weeks.(7–9) It is reported that, pre-operatively vaginal estrogen application for 4–6 weeks improved the post-operatively matrix restore and maintaining the integrity of pelvic floor connective tissues(10) through increased the collagen synthesis and vaginal epithelium thickness,(10) enhanced the blood circulation and elasticity,(11) and reduced the degrading enzyme activity and the bacteriuria frequency and the cystitis incidence,(12) otherwise, post-operative estrogen treatment reduced the incidence and severity of urinary frequency and urgency, (13, 14) and decreased the appearance of granulation tissue and other objective atrophy symptoms,(15) and without obvious adverse events. Although the local E2 treatment has been shown to be effective in alleviating POP symptoms after surgery, however, The International Urogynecological Association research and development committee point out that the current evidence in POP local estrogen therapy is still controversial, the duration, optimal dosage, long-term effects and cost-effectiveness of LET are still unclearly.(16) In addition, patients are still very cautious about estrogen therapy, include the long-term safety concerns, complication and the compliance.(17, 18)

The anti-senescence effect of estrogen is not only been widely used in clinical, but also widely studied at the cell and animal level, many studies reported on the senescence regulating effect of estrogen, owing to the lack of estrogen, dermal aging is accelerated immediately after menopause.(19) E2 also regulating bone development and metabolism, calcium balance, cell growth and differentiation to resist age-related bone resorption and stimulate bone formation through enhanced the biological activity of bone vitamin D receptor (VDR).(20) Recently, substantial evidence has shown that E2-mediated activation of the sirtuin family, especially SIRT1 (a mammalian NAD+-dependent histone deacetylase), contributes to the anti-aging of skeletal system and vascular system and the repair of neurodegenerative diseases by increasing endothelial nitric oxide synthase (eNOS) activation and autophagy, reducing oxidative stress, inflammation and DNA damage.(21, 22) Animal studies have proved that SIRT1 has a protective effect on atherosclerosis, at least in vascular endothelial cells and smooth muscle cells.(23, 24) Besides, 17β-E2-induced upregulation of SIRT1 can promote autophagy through the AMPK-mTOR pathway and inhibit osteoblast and chondrocyte apoptosis, thus, become a target in the treatment of osteoporosis.(25, 26) However, the relationship between estrogen and SIRT1 in the background of menopause-induced POP development is unclear, the relative anti-senescence mechanism of estrogen on POP mediated by SIRT1 has not been reported yet.

With the extension of human longevity, women are lives in a long stage of estrogen-deficient state after menopause,(27) the ovarian steroid hormone 17β-estradiol (E2) deficient, which will result to a series of age-related diseases especially the pelvic floor organ prolapse (POP).(28, 29),(30, 31) Pelvic organs including uterus, vagina, bladder, urethra and pelvic floor muscles are generally contained estradiol receptors α and β (ESRα/β), which are very sensitive to estrogen level, the decreased of estrogen levels may have a significant effect on reproductive organs and lower urinary tract function.(32) In addition, estrogen also have a profound impact on the synthesis and metabolism of the components of pelvic connective tissue such as collagen, elastin and fibroblasts.(33) The atrophy of these tissues would weaken the capacity of the pelvic floor muscles, connective tissues and ligaments tissues to support the pelvic organs, thus caused these organs descent to the vagina, eventually lead to POP.(34, 35)

Although, the estrogen is generally used in POP clinical treatment, however, the certain effect and mechanism of estrogen against POP senescence have not been reported yet and the current data are not enough to guide the clinical local estrogen treatment and patients are still very cautious about estrogen therapy. Thereby, in our study, we provide preliminary evidence that anti-aging effect and mechanism of estrogen on POP fibroblasts, hoping to provide a theoretical basis for the study of estrogen against POP senescence, and guide the clinical application and local administration of estrogen on POP treatment.

Materials And Methods

2.1. Sample capture.

All Pelvic floor vaginal anterior wall prolapse samples (n = 8, age from 50–75) were obtained from surgical patients at the department of obstetrics and gynecology department, West China Second University Hospital (Chengdu, China). All patients received written informed consent, which was approved by the Ethics Committee of Sichuan University. The patient was clinically diagnosed with serous III to IV degree of vaginal anterior wall prolapse and did not receive any other treatment 6 months prior to harvesting the sample.

