Moxibustion regulates hypothalamic microglia M1-M2 polarization to against the aging of GnRH neurons (2025)

mild moxibustion

microglia

polarization

GnRH

aging

The world has entered a serious aging society, with the increase of age, the organism's response to environmental stimulation decreases, and the susceptibility to disease and death increases. So aging is easy to induce a variety of disorders and diseases^[1], so it is very necessary to actively seek effective health care anti-aging countermeasures in the early stage of aging. Aging is not determined by a single factor, but by a variety of factors, the decline of sex hormones is an important sign of aging^[2]. Although the functional decline caused by androgen deficiency in middle-aged and elderly men is relatively mild and hidden, the research on "male menopause"^[3] has been gradually paid attention to and strengthened. It was found that older androgen deficiency easier with a variety of geriatrics and function decline, with the period of a decade of research shows that 29% of the health level of low reproductive function of male recession, most of them have cancer^[4], visible, the prevention and treatment of senile sex hormone lack cannot because no reproduction need can be ignored.

Hypothalamic gonadotropin-releasing hormone (GnRH) neurons are the key output cells of the central neural network controlling the reproductive capacity of mammals, rodents, and other animals, and are responsible for integrating multiple internal homeostasis and external environmental signals to maintain individual fertility. It is the main central pathway of testosterone (T) regulation in men. As a key factor in reproductive regulation, GnRH is not only involved in reproductive aging, but also plays a crucial role in overall aging, especially in cell proliferation^{[5, 6]} and neurodegenerative diseases^[7]. Several recent studies have suggested that the hypothalamus plays a key role in life span control and may act as a regulator of systemic aging. Hypothalamic inflammation was manifested by the activation of inflammatory pathways and glial cells^[8], and the inflammatory microenvironment would directly affect GnRH neurons^[9], resulting in functional disorders of the hypothalamic pituitary axis (HPG). Hypofunction of the HPG axis may promote the occurrence and progression of various senile diseases^[10]. Clinically, treating sex hormone decline with testosterone replacement therapy (TRT) is commonly used, but it still poses potential risks for patients with cardiovascular disease, prostate cancer, and sleep apnea^[11] Therefore, it is of great significance to explore a safe and effective treatment to cure male reproductive aging.

Microglia are the primary immune cells of the central nervous system (CNS)^[12], and they are similar in nature to macrophages. Activated microglia can be divided into two phenotypes:M1-type pro-inflammatory microglia and M2-type anti-inflammatory microglia^[13]. In the brain of neurodegenerative or aging animals, microglia are in a "pre-excited" or "activated" state (M2-type)^[14], and the abnormal activation of senescent microglia exacerbates the central neuroinflammatory response, thus triggering neuropathy in old age^[15] .Multiple lines of evidence suggest that microglia are the primary factor that exacerbates neuroinflammation in the hypothalamus, thereby leading to impaired function of other glial cells and neurons^{[16, 17]}, and that the inflammatory microenvironment resulting from microglia senescence directly affects the function of GnRH neurons.

Mild moxibustion is a traditional Chinese medicine treatment method, and its mechanism of action on aging is mainly reflected in seven aspects: anti-oxidation of free radicals, regulation of lipids, regulation of endocrine, improvement of blood flow, regulation of neurotransmitters in the brain, enhancement of immunity, and enhancement of memory^[18], especially the benign regulation of HPG axis. The effects of acupuncture on the pituitary-target gland axis of the aged Yang deficiency model rats were observed, and it was found that acupuncture could regulate β-endorphin levels in the hypothalamus-pituitary gland and promote the expression of third messengers such as c-Fos, thus promoting the secretion of gonadotropins^[19]. And some reports in the literature suggest that mild moxibustion can regulate inflammatory factors and signaling pathways and improve central nervous function through both central and peripheral mechanisms^[20]. Using 20-month-old male rats as a model of aging, in this study we examined serum sex hormone levels and hypothalamic GnRH neuronal activation levels in rats to assess the efficacy of mild moxibustion on the modulated HPG axis. In addition, we observed the expression of M1 and M2 types of hypothalamic microglia and the levels of microglial inflammatory and anti-inflammatory factors to investigate whether this effect was mediated through pro-microglia polarization.

