The regulatory mechanisms of acupuncture on immune function primarily involve its neuroregulatory effects, which are mediated through stimulation of the hypothalamic-pituitary-adrenal (HPA) axis and activation of autonomic nerve pathways. Specifically, acupuncture enhances the secretion of certain neuromodulators and increases the expression of glucocorticoids and their receptors, thereby modulating immune function (77). Furthermore, acupuncture exhibits immunomodulatory effects by regulating the pathophysiological microenvironment in inflammatory lesions. This therapeutic intervention downregulates the expression of key proinflammatory mediators including IL-6, IL-1β, IL-18, and TNF-α, while simultaneously upregulating the bioactivity of anti-inflammatory regulators exemplified by IL-10 and TGF-β (78).

Existing clinical investigations have demonstrated that the application of acupoint stimulation therapy exhibits notable efficacy in managing various inflammatory pathologies. Specifically, this therapeutic modality has been shown to improve rheumatic joint disorders, nasal hypersensitivity reactions, and gastrointestinal inflammatory conditions in both acute and chronic stages. Mechanistic studies suggest its anti-inflammatory properties primarily stem from the downregulation of key pro-inflammatory signaling molecules, such as IL-6 and TNF-α, thereby modulating immune-mediated pathological processes. The observed cytokine profile alterations correlate with significant attenuation of clinical manifestations in multiple trial cohorts. Additionally, acupuncture has been demonstrated to slow disease progression and improve symptoms in animal models of autoimmune diseases, including multiple sclerosis (79, 80).

The mechanisms underlying the activation of immune responses through acupuncture involve promoting the phenotypic transition of macrophages from proinflammatory M1 variants to immunoregulatory M2 subtypes, while simultaneously reducing the levels of pro-inflammatory factors such as TNF-α and IL-6 (81).It also increases the concentration of anti-inflammatory factors such as IL-10 and TGF-β. In addition, scientific investigations have demonstrated that acupuncture exerts immunomodulatory effects by suppressing the activity of pro-inflammatory lymphocyte subsets, particularly Th1 and Th17 populations, along with their corresponding interleukin secretions. Furthermore, this therapeutic intervention enhances the activity of immunosuppressive Tregs and stimulates the production of anti-inflammatory mediators including TGF-β. These coordinated cellular responses effectively restore the equilibrium between Th1 and Th2 cell populations within the immune system (79, 80). These effects contribute to maintaining the immune system’s steady state against pathogen invasion and abnormal immune responses.

Jia Li et al. explored the effects of acupuncture treatment on d-galactose-induced vaccine-induced immune responses in senescent rats. The protective efficacy of vaccination is primarily correlated with the strength of immunological responses elicited in recipients. Notably, immunodeficient populations exhibit heightened susceptibility to suboptimal immunization outcomes, particularly among elderly individuals experiencing age-associated declines in adaptive immune functions (82–84).This vulnerable demographic frequently demonstrates compromised responsiveness in both lymphocyte-mediated defenses and antibody production mechanisms, resulting in diminished capacity to establish sufficient immunological memory against pathogenic challenges (85). Immunosenescence, a key pathological feature in aging, refers to the deterioration of the immune system with age, manifested by a progressive weakening of the adaptive immune response to external antigenic stimuli. The clinical manifestations of this pathological state are multidimensional: not only does it significantly weaken the body’s defense against exogenous pathogens, but it is also positively correlated with the risk of malignant tumors; at the same time, it may induce abnormalities in the immune tolerance mechanism, leading to an increase in the incidence of autoimmune diseases. It is worth noting that the imbalance of the immune microenvironment homeostasis also leads to a reduction in the efficacy of the immune response to vaccines, which is reflected in the molecular biology of decreased antigen presentation efficiency and insufficient production of protective antibodies (86). To overcome immune senescence, current research efforts are focused on the design and synthesis of novel immunological enhancers for vaccines (87) The team initially proposed acupuncture as an adjuvant for vaccine response. Acupuncture is considered to be one of the basic methods of preventing infectious diseases. Moreover, the mechanism by which acupuncture enhances the action of antigen-presenting cells and lymphocytes (88) is similar to that of adjuvants. Experimental data showed that moxibustion intervention significantly modulated the body’s immune response to Staphylococcus aureus (S. aureus) antigens in the vaccine, as well as promoting the increase of specific antibody potency in experimental animals. Notably, this immunomodulatory effect showed a dose-dependent feature in the rabbit model, and the mechanism may be closely related to the activation of intrinsic immune cells and the synergistic effect of humoral immune response (89).