2.2. Pelvic floor fibroblasts isolation and culture.

The obtained surgical sample was immediately cut into small pieces, digested with type-I collagenase at a concentration of 1 mg/ml, placed in a 37°C water bath shaker at 150 rpm for 2 hours, and then passed through 70µm and 40µm cell filters filtration in sequence, the obtained single cell suspension were cultured in DMEM medium (Hyclone) supplemented with 15% fetal bovine serum (Gibco) and 1% penicillin-streptomycin (Hyclone), and all cell cultured in a humidified incubator (Heraeus) with 5% CO2 saturation at 37°C, the medium was changed for every 3 day, the first passage cell confluence at 80% takes about 15 days and can be sub-cultured for further test, and the cells used in our experiment in the third or fourth passage.

2.3. Immunohistochemistry.

Immunocytochemistry was used to characterize fibroblasts specific surface markers, the cells were digested by trypsin-EDTA and seeded into 6 cell plates at the concentration of 1×105 cells/well, and incubated in a 5% CO2 saturation at 37°C. When the cell confluence at 80%-90%, washed with PBS and fixed in 4.00% paraformaldehyde for 15 minutes, then, 0.50% Triton X-100 and 5.00% normal goat serum was used to permeate and block for 5 minutes, respectively. After that, the cells were incubated with the primary antibodies including the use of anti-Cytokeratin, anti-α-SMA and anti-Vimentin (1:200, Abcam) antibodies at 4°C overnight, and the second antibody was incubated at room temperature for 1 hours. Finally, the cells were counterstained with haematoxylin and visualized with the DAB.

2.4. Administration.

The effect of different concentration (range from 10− 5-10− 10 mol/L) of E2 on pelvic floor fibroblasts proliferation were tested by CCK-8 assay at 12h, 24h and 48h, After obtain the best administration concentration, cells are administrated with E2 (with the best concentration) and rapamycin (with the concentration of 10− 8 mol/L) for further detected, besides, cells are treated with lysosomotropic reagents chloroquine (with the concentration of 40 µmol/L for measuring "autophagy flux",(36) and all drugs are purchased from solarbio, China.

2.5. Proliferative assay.

Colorimetric Cell Counting Kit (CCK8, Beyotime, China) was used to test the cell proliferative capacity. The cells were seeded at a density of 1×104/well into the 96-well plate, 10 µL CCK-8 reaction solution was added to each well after administration for 12h, 24h and 48h and incubated for 2 h. The microplate reader was used to measure the absorbance at 450 nm. The final absorbance value is calculated from the absorbance value of the test well minus the absorbance value of the reagent background.

2.6. Senescence-Associated β-Galactosidase (SA-β Gal) Staining.

The senescence of the pelvic floor fibroblasts was evaluated by SA-β gal staining kit (Beyotime, China). The cells were seeded at a density of 1×105 cells/well on coverslip, when the cell confluence at 80%-90%, fixed with 4% paraformaldehyde for 15 min and washed three times with PBS, finally, incubated the cells with SA-β-gal reaction solution at 37° C for 12 h according to the manufacturer’s protocol, then washed three times with double-steaming water, SA-β gal positive cells were counted under the optical microscope.

2.7. Western blotting analysis.

Western blots were used to measure protein expression. The concentration of protein was determined by the BCA Kit (Beyotime, China). The total protein equivalent (30 µg) of each sample was separated by 10% sodium dodecyl sulphate-polyacrylamide gel (SDSPAGE) and polyvinylidene fluoride (PVDF) membranes (Invitrogen Life Technologies, Inc) in the membrane transfer system. Then, the membrane was placed in 5% fatfree milk and sealed at room temperature for 2 hours. After washing with TBST (Tris Buffer Saline-Tween 20), Followed by incubated with primary antibodies against p16INK4A, p21, p-53 sirt-1(Cell Signaling Technology, Inc), COL-I & COL-III (Proteintech, Inc), LC3-I/II and GAPDH (Signalway Antibody, Inc) (1:1000 dilution) at 4 ℃ overnight, and then incubated with secondary antibody (anti-rabbit or anti-mouse IgG-conjugated with HRP) (1:3000 dilution) at room temperature for 1 hours. the membranes were visualized in Molecular Image® ChemiDocTM XRS + system (Bio-Rad Inc., USA) with Image LabTM Software and analyzed by Image J 1.44p software (National Institutes of Health, USA).