Experimental animals

Twenty-four 20-month-old male SD rats (weighing 500-600g) and eight 3-month-old male SD rats (200-250g) were obtained from the Animal Experimentation Centre of the Chongqing Medical University in China. The animals were kept together (four per cage) in the same environment (a room maintained at a temperature of 24±2°C and a relative humidity of 55%, under a 12-hour light/dark cycle. Access to food and water was unrestricted) from weaning until the day before the experimental injury. This study was approved by the Ethics Committee of the Chongqing Medical University (NO.2021032).

Experimental design

Twenty-four 20-month-old male SD rats were randomly divided into three groups (n=8 per group): aging control group (AC); Medicine group (MC) ; mild moxibustion treatment group (MM), and another eight 3-month-old male SD rats were chosen as young control group (YC). Rats in the MM group received mild moxibustion using a moxa stick held over CV4 (Guanyuan) and BL23 (Shenshu) acupoints (Figure 1) for 20 min while being restrained in a tubed-shape holder, with the central part of the abdomen and the four limbs exposed. The moxa stick (0.5 cm ×20 cm, Henan China) was ignited and held 2–3 cm above CV4 (located on the midline of the lower abdomen, 10 mm inferior to the umbilicus) and BL23(Located in the lower back, 7 mm next to the second lumbar spine) according to a standard atlas of rat acupuncture points. All treatments were performed 20 min per day, 5 days per week for 8 weeks. Rats in the MC group were injected subcutaneously with testosterone propionate (7mg/kg) every 3 days for 8 weeks at alternating injection sites. Rats in the YC and AC groups were injected subcutaneously with physiological saline (7mg/kg) every 3 days for 8 weeks at alternating injection sites. All rats were fasted the night of the last treatment in the experiment, then weighed and anaesthetized using an intraperitoneal injection of Sodium pentobarbital (2%) at 0.3ml/100g the next morning.

Exhaustive swimming experiment

The Exhaustive swimming experiment was used to evaluate the physiological conditions of rats. Before and after treatment, we used a plastic bucket with a height of 80cm and a diameter of 50cm. The water depth was maintained at 50cm, and the water temperature was controlled at (25±1) ℃. The rats' exhaustive swimming time was recorded, and it was judged that they could not return to the surface after 10 seconds of swimming.

Enzyme-Linked Immunosorbent assay

5mL of blood was collected from the abdominal aorta, spun at 12000rpm for 15min at 4℃, and supernatants were collected. The serum concentrations oftotal testosterone (TT), free testosterone (FT), Follicle-stimulating hormone (FSH), luteinizing hormone (LH) and Gonadotropin-releasing hormone (GnRH) were measured according to the instructions in the ELISA kits,Experiments were repeated in triplicate, with three replicates per sample.

Western blot (WB) analysis

The protein was extracted from the hypothalamus tissue and its concentration was determined by the BCA assay kit. The proteins were placed in 10% /12% sodium dodecyl SDS-PAGE gel for electrophoresis. The proteins were then transferred to a PVDF membrane, then, Sealed with Fast Blocking Western at room temperature for 15 minutes, and the membrane was incubated with primary antibodies, IL-10 (1:1000; BIOSS, China), TGF-β (1:1000; Abcam, China), GDNF (1:1000; BIOSS, China), IL-12 (1:1000; BIOSS, China), and IL-1β (1:1000; BIOSS, China) overnight at 4 °C. The expression levels of proteins were detected with an enhanced chemiluminescence ECL reagent after secondary antibody GAPDH (1:10000; Affinity Biosciences, USA) was shaken at room temperature for 1 h, and densitometric quantification was carried out using Image J software.