The team allocated 40 female Wistar rats (230-280g, 10 weeks old) into four groups of 10 rats each. Group A simulated immunity plus acupuncture, Group B simulated immunity plus control, Group C normal immunity plus acupuncture, Group D normal immunity plus control. Rats in groups A and B were injected subcutaneously with d-galactose (350 mg/kg day), and groups C and D were injected with saline for 6 weeks. The model induced by d-galactose has been extensively employed in scientific investigations into aging mechanisms and related pathological conditions in rodents (90). Excessive accumulation of d-galactose induces oxidative stress through ROS generation while simultaneously impairing the activity of endogenous antioxidant defense systems within cerebral tissues. This dual mechanism subsequently triggers a cascade of neurophysiological alterations, manifesting as cognitive impairment, accelerated brain aging phenotypes, progressive declines in motor coordination, and diminished lifespan expectancy in experimental murine models. Notably, this pathophysiological progression effectively recapitulates key aspects of physiological aging observed in mammalian organisms (91). In this study, experimental rodent models were established through chronic administration of D-Galactose to induce physiological alterations. At the seventh week of the experimental timeline, all subjects received intramuscular administration of acellular pertussis-containing vaccine (DTaP) at a dosage equivalent to one-twentieth of the standard human immunization quantity. The DTaP immunization protocol implemented in this investigation has demonstrated clinical efficacy in both adolescent and adult populations (92); however, antibody titers in vaccine recipients diminish at a significant rate (93), In addition, the administration of DTaP vaccine has been linked to various adverse effects impacting the digestive system, such as nausea, vomiting, diarrhea and stomach pain. The research group hypothesizes acupuncture may improve the body’s immune response to vaccines by functioning in an adjuvant-like manner. This hypothesis is grounded in traditional Chinese medical principles regarding meridian regulation and empirical evidence from prior experimental investigations. Specifically, the therapeutic protocol involving simultaneous stimulation of Zusanli (ST36, Leg Three Li) (94), Guanyuan (CV4, Cinnabar Field), and Baihui (GV20, Hundred Convergences) acupoints - termed “dual tonification and single regulation” therapy - demonstrates particular potential, has a special effect on regulating the immune system and delaying immune aging in rats (95–97). Rats in groups A and C underwent electroacupuncture and moxibustion at specific acupoints (ST36, CV4, GV20), and the control group was fixed only.

For the assessment of their immune response, diphtheria antibody titers were detected using the Vero cell neutralization test (98). The experimental data revealed significant variations in diphtheria antitoxin levels among the different intervention groups. Quantitative analysis revealed that the immunization model control cohort exhibited markedly reduced antitoxin concentrations relative to the standard immunization control group, with statistical significance confirmed. Contrastingly, subjects receiving combined low-frequency electroacupuncture stimulation and thermal moxibustion therapy in the model intervention group displayed substantially enhanced antitoxin production, achieving statistical significance. Furthermore, the conventional acupuncture intervention group demonstrated a measurable improvement in the immunological response, although the effect size was relatively modest.

Previous studies have demonstrated that the liquid formulations of the DTaP vaccine exhibit cytotoxic effects on cells. Consequently, researchers employed quantitative analysis of lymphocyte subpopulations to indirectly assess the effects mediated by cellular immunity. Experimental observations revealed enhanced apoptotic tendencies in T-lymphocytes within aging organisms, with CD4+ lymphocytes demonstrating higher apoptotic susceptibility compared to CD8+ counterparts. This selective depletion resulted in marked numerical reduction of CD4+ populations, consequently inverting the typical CD4+/CD8+ ratio within the immune cell repertoire (99). Quantitative analysis of lymphocyte subpopulations was conducted using flow cytometry to assess the distribution patterns of CD3+, CD4+, and CD8+ cells. Comparative analysis revealed a marked disparity in CD4+/CD8+ T lymphocyte ratios among CD3+ populations across experimental cohorts. The model immunization control cohort exhibited a statistically significant reduction in this immunoregulatory index relative to the standard immunization control group (p<0.01). Subsequent therapeutic application of electroacupuncture combined with moxibustion modalities in the model intervention group demonstrated a notable elevation in this critical immune parameter, reaching statistical significance at p<0.05 when compared with pretreatment baseline measurements. The CD4T/CD8T cell ratio was significantly lower in group B model immunized controls compared to group D normal immunized controls. The group A model immunized with acupuncture had a higher CD4T/CD8T cell ratio compared to the group B model immunized control. The CD4T/CD8T cell ratio was increased in normal-immunized acupuncture compared with normal-immunized controls (p < 0.05). The assessment of diphtheria antitoxin titers and CD4+ to CD8+ T lymphocyte ratios currently serves as validated biomarkers for evaluating immune responsiveness in humans following DTaP immunization. Nevertheless, the therapeutic potential of combined electroacupuncture and moxibustion to modulate these immunoregulatory parameters remains insufficiently explored in clinical research (100, 101).