2.8. Ultrastructure of fibroblasts observed under transmission electron microscope (TEM).

Take the logarithmic growth phase fibroblasts, expand culture and collect cells, so that the total number of cells reaches 2×1010, washing with PBS and centrifuge at 1,000 rpm for 5 minutes, then resuspend the cells with 1:6 diluted fixating solution (3% glutaraldehyde: PBS) and stand at 4 ℃ for 5 min, after that, centrifuge at 10000 rpm for 15 min, discard the supernatant, and then fix with 3% glutaraldehyde at 4 ℃ for 2 h. after dehydration, immersion, embedding, slicing and staining, the cells are observed under transmission electron microscope (TEM).

2.9. Statistical analysis.

Results were analyzed by GraphPad Prism 9.0 Software (GraphPad, San Diego, CA, USA). The values are expressed as mean ± SEM at least three independent experiments performed. The data were analyzed using one-way analysis of variance (ANOVA) and post-hoc analysis, Pvalues < 0.05 were considered to indicate a statistically significant difference, the significance was calculated by comparing the controls with experimental groups.

Results

3.1. Identification of pelvic floor fibroblasts.

The isolated primary cultured fibroblasts of human vaginal anterior wall generally need about 15 days to reach 80%-90% confluence, the growth process of these primary fibroblasts as shown in Supplementary-Fig. 1.The confluence fibroblasts are mostly long fusiform, flat star, some are triangular, and arranged in clusters under the inverted microscope, as shown in Fig. 1. We performed cellular immunohistochemistry to identify the isolated and cultured fibroblasts. Figure 1 shows that the specific structural proteins of fibroblasts, including vimentin and α-smooth muscle protein (α-SMA), are stained as brown granular substances in the cytoplasm, and epithelial cell markers Cytokeratin staining was negative, Indicating the isolated cells were verified as fibroblasts.

3.2. The effect of 17β-estrogen on human pelvic floor fibroblasts proliferation.

As shown in Fig. 2-A, the absorbance of primary fibroblasts after treated with E2 for 48h were obviously higher than that of 12h and 24h, indicating that E2 treatment for 48h has a stronger ability to promote the proliferation of fibroblasts. In addition, the absorbance value of fibroblasts increased with the E2 concentration until 10− 7 mol/L in each treated time point, after that, gradually decreased, and the absorbance value has a significant difference between the concentration of 10− 7 mol/L and control (*P < 0.05). Therefore, we choose E2 treatment for 48h and concentration for 10− 7 mol/L for further research. We further illustrated the proliferation promote ability of E2 and found that E2 can significantly enhanced the proliferation of fibroblasts, however, rapamycin as a positive control seems to have no effect on cell proliferation, as shown in Fig. 2-B.

3.3. SAL-β-gal of human pelvic floor fibroblasts.

In order to detect the anti-aging effect of E2, the β-galactosidase staining was tested. As shown in Fig. 3-A, the cells stained blue are β-galactosidase staining positive cells. Compared with the control, estrogen administration significantly decreased the number of positive cells (**P < 0.01), and there is no significant difference between the E2 and rapamycin group. Figure 3-B shows the statistic results of the staining positive rate, the staining rate of E2 group is significantly reduced by 3.9% compared with the control (**P < 0.01), the staining results of all samples are shown in supplementary-Fig. 2, indicating that E2 has a significant anti-aging effect.

3.4. The expression of senescence-related function and marker proteins of human pelvic floor fibroblasts.

Collagen fibers as the main component of the ligament tissue, mainly composed of type-I and III collagen. The decrease in the quantity and quality of collagen fibers and the change in the ratio of collagen subtypes would result to the flabby of the pelvic floor tissue, ultimately lead to pelvic floor organ prolapse. The senescent cells usually highly expressed the senescence marker protein P16INK4a, indicating that P16INK4a plays a crucial role in regulating cell aging. As shown in Fig. 4, our results showed that the expression level of COL-I and COL-III in the E2 treated fibroblasts are remarkedly increased by 1.79 (*P < 0.05) and 1.93 (**P < 0.01) times respectively, and the senescence marker protein P16INK4a significantly decreased by 2.27 (**P < 0.01) times compared to control, and a similar P16INK4a expression trend was also observed in the rapamycin group, indicating that E2 may improve the secretion of functional proteins of senescent cells to remodel the ECM, and remit the cells senescence by reduce the ageing protein expression, thereby exerting anti-senescence effects.

