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.