Heat shock proteins (HSPs), serving as essential molecular chaperones, are crucial for maintaining proteostasis and structural integrity within cellular systems. These conserved polypeptides demonstrate significant immunomodulatory capacities through their involvement in coordinating diverse immune cell populations, while concurrently facilitating antigen processing and presentation mechanisms essential for adaptive immunity (102, 103). The secretion of HSPs is triggered by cellular stress and exposure to immune danger signals. Once released into the extracellular compartments or systemic circulation, HSPs interact with cellular membranes via receptor-mediated mechanisms, triggering intracellular signaling pathways that promote the internalization of immunogenic polypeptide. This biological process initiates a cascade of molecular events, ultimately promoting the efficient processing and presentation of antigenic determinants within the host immune system (104). HSP70-bound peptides can be effectively internalized by antigen-presenting cells (105) via CD91, LOX-1 (106, 107), thereby facilitating the presentation of the antigenic peptide via major histocompatibility complex (MHC) class I molecules on the cell surface. The team determined the expression level of HSP70 mRNA in spleen tissues by real-time RT-PCR, and used GAPDH as an internal reference to analysis gene expression. The analysis of splenocyte gene expression profiles revealed a marked reduction in HSP70 transcriptional activity among subjects subjected to model immunization protocols when contrasted with counterparts receiving standard immunization regimens (p < 0.01). However, the relative expression of HSP70 mRNA in splenocytes was elevated in the immunoacupuncture group after the model immunoacupuncture treatment (P < 0.05). Additionally, the relative expression of HSP70 mRNA in splenocytes showed a slight increase in the normal-immunized acupuncture C group compared to the normal-immunized control group D (P > 0.05).

The results showed significant increases in diphtheria antitoxin titer, CD4T/CD8T cell ratio and HSP70 mRNA expression after acupuncture intervention, suggesting that these practices could be used as novel vaccine adjuvants. Importantly, the DTaP vaccination protocol employed in this study utilized aluminum hydroxide as an immunostimulant, facilitating the concurrent evaluation of acupuncture’s potential adjuvant compatibility. These findings suggest that acupuncture may synergize with aluminum-based adjuvants in therapeutic applications, based on the experimental parameters employed. Nevertheless, constrained by the experimental parameters, this research presents two principal methodological limitations: first, the investigation did not include quantitative analysis of HSP70 protein expression levels through standardized immunoassay techniques; second, the temporal dynamics of immune activation processes remained unexplored across critical immunological phases.

Ulcerative colitis (UC), a persistent inflammatory disorder of the colonic mucosa, manifests as a chronic condition characterized by intermittent phases of mucosal inflammation and tissue damage. UC patients usually exhibit disruption of the Treg/Th17 immune axis. The therapeutic mechanism of moxibustion in traditional Chinese medicine involves suppression of SIRT1 protein activity, thereby modulating the immune homeostasis mediated by Th17/Treg cell differentiation. Experimental evidence indicates that concurrent administration of EX-527, a selective SIRT1 enzymatic antagonist, synergistically enhances the clinical efficacy of moxibustion intervention through coordinated immunoregulatory pathways (108). In addition, a study showed that acupuncture combined with bloodletting effectively inhibited mast cell degranulation and improved histopathological morphology in sensitized skin tissues on the back of rats with urticaria. The mechanism of action may be related to the inhibition of the differentiation and proliferation of helper T cells 2, as well as the regulation of humoral immune response (109).

Notably, a randomized controlled clinical trial (NCT04844710) of mild-to-moderate COVID-19 hospitalized patients has explored the immunomodulatory effects of acupuncture in combination with standard of care. The study directly assessed acupuncture’s ability to modulate the human immune system by testing laboratory markers (e.g., lymphocyte subsets, NK cell activity, and inflammatory factor levels). Although the final results are not yet available, its framework of research focusing on immune parameters provides an important rationale for the clinical translation of acupuncture to enhance vaccine response, especially in a population of recovering infections with poor vaccine response.

Near-infrared (NIR) light therapy, also known as low-level laser therapy or photobiomodulation (PBM), is a non-invasive treatment that utilizes light at specific wavelengths to promote cellular healing and tissue restoration. NIR light radiates in a non-ionizing manner and does not cause significant tissue damage or genotoxicity (110). Equipment for NIR is much cheaper compared to