3.5. Changes of autophagy flux and ultrastructure of pelvic floor fibroblasts.

To obtain further insights into the anti-senescence capability of E2 on fibroblasts, we measured the cell autophagy flux-LC3 turnover rate. The difference of the ratio of LC3-II/I with and without chloroquine between the different treatment groups represents the degradation amount of LC3 delivered to the lysosome. As shown in western blot analysis (Fig. 5), the ratio of LC3-II/I (the increase of the ratio indicates an increase of autophagic flux) with or without chloroquine were 2.357 ± 0.03 and 1.753 ± 0.19 under the control conditions, while the ratio of LC3-II/I with or without chloroquine increased to 4.382 ± 0.20 and 2.136 ± 0.14 under the E2 treatment conditions (Fig. 5-B). The ratio of LC3-II/I in the E2 with chloroquine group were significantly different in the control (with chloroquine) (*P < 0.05) and estrogen (without chloroquine) (**P < 0.01) group. Indicating that E2 regulated the formation and degradation of autophagy and increased phagocytic flux to plays an anti-senescence role.

Ultrastructure of fibroblasts were examined by transmission electron microscopy (TEM). As shown in Fig. 5-C, in the control group, mitochondria were swollen, vacuolated, and enlarged, mitochondria cristae ruptured and disappeared, endoplasmic reticulum expanded and a small number of autophagosomes and autolysosomes were observed. in the E2 treatment group, mitochondria quantity increased accompany by swelling decreased, mitochondria cristae were distinct, autophagosomes and the autolysosomes increased, and the similar results were also observed in rapamycin treated group. Implying that E2 inhibit senescence by promoting the cell metabolic activity.

3.6. Changes in the SIRT1/p53/p21 axis in fibroblasts after E2 treatment.

Silent Information Regulator 2 Related Enzyme 1 (SIRT1) is a histone deacetylase, which regulates cell proliferation, differentiation, metabolism, aging and apoptosis to play a crucial role in anti-senescence process. In our study, SIRT1 was markedly down-regulated in senescent fibroblasts, accompanied by markedly up-regulation of p53 and p21. However, after E2 administration, The SIRT1 was significantly up-regulated by 1.50 times (**P < 0.01), and p53 and p21 were significantly down-regulated by 2.40 and 1.59 times (**P < 0.01) compared to the control, and a similar trend of the level of p21 and p53 was also observed in the rapamycin group (Fig. 6). The SIRT1/p53/p21 axis is a typical aging regulation pathway, these results indicated that the SIRT1/p53/p21 pathway was involved in regulating the E2-mediated anti-aging process of fibroblasts.

Discussion

With the increase of age, women are in a status of long-term estrogen deficiency after menopause.(27) The function of various organs and tissues experienced senescent changes, especially the pelvic floor organs and tissues, lead to severe vaginal anterior and posterior wall prolapse, uterine prolapse, stress urinary incontinence, etc.(28, 29) The increase of senescent cells was believed to have a profound negative impact on tissue function in the elderly animal tissues.(37, 38) Thus, the basic biological mechanisms mediated by cellular senescence may be at least partly responsible for age-related tissue dysfunction, degeneration and pathological changes.(39) The main functional cells in the connective tissue of the pelvic floor are fibroblasts, alter of their quantity, activity, secretion and synthesis capability of ECM may contribute to the damage of the elastic and strength of the tissue and fascia. Although estrogen was often used as local treatment after pelvic organ prolapse surgery, its specific role is unclear. In order to verify the anti-aging effect and mechanism of estrogen, we cultured primary fibroblasts and identified the estrogen-mediated improvement of the aging phenotype. The results showed that, compared with control, after treated with estrogen, the senescent fibroblasts proliferation activity, autophagy flux and metabolic activity was enhanced and β-galactosidase activity was decreased, the expression of functional protein include types-I and -III collagen were significantly increased and senescence-related marker p16INK4a was significantly reduced. These results were consistent with several key anti-senescence phenotype which displayed in neurons, liver cells, and adipocytes.(40, 41) In addition, we illustrated that estrogen exerts anti-senescence effects by mediating the sirt1 axis. Our results showed that estrogen improved the senescent fibroblasts in the pelvic floor tissue, providing a theoretical basis for local estrogen administration after clinical POP surgery.

Our study showed that, the 17β-estradiol has the most significant ability to promote proliferation at the concentration of 10− 7mol/L, which is consistent with other research of 17β-estradiol (10− 7 mol/L) decreased senescence and improved the osteogenic ability of mini-pigs-BM-MSCs. Also, 17β-estradiol (10− 7 mol/L and 10− 9 mol/L) prevented telomere shortening by reducing oxidative stress and decreasing h-MSC senescence (42).