Real-Time PCR

Real-time quantitative PCR analysis of iNOS, CD16, Arg1, HO-1, IGF-1 and GnRH. Total RNA was extracted using TRIzol reagent(Servicebio, China).According to the manufacturer-s instructions Real-time PCR was carried out using the following primers:CD16(forward:5’-CAGAATGGCAAAGGCAAGAAGTA-3’;reverse:5’-GTT TGGGTTCCTCATAGACTGCC-3’).iNOS(forward:5’-TTTTCACGACACCCTTCACCA-3’;reverse:5’-TCTATTTCCTTTGTTACGGCTTCC-3’).Arg1(forward:5’-TGGACCCTGGGGAACACTAT-3’;reverse:5’-CTTCCTTCCCAGCAGGTAGC-3’).HO-1(forward:5’-CAGCATGTCCCAGGATTTGTC-3’;reverse:5’-CCTGACCCTTCTGAAAGTTCCTC-3’).IGF-1(forward:5’-CTCTGCTTGCTCACCTTTACCA-3’reverse:5’-CTCATCCACAATGCCCGTCT-3’).GnRH(forward5’-CCGCTGTTGTTCTGTTGACTGT-3’;reverse:5’-GTTCTGGGGTTCTGCCATTTG-3’).The reaction conditions were 95 ℃ for 10 min, followed by 40 cycles of 95 ℃ for 15 s and 60 ℃ for 32 s. Gene expression was calculated as the fold-change normalized to endogenous GAPDH using the 2^−ΔΔCtmethod and then expressed as relative expression levels using the control as a reference.

Immunofluorescence staining

Rats were infused cardiologically with PBS, followed by a cold 4% paraformaldehyde solution. Brains were removed and post mixed in the same fixative overnight at 4°C with subsequent replacement with 30% sucrose in PBS for 72 h. The brains were coronally sectioned at a thickness of 10μm.0.3% polyethylene glycol octyl phenyl ether (TritonX-100) at room temperature to rupture membrane, 3% hydrogen peroxide to inactivate endogenous peroxidase, serum blocking, add CD68/Iba1 (200:1;Servicebio, China), CD206/Iba1 (100:1;Servicebio, China),add Cy3-labeled goat anti-mouse/donkey anti-rabbit and FITC-labeled goat anti-rabbit/donkey anti-goat secondary antibody (1:100;Servicebio, China) overnight at 4°C, incubate at room temperature in the dark For 1 h, mount the slides with a mounting medium containing 4',6-diamidino-2-phenylindole (DAPI). For each section, select 3 visual fields with non-repetitive tissue in the peripheral area of cerebral ischemia, observe and take pictures under a fluorescence microscope (Olympus, Japan), count the number of double-labeled positive cells, and take the average value. GnRH/ c-Fos was detected by the same method.

Statistical analysis

Statistical analysis was carried out using SPSS 22.0. All quantities are presented as mean ± SEM. The two-group comparison was determined using the unpaired Student-s t-test, and one-way ANOVA made multiple-group comparisons. P< 0.05 was considered statistically significant.

Mild moxibustion and testosterone improvedphysiological conditions

All the rats were subjected to exhaustive swimming tests before and after the intervention, and the time they spent was recorded for comparison. Before the intervention, the exhaustion time of swimming in the young control (YC) group was significantly higher than that in the other groups(P<0.01),andthere was no significant difference in time among the aging control (AC) group, medicine (MC) group, and mild moxibustion (MM) group(P>0.05) (Figure 2A).After mild moxibustion treatment and medicine treatment for 8 weeks, the exhaustion time of swimming was improved in both MC group and MM group compared to the AC group(Figure 2B).

SerumTT, FT, FSH, LH and GnRH levels in the aging rats.

