The delaying effect of Qingxin Zishen decoction on ovarian aging: Examining regulation of ovarian mitochondria apoptosis in aged rats

Ovarian aging is a significant challenge in gynecology, and there is currently no effective treatment for it. However, the medicinal agent Qingxin Zishen decoction (QZD) has shown potential in the treatment of ovarian dysfunction. The present study aimed to evaluate the mitochondrial apoptotic mechanism of delayed ovarian aging in QZD in aging rats. The healthy female Sprague-Dawley (SD) rats ( n = 40, 350 ± 20 g) were randomly assigned to different dosage groups and 4-month-old SD rats ( n = 10) were assigned to the control group. QZD groups were treated with QZD for four weeks


Introduction
Menopause is a natural physiological process affecting women.Clinical conditions such as ovarian aging and premature failure pose a serious threat to female reproductive health.The common causes of ovarian aging include genetic factors (chromosomal abnormalities and gene variations), iatrogenic factors (surgery, radiotherapy, and chemotherapy), immune factors (autoimmune dysfunction may cause ovarian function damage), and environmental factors (Yu and Chen, 2023).As ovarian function declines, fluctuations in sex hormone and central neurotransmitter levels can result in various physiological and psychological changes and discomfort (Monteleone et al., 2018).One of the most challenging issues in gynecology is identifying effective ways to treat and delay ovarian aging and failure.Despite the expansion of the concept of women's health to encompass physical, mental, and emotional well-being, concerns regarding pregnancy, menopause, and ovarian health remain significant (Stuenkel and Manson, 2021).Consequently, management of the female life cycle in relation to ovarian aging has become a critical research focus worldwide.
Moreover, the incidence of premature ovarian failure (POF) is closely correlated with apoptosis.Oocyte maturation depends on the maintenance of ovarian and follicular microenvironment homeostasis.Apoptosis plays a crucial role in the elimination of germ cells at all stages of oocyte formation and post ovulation (Bai and Wang, 2022;Liang et al., 2021).The majority of germ cells are eliminated through follicular atresia, which is brought about by apoptosis, while <1 % ultimately transition to oogonia (Coticchio et al., 2015).Thus, any disturbance in apoptotic mechanisms can lead to follicular atresia without oocyte maturation, resulting in premature depletion of the ovarian reserve.
Given its advantages in improving ovarian function and preventing ovarian aging, with fewer side effects, traditional Chinese medicine (TCM) has gradually gained attention.Professor Xia Guicheng, a pioneer in TCM with over 40 years of clinical experience, has created the "Qingxin Zishen decoction" (QZD), which has achieved beneficial effects in treating ovarian dysfunction and POF while also improving serum sex hormone and central neurotransmitter levels (Meng et al., 2017).Specifically, menopausal-related symptoms, such as hot flashes, sweating, upset, and insomnia, can also be significantly improved (Sin et al., 2023).Moreover, compounds extracted from TCM have been observed to effectively alleviate oxidative stress in the ovary and decrease apoptosis in ovarian granulosa cells and oocytes (Yan et al., 2022).Consequently, these compounds regulate the function of ovarian granulosa cells by controlling follicle development, prolonging their lifespan, and delaying the onset of ovarian aging, thereby reducing the incidence of POF (Liang et al., 2021;Xie et al., 2023).However, mitochondrial apoptotic pathways following QZD treatment have not yet been evaluated.Therefore, this study aimed to investigate the molecular mechanisms underlying the therapeutic effects of QZD in the ovaries of aged female rats.

Animals
Female Sprague-Dawley (SD) rats (n = 50) were housed under controlled conditions (24 ± 2 • C and 45 %-65 % humidity) under 12hour light/dark cycle.Food and water were provided ad libitum.There was no restriction on access to water and food.Healthy female SD rats (n = 40, 350 ± 20 g) were randomly assigned to different dosage groups and 4-months-old SD rats (n = 10) were assigned to the control group.All rats were fed adaptively for two weeks before QZD treatment.All animal experimental procedures were approved by the Nanjing University of Chinese Medicine Committee on Laboratory Animal Care and strictly followed rules and regulations.The experiments were performed according to the international guidelines of the National Institutes of Health.