Since cell senescence is accompanied by the inhibition of cell proliferation,(43) our results showed that E2 promoted the proliferation of senescent fibroblasts. Paola et al,(44) also found that E2 improved the proliferation activity of human skin keratinocytes/fibroblast and prevented the cells from UV damage. SA-β-Gal is a method used by other studies to verify the reversal of aging.(45) In our study, we used SA-β-Gal to verify the reversal effect of estrogen on fibroblast senescence, and found that E2 was able to inhibit the senescence of fibroblasts.

Changes in the collagen content of the pelvic floor connective tissue, such as collagen degradation, structural changes and proportions imbalanced also play an important role in POP. Kobak et al,(46) found that collagen and elastic fibers were immaturity and weakened the mechanism support effect of pelvic floor, which increased the incidence of uterine prolapse. Moalli et al,(47) found that the decrease of the type-I/-III ratio of POP patients’ fascia caused a significant weakened of the pelvic floor tissue. In estrogen-deficient women, Types-I and -III collagens are considered to reduced, and the difference in collagen subtypes is also recognized in postmenopausal women, manifested as a decrease in type-I and -III collagen and a reduce of type III/I ratio.(48) The clinical results of pre-operatively vaginal estrogen application for 6 weeks increased the synthesis of collagen, improved the mature of collagen and increased the thickness of the vaginal wall, thereby improving the postoperatively matrix repair and maintaining the organizational integrity of pelvic floor connection,(10) our results also showed that E2 may improve the secretion and synthesis of extracellular matrix of fibroblasts, and promoted the expression of type-I/-III collage, as Fig. 7 shown. In addition, as well as increased the expressions of collagen type I, collagen type IV, elastin, and fibrillin-1.(49) Researchers had illustrated that E2 increased collagen maybe by induced VEGF and improved the TGF-β, and reduced collagen degradation by inhibited MMPs and increased TIMP in the dermal fibroblast.(49–51)

It is generally believed that E2 plays a crucial role in mitochondrial biogenesis and macrophage/autophagy function through estrogen receptors.(52) The LC3 turnover rate is one of the useful parameters for measuring autophagy flux. Cells treated with chloroquine would increase the pH of lysosomes or inhibit the fusion of autolysosomes, thus inhibit the degradation of LC3-II, measuring the accumulation of LC3-II so that to test the autophagy flux.(53) As shown in Fig. 7, we tested the changes of cell autophagy flux, mitochondria and autophagosomes after E2 treatment and found that E2 play an anti-aging effect by mediating the increase of autophagy flux and cell metabolic activity. Similar results have found by others researchers(53) found that estrogen receptor alpha (ERα) promotes intestinal homeostasis and protects the host from harmful inflammation and mitochondrial dysfunction through autophagy activation, Singh and Preciados et al.(54, 55) found that hormone-induced activation of ERRα regulates cell autophagy and mitochondrial division and biogenesis, ERβ can also induced autophagy to inhibit the migration and invasion of breast cancer cells, Song and Gavali et al.(52, 56) found that E2 promoted autophagy during osteoblast differentiation by up-regulating rab3gap1, so as to increase the survival and mineralization capacity of osteoblasts. In addition, mitochondria and autophagy play a central role in cell energy metabolism, its dysfunction leads to metabolic disorders and pathological features of aging.(57) Estrogen-related receptor is a nuclear receptor (NRs) that regulates gene transcription of mitochondrial biogenesis in a tissue-specific manner.(58, 59) Indicating that hormone or pharmacologic-induced activation of Estrogen-related receptor can be used to improve the cell autophagy and mitochondrial function in metabolic and neurodegenerative diseases and aging.