Male senescence is mainly reflected in reproductive function ,assay kits were used to evaluate the levels of total testosterone (TT),free testosterone (FT) ,Follicle-stimulating hormone (FSH) ,luteinizing hormone(LH) and Gonadotropin-releasing hormone (GnRH).The result showed that levels of TT, FT, FSH, LH, GnRH in the YC group were significantly higher than those in the AC groups(TT:7.32±0.95 vs. 3.91±1.17;P<0.01.FT:9.23±0.37 vs. 5.34±0.10;P<0.01.LH:46.19±3.19 vs. 22.95±2.28;P<0.01.FSH:10.61±0.44 vs. 5.11±0.11;P<0.01.GnRH:58.73±2.41 vs. 24.89±1.53;P<0.01)(Figure 3A-E).Compared with the AC group, the TT, FT, LH, FSH, GnRH levels in the MM group were significantly increased (TT:3.91±1.17 vs. 6.29±0.93;P<0.01.FT:5.34±0.11 vs.7.79±1.36;P<0.05.LH:22.95±2.28 vs.38.93±2.71;P<0.01.FSH:5.11±0.11 vs. 7.65±0.45;P<0.01.GnRH:24.89±1.53 vs. 43.45±2.16;P<0.01),and there was no significant difference in TT, FT, FSH, LH among the MC group and MM group (TT:5.97±1.33 vs. 6.29±0.93;P>0.05.FT:8.25±1.16 vs.7.79±1.36;P>0.05.LH:34.21±1.63 vs.38.93±2.71;P>0.05.FSH:7.10±0.24 vs. 7.65±0.45;P>0.05.GnRH:35.51±1.35 vs. 43.45±2.16;P<0.01).The data showed that both mild moxibustion treatment and testosterone replacement therapy could elevate TT, FT, LH, FSH, GnRH expression.

Mild moxibustion and testosterone improved hypothalamic inflammatory microenvironment

It is demonstrated that the hypothalamic inflammation is the main factors of aging. Microglia are the primary immune cells of the central nervous system (CNS). Activated microglia can be divided into two phenotypes: M1 type pro-inflammatory microglia and M2 type anti-inflammatory microglia. Various evidence indicate that hypothalamic microglia are actively involved in the occurrence and deterioration of hypothalamic inflammation. To explore the effects of testosterone and mild moxibustion on the hypothalamic inflammation, western blot was used to detect the expression of IL-12, IL-1β, IL-10, TGF-β, GDNF proteins in the hypothalamus. The results showed that AC group had the higher IL-1β and IL-12 expression than YC group, the expression levels of IL-1β and IL-12 expression in MC and MM group decreased gradually (Figure 4D-E). Moreover, the levels of anti-inflammatory factors IL-10, TGF-β and neurotrophic factor GDNF increased in MC and MM group, and the effect of MM group was better than that of MC group (Figure 4F-H).

Effect of mild moxibustion on activation rate of GnRH neurons in the hypothalamus

In the previous experiment, the GnRH mRNA level in the hypothalamus was detected by RT-PCR. After mild moxibustion intervention, the GnRH mRNA level in the aging rats showed a significant trend of increase. In order to further reveal the effect of mild moxibustion on GnRH, the activation of GnRH neurons was detected by immunofluorescence double standard method (GnRH/ c-Fos). GnRH neurons co-labeled with c-Fos are activated GnRH neurons indicated by white arrows (Figure 4A). The GnRH neurons in the aged rats decreased significantly compared with the young rats, while the activation rate of GnRH neurons in the MM group increased significantly after intervention (Figure 5A-B), suggesting that mild moxibustion can promote the activation of GnRH neurons and inhibit the aging of GnRH neurons.

Effect of mild moxibustion on microglia polarization

Mild moxibustion can significantly improve hypothalamic inflammatory microenvironment, but whether its mechanism of action is related to the polarization of microglia and GnRH neuron is unclear. To reveal its potential mechanism of action, the positive expressions of M1 microglia marker CD68 and M2 microglia marker CD206 were detected by immunofluorescence double-label method (Figure 6A-B). There were no CD68⁺/Iba1⁺ cells and fewer CD206⁺/Iba1⁺ cells in YC group. However, the overactivation of CD68⁺/Iba1⁺ cells in aging rats were not improved by exogenous drug treatment. The CD68⁺/Iba1⁺ cells in MM group were significantly decreased, and the CD206⁺/Iba1⁺ cells were significantly increased compared with those in AC group, indicating that mild moxibustion played a positive role in regulating the polarization of microglia from M1 to M2 type. We also examined the expression levels of GnRH, M1 microglia marker iNOS, CD16, HO-1, and M2 microglia marker IGF-1, Arg1 mRNA. Figure 6C-H show mRNA expression of iNOS, CD16, Arg1, HO-1, IGF-1 and GnRH in the hypothalamus. Compared with the YC group, the AC group showed a significant increase in the hypothalamus iNOS and CD16 mRNA expression (P<0.01), and decrease HO-1, IGF-1 and GnRH mRNA expression. Compared with the AC group, the MM, and MC groups all showed a significant decrease in the hypothalamus iNOS and CD16 mRNA expression (P<0.01). The MM and MC groups also showed a significant increase in HO-1, IGF-1 and GnRH mRNA expression(P<0.01). This result confirms that mild moxibustion has a promoting effect on GnRH neurons and that its mechanism of action is related to microglia.