Experimental design and drug treatment
Qingxin Zishen decoction was prepared by the traditional Chinese Medicine Pharmacy of Jiangsu Provincial Hospital of traditional Chinese Medicine and is composed of the following eight herbal medicines: Gouteng (Ramulus Uncariae Cum Unicis) 15 g, Lianzixin (Plumula Nelumbinis) 5 g, Huanglian (Rhizoma Coptidis) 3 g, Suanzaoren (Semen Ziziphi Spinosae)15 g, Gandihuang (Radix Rehmanniae Recens) 10 g, Shanzhuyu (Cornus Officinalis) 9 g, Danshen (Salvia miltiorrhiza Bunge) 10 g, Fuxiaomai (Triticum aestivum L) 30 g.The quality and dosage of these herbs were strictly guaranteed and processed according to the Chinese Pharmacopoeia (2015) (Dan et al., 2016).All herbs were ground and dissolved in distilled water, subsequently decocted, and concentrated into crude medicine at a concentration equivalent to 3.2 g/ ml.The samples were stored at 4 • C until future use.
Vaginal exfoliated cell smearing was performed for 14 days to confirm the estrous cycle disorder in the animals.The rats were then randomly divided into four groups (n = 10 per group): aging rat model group, low-dose, medium-dose, and high-dose QZD groups.Ten 4month-old female SD rats with regular estrous cycles were used as the young control group.Animals in the low-dose, medium-dose, and highdose groups were administered QZD at 8 g/kg, 16 g/kg, and 32 g/kg, respectively, through oral gavage, while the model group and young control group were administered 10 ml/kg 0.9 % saline.The rats were administered QZD once a day at 0830-0930 according to the above doses.The weight of the animals was measured every other day to adjust the dosage for four weeks.Twenty-four hours after the last administration, all the animals were sacrificed by cervical dislocation and fixed in the abdominal position.The last rib, mid-axillary line, and intersection approximately 2 cm from the outside of the spine were taken.Following hair removal, the animal skin and dorsal muscles were dissected to expose the white cellulite surrounding the ovary.Upon careful separation of the cellulite, ovaries were quickly extracted, snap-frozen in liquid nitrogen, and stored at − 80 • C for future use.
Total RNA was extracted from ovary tissue using TRIZOL (Invitrogen, USA) and converted into cDNA using a reverse transcription kit (Invitrogen, USA) according to the manufacturer's instructions.Two micrograms of total RNA were used for reverse transcription to produce cDNA.Polymerase chain reaction (PCR) was then performed on a Multi Gene Gradient TC9600-GPCR thermocycler (Labnet, USA) to detect the expression of Bcl-2, Bax, cyto-C, Apaf-1, caspase-9, and caspase-3 in ovary tissues using TRIzol, cDNA kit, and other related materials (Invitrogen, USA), according to the manufacturer's instructions.Primers were designed using the Primer5 software, and their sequences are listed in Table 1.The reactions were performed under standard conditions (95 • C for 5 min; 32 cycles of 95 • C for 40s, 56 • C for 40s, 72 • C for 45 s).Gel electrophoresis was then performed using the PCR products and BandScan4.3was used for the analysis of electrophoretic bands, with

Table 1
Primer sequence and PCR product size.

Statistical analyses
All statistical analyses were performed using SPSS software version 25.0 for Windows (IBM Corp., Chicago, IL, USA).The pairwise comparison of measurement data between groups was analyzed using oneway analysis of variance (ANOVA) to test for significant differences among the five groups.All data are expressed as the mean ± standard deviation (SD), and statistical significance was set at p < 0.05.