We also explored the mechanism of estrogen-mediated inhibition of fibroblast senescence. SIRT1 is a mammalian NAD+-dependent histone deacetylase. SIRT1 has been shown to deacetylate p53 to regulate apoptosis, stress response, cell metabolism, DNA repair and cell aging,(60, 61) thus eliminate cell senescence and apoptosis. Once p53 was acetylated, would aggravate the expression of growth-suppressing genes and contributed to cell senescence,(62) Sasaki’s data showed that E2 up-regulates the expression of SIRT1 in ovariectomy (OVX) induced senescent vascular endothelial cells and deacetylates p53, thereby, remit arterial senescence and atherosclerosis caused by menopause, and these results was consistent with our research.(23) Estrogen usually mediates downstream reactions through Estrogen Relative Receptor (ERR) activation, clinical local estrogen therapy increased the ERα expression to mediate the proliferation of posterior vaginal tissues in postmenopausal women has proved this conclusion.(63) Studies have also found that 17β-estradiol can eliminate oxidative stress in an ERα/sirt1-dependent manner to improve memory impairment, neuroinflammation, and neurodegeneration in adult mice.(21) In addition, both estrogen and hypothalamic ERα are related to aging (64) and long-term estrogen therapy may prolong the healthy lifespan of postmenopausal women.(65) In our study, the expression level of Sirt1 was significantly up-regulated after estrogen treatment. More importantly, estrogen not only up-regulate Sirt1, but also inhibit p53 by deacetylating it and further inhibit the expression of p21 to inhibit the senescence of primary fibroblasts in the pelvic floor tissue, which support the view that estrogen inhibit fibroblast senescence through the Sirt1/p53/p21 axis. Similar results were found in research of Wen et al, that the Sirt1/p53/p21 signaling pathway plays an important role in inhibiting the senescence of osteoblasts in aged rats.(66, 67) As shown in Fig. 7, in conclusion, our study illustrated that the estrogen mediated the signal pathway of sirt1/P53/P21 axis through activated the estrogen relative receptors to decrease the number of senescent cells and promote the proliferation of fibroblasts, enhance the synthesis, secretion and maturation of ECM and improve the fibroblasts metabolic function, thereby alleviating the pelvic floor muscle content, muscle fiber atrophy and degeneration to inhibit the POP progress.

Limitation

At present, our results limited focused on the role of estrogen in regulating the Sirt1/p53/p21 axis, and the system anti-aging mechanism still needs to be supplemented. In addition, E2 exerts anti-aging effects by regulating other signaling pathways such as PI3K-Akt/mTOR and MAPK et al, which also need further in-depth study.

Conclusion

The present study indicated the estrogen promoted the proliferation and autophagy activity of fibroblast, as well as enhanced the synthesis, secretion of extracellular matrix such as COL-I and COL-III. In addition, the current study observed that the estrogen played its improvement role by activating the sirt1/P53/P21 axis in fibroblasts. These findings provide a theoretical basis for the anti-aging effect and mechanism of estrogen on POP fibroblasts, which indicated that the clinical application and local administration of estrogen on POP treatment may be helpful for long-term maintenance and rejuvenation of connective tissue of the pelvic floor.

Declarations

Ethics approval and consent to participate

All Pelvic floor vaginal anterior wall prolapse samples (n = 8, age from 50-75) were obtained from surgical patients at the department of obstetrics and gynecology department, West China Second University Hospital (Chengdu, China). All patients who volunteered to participate were required to complete an informed consent form to be enrolled in the study. This study was approved by the Ethics Committee of Sichuan University.

Ethical guidelines

all methods were carried out in accordance with relevant guidelines and regulations and all experimental protocols were approved by a named institutional and/or licensing committee.

Consent for publication

not applicable

Availability of data and materials

The datasets used and/or analysed during the current study available from the corresponding author on reasonable request and part of the datasets are included in the supplementary information files.

Competing interests

The authors have declared that no competing interest exists. The manuscript has been read and approved by all the authors and each author believes that the manuscript represents honest work.

Funding

Thanks for the Funding as the following: 1. Foundation of Sichuan Provincial Science and Technology Program (Grant Nos. 2022YFS0084, 2019YFH0147 and 2019YFH0158). 2. Cooperation project for academician & experts workstation (Grant no. HX-Academician-2019-06) and 1.3.5 project for disciplines of excellence (Grant No. ZYJC18016) provided by the West China Hospital, Sichuan University.

Authors' contributions

Juan Cheng, Jiang Wu, Zhiwei Zhao, Yali Miao propose the research conception and design; Juan Cheng, Zhiwei Zhao, Ling wang capture, analysis and interpret the study; Juan Cheng, Zhiwei Zhao draft the article; Jiang Wu, Yali Miao, Ling wang, Jirui Wen revise the important intellectual content; Yali Miao, Jiang Wu Final approval of the version to be published; Jiang Wu, Yali Miao agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the article are appropriately investigated and resolved.

Acknowledgements

Thanks for the Funding as the following: 1. Foundation of Sichuan Provincial Science and Technology Program (Grant Nos. 2022YFS0084, 2019YFH0147 and 2019YFH0158). 2. Cooperation project for academician & expert workstation (Grant no. HX-Academician-2019-06) and 1.3.5 project for disciplines of excellence (Grant No. ZYJC18016) provided by the West China Hospital, Sichuan University. 

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