Studies have shown that acupuncture can regulate the overall function and delay the aging process of the body by regulating the neuro-endocrine-immune system^[20]. The effect of mild moxibustion on delaying aging in men has been reported in previous studies, and mild moxibustion has shown better efficacy than testosterone therapy^[21]. There is increasing evidence that hypothalamic dysfunction is associated with aging metabolism^[22],activation of microglia may be involved in hypothalamic inflammation and play an important role in systemic aging^[23]. Our experiment showed that compared with young rats, old rats showed obvious peripheral inflammation and hypothalamic inflammation, which activated glial cells and inhibited GnRH secretion. Mild moxibustion can reduce systemic inflammation and metabolism, improve hypothalamus dysfunction, improve GnRH neuronal activity, and promote GnRH secretion. However, the mechanism of mild moxibustion improving hypothalamic inflammation and aging has not been clarified.

Microglia is a kind of glial cells, accounting for 5%-12% of the total number of brain and spinal cord cells. Microglia is the first and most important immune defense line in the central nervous system and plays an important role in physiological and pathological conditions^[24]. In the resting state, microglia mainly play an immunological surveillance role. When the central nervous system is stressed, microglia activation has both pro-inflammatory (M1) and anti-inflammatory (M2) polarization directions, depending on the intercellular environment and induced stimulation^[13]. When CNS is in normal activity, neurons can release TGF-β, IL-10, GDNF and so on, so that microglia can be polarized in the anti-inflammatory direction, cell debris can be cleared through phagocytosis, and nerve growth factor and anti-inflammatory factor can be released to reduce nerve damage, promote tissue repair, and play a neuroprotective role. However, when CNS is stimulated by inflammation, infection, trauma and other factors, the released CCL21, ATP, UTP, glutamate and other factors can over-activate microglia, induce inflammatory response and release a large number of pro-inflammatory factors (TNF-α, NO, IL-1β, IL-12, etc.) and cytotoxic substances, resulting in neuronal inactivation and cell death. Causes neurological dysfunction^{[25, 26]}. Physiological aging is accompanied by neuron loss and synaptic density reduction. Microglia, as the main macrophage of hypothalamus, can induce the release of a large number of inflammatory factors and a series of inflammatory reactions when stimulated and activated^[27], and the inflammatory microenvironment directly affects GnRH neurons, resulting in the dysfunction of HPG axis. How to inhibit the inflammatory activation of M1-type microglia, reduce the inflammatory response, increase the neuroprotective effect of M2-type microglia, and maintain the positive balance of homeostasis has become the direction of delaying aging.

In our experiment, immunofluorescence double-label method was used to detect the cell number of M1 microglia marker CD68+/Iba1 + and M2 microglia marker CD206+/Iba1 + in the hypothalamus. We found that the number of CD68+/Iba1 + cells in elderly rats was significantly higher than that in young rats, while the number of CD206+/Iba1 + cells was lower than that in young rats, and microglia morphologies were changed and synapses became shorter and fewer, suggesting that microglia play an important role in nervous system aging, which is consistent with previous research results. At the same time, we also detected the mRNA expression levels of M1 microglia markers CD16 and iNOS and M2 microglia markers IGF-1, HO-1 and Arg1 in hypothalamus. Compared with the young group, the mRNA expression levels of CD16 and iNOS in the elderly group were significantly increased. The mRNA expression levels of IGF-1, HO-1 and Arg1 decreased. After treatment, the mRNA expression levels of CD16 and iNOS decreased in the mild moxibustion group compared with the elderly group, while the mRNA expression levels of IGF-1, HO-1 and Arg1 increased in the mild moxibustion group, which was significantly higher than that of the MC group. These results suggest that mild moxibustion may be more beneficial to improve the function of hypothalamus and promote the m2-type polarization of microglia, which provides a new idea for treatment. Mild moxibustion can be used as an alternative therapy for testosterone.