Effects of QZD on mRNA levels of pro-and anti-apoptotic genes
To determine the molecular pathways that mediate the therapeutic effects of QZD on ovarian aging, we examined the mRNA expression of anti-and pro-apoptotic markers in the ovarian tissues of naturally aged rats and their corresponding young control groups (Fig. 1).
Bcl-2 mRNA expression was significantly lower in the ovaries of the aging rat model group than in young rats (P < 0.001).Similarly, rats that received different dosages of QZD showed significantly higher Bcl-2 mRNA expression levels than aged rats that received a normal diet (P < 0.001) (Fig. 1A).In contrast, the mRNA expression of Bax, a proapoptotic gene, mRNA expression significantly decreased in young rats or those that received different QZD dosages (P < 0.001).Moreover, rats administered high-dose QZD demonstrated significantly lower Bax mRNA expression levels than the low-dose and medium-dose groups (P < 0.05) (Fig. 1B).Significantly higher mRNA expression levels were found in the Bcl-2/Bax ratio in aged rats that received high-dose and medium-dose QZD than in aged rats fed a normal diet (P < 0.001 and P < 0.05, respectively).In addition, a higher dose of QZD was significantly upregulated compared with that in the low-and medium-dose groups (P < 0.05) (Fig. 1C).
The other four pro-apoptotic genes, caspase-3, caspase-9, Cyto-c, and Apaf-1, showed the same pattern.All four genes had significantly lower mRNA levels in the ovaries of young rats than in the aged model groups (P < 0.001).Furthermore, the three aged groups that received different doses of QZD had significantly lower pro-apoptotic mRNA levels than those with a normal diet (P < 0.001), with significantly lower levels in the high-dose group than in the low-and medium-dose groups (P < 0.05) (Fig. 1D-G).

Effects of QZD on protein levels of pro-and anti-apoptotic genes
We also investigated whether the changes observed in the apoptotic mRNA levels were mediated by the modulation of protein gene levels (Fig. 2A).
Bcl-2 protein expression was significantly downregulated in the aging rat model compared to that in control rats (P < 0.001).The expression level of Bcl-2 in the aging rat model group was lower than that in the QZD-treated rats, with significantly higher levels in the highdose group (P < 0.001) (Fig. 2B).On the other hand, protein expression of the pro-apoptotic gene Bax was significantly higher in aged rats than in control rats (P < 0.001).The three different QZD dosages downregulated Bax protein expression compared to that in aged rats fed a normal diet, with significant differences in the medium-and high-dose groups (P < 0.001).Bcl-2/Bax protein expression was significantly higher in the young control group than in the normal diet-aged rats (P < 0.001).The QZD-treated groups had higher levels than the aging rat model group, with a significant difference in the high-dose group (P < 0.001) (Fig. 2C).
The expression of other pro-apoptotic genes, including caspase-3, caspase-9, cyto-c, and Apaf-1, was significantly higher in the aged model group than in the young control group (P < 0.001) (Fig. 2E-H, respectively).The protein expression of these genes in the ovaries of rats in the QZD groups decreased to varying degrees compared with those in the aging rat model group and showed a dose-response relationship.
Significantly lower levels of caspase-3 and caspase-9 genes were observed in the high-dose QZD groups compared to aged rats fed a normal diet (P < 0.05 and P < 0.001, respectively).The expression of Cyto-c and Apaf-1 in the high-dose QZD group was significantly higher than that in the aged rats administered a normal diet (P < 0.001).