Inflammation is an important risk factor for age-related diseases. Zhang et al. reported that the NF-κB pathway in the hypothalamus is associated with systemic aging. overexpression of TNF-ɑ and NF-κB occurs in the activation of hypothalamic microglia, which engage in the aging process^{[28, 29]}. It has been shown that inhibition of inflammatory pathways in the hypothalamus increases the secretion and release of GnRH and prolongs the lifespan of rats, and that elevated levels of GnRH in rats partially alter the aging process and suppress hypothalamic inflammation^[30]. Our study found that GnRH expression and GnRH neuronal activation were reduced in the hypothalamus of aged rats, while the expression of anti-inflammatory factors TGF-β and IL-10 was much lower than that of pro-inflammatory factors IL-1β and IL-12, and the hypothalamus was in an inflammatory microenvironment. mild moxibustion could improve the expression of GnRH in hypothalamus as well as increase the activation rate of GnRH neurons and improve hypothalamic inflammatory environment. After treatment, the expression of GnRH, TGF-β, IL-10 and the activation rate of GnRH neurons in the hypothalamus of mild moxibustion rats increased, and the expression of IL-1β and IL-12 decreased significantly compared with that of the aged group. This suggests that mild moxibustion can induce GnRH secretion in the hypothalamus to counteract aging-related physiological changes, which is closely related to the inhibition of Inflammatory pathways to promote microglia polarization.

The core of the "neuro-endocrine-immune network regulation" theory of aging is the "hypothalamic-pituitary-gonadal axis" (HPG axis)^[31], GnRH levels are related to aging^[32], we used exhaustive swimming test to detect the swimming time of rats before and after treatment, as well as the serum sex hormone levels of rats. To evaluate the physiological status and reproductive senescence of rats at different stages. In this study, it was found that the exhausted swimming time and serum sex hormones GnRH, LH, FSH, TT and FT of the elderly rats were significantly lower than those of the young rats, indicating that the physical and reproductive functions of the elderly rats were decreased. The exhaustive swimming time and GnRH, LH, FSH, TT and FT levels in the Medicine group and mild moxibustion group were increased. Compared with the Medicine group, the increase of exhaustive swimming time was more significant in mild moxibustion group, and the serum sex hormone level was generally higher than that in the Medicine group. Studies have shown that hypothalamic inflammation associated with aging inhibits GnRH expression, thereby affecting physiological functions. However, testosterone and mild moxibustion can improve the physical strength and delay reproductive aging of rats, which may be related to promoting the activation of GnRH neurons in the hypothalamus and improving the function of HPG axis.

Ethics approval and consent to participate

This study was approved by the Ethics Committee of the Chongqing Medical University (NO.2021032).

Consent for publication

The manuscript is approved by all authors for publication.

Availability of data and materials

The datasets used in the current study are available from the corresponding author on reasonable request.

Competing interests

The authors declare that they have no competing interests.

Funding

“Chongqing Basic Science and Frontier Technology Special Project (No. cstc2017jcyjAX0213)” and “Apricot Grove Program of College of Traditional Chinese Medicine, Chongqing Medical University-Youth Seedling Project (2021ZDXKM09)”.

Authors' contributions

XH and XL designed the study; XH and YL performed the animal experiments; XH and QW analyzed the data and prepared the figures; XH wrote the main manuscript text; XL and LL contributed to revisions of the manuscript. All authors read and approved the final manuscript.

Acknowledgements

We are grateful for the support of the “Chongqing Basic Science and Frontier Technology Special Project (No. cstc2017jcyjAX0213)” and the “Apricot Grove Program of College of Traditional Chinese Medicine, Chongqing Medical University-Youth Seedling Project (2021-ZDXK-M09)”.

Author details

College of Traditional Chinese Medicine of Chongqing Medical University, Chongqing Key Laboratory of Traditional Chinese Medicine for Prevention and Cure of Metabolic Diseases, Chongqing 400016, China.

Xuezhi Li, Email: [emailprotected]

^*Corresponding author.

⁺Contributed equally.

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