Discussion
To the best of our knowledge, this is the first study to assess the apoptotic mechanisms of QZD in the ovaries of aged rats.Findings revealed that the mRNA and protein expression levels of pro-apoptotic markers, including Bax, caspase-3, caspase-9, cyto-C, and Apaf-1, in the ovaries of rats treated with QZD were downregulated in a dosedependent manner, with animals treated with high-dose QZD exhibiting the lowest expression levels.Moreover, BCL-2 and Bcl-2/Bax ratio were upregulated in the ovaries of aged rats following QZD administration.
It is widely posited in the field of TCM that the decline in ovarian function is mainly caused by heart dysfunction, which leads to a decline in kidney function, ultimately leading to impaired female reproductive function.Therefore, cardionephrosis pathogenesis is believed to play a dominant role in ovarian aging (Miao et al., 2015;Xia and Liu, 2022).By adopting a method of clearing the heart and nourishing the kidney to regulate yin and yang, the combination of water and fire, as well as the coordination of viscera, can effectively alleviate and control the symptoms of menopausal syndrome (Dong et al., 2017;Yu, 2018).Modern medical research leads to the hypothesis that the accelerated reduction in depletion of finite oocytes contained in the follicle pool in the ovary may underlie female reproductive aging (Park et al., 2021).Studies have shown that apoptosis is a crucial reason for the aggravation of perimenopausal follicular atresia leading to ovarian dysfunction (Matsumine et al., 2008).Follicle atresia, a key event leading to ovarian aging, exhibits many pathophysiological changes associated with physiological aging, such as mitochondrial dysfunction (May-Panloup et al., 2016).
Mitochondria are susceptible to multiple factors, and their apoptotic pathway is one of the most sensitive indicators of cytopathicity or senescence (May-Panloup et al., 2016), which plays a vital role in the signal transduction cascade leading to intrinsic apoptosis (Galluzzi et al., 2016).Mitochondrial outer membrane permeability (MOMP) is a key step in the intrinsic pathway (Galluzzi et al., 2018) and is regulated by Bcl-2 family proteins (Kalkavan and Green, 2018).Bcl-2 and Bax play crucial roles in mitochondrial stress-induced apoptosis.In general, mitochondria release cyto-C, which forms a polymer complex with Apaf-1.These complexes recruit caspase-9 precursors to the cytosol and subsequently initiate the caspase cascade reaction, leading to the activation of executioner caspases-3.caspase-3 plays a central role in the execution of apoptotic programs initiated by various factors (Kasture et al., 2021;Yuan et al., 2020).This series of reactions ultimately leads to cell apoptosis (Green, 2022).
In the present study, by measuring the mRNA and protein expression of key components in the mitochondrial pathway sensitive to cell senescence, we showed that QZD exhibited anti-apoptotic effects by significantly upregulating the upstream signal anti-apoptosis factor Bcl- 2 and simultaneously suppressing pro-apoptotic protein Bax expression.This was further supported by the Bcl-2/Bax ratio, a commonly used parameter for evaluating the degree of apoptosis.Significant changes were also detected in the proteins involved in the midstream of the apoptotic pathway, namely cyto-C and Apaf-1.We also confirmed the anti-apoptotic effect of QZD by measuring the expression levels of caspase-3 and caspase-9, both of which are crucial for apoptosis.Notably, we observed a dose-dependent effect of QZD-induced changes in the expression of these markers at both the mRNA and protein levels.Further investigation of the optimal effective dose and isolation of the active biochemical compounds from this decoction may be warranted to identify key ingredients and effective regimens to devise optimal therapeutic strategies against ovarian aging.
Meng et al. compared scrapping therapy with QZD in patients with perimenopausal syndrome (PMS) with a pattern of fire excess from yin deficiency.Both observation, received scrapping therapy and QZD, and control received QZD, groups showed significant decrease in the menopausal quality of life (MENQOL), while other factors, including vasomotor symptoms, psychosocial and physical conditions were significantly lower in the observation group compared to the control group.Moreover, the serum level of estrogen (E2) increased following treatment, while circulating levels of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) decreased in the combined therapy group.They found that the integration of scrapping therapy in conjunction with QZD, or the sole administration of QZD, has the capacity to enhance PMS symptoms and elevate the standard of living while also deferring ovarian atrophy (Meng et al., 2016).
Juan et al. investigated the efficacy and mechanism of QZD in premature ovarian insufficiency (POI) of the heart-kidney non-interaction type under the guidance of "heart-kidney-uterus axis" theory.The control group received estrogen and progesterone treatment while the case group had the same hormone treatment and modified QZD treatment based on "heart-kidney-uterus axis" theory for three months.This study aimed to observe and compare the variations in the average diameter of the ovary, ovarian area, and antral follicle count as determined by Bultrasound, alterations in quality of life, and serum concentrations of LH, FSH, E2, and anti-Mullerian hormone (AMH) prior to and following treatment.The use of QZD for the treatment of POI has shown promising results in improving estrogen levels, optimizing ovarian function, and enhancing the quality of life (Li et al., 2019).

Conclusion
Our findings demonstrated that QZD modulates the expression of genes involved in ovarian mitochondrial apoptotic pathways by upregulating anti-apoptotic genes, including Bax, caspase-3, caspase-9, cytoc, and Apaf-1, and downregulating pro-apoptotic genes, such as Bcl-2 and Bcl-2/Bax ratio.This study suggests that QZD might have therapeutic potential in delaying ovarian function decline and in preventing and treating ovarian aging-related diseases.Nevertheless, this intervention lacks clinical evidence, suggesting that additional clinical investigation is necessary to validate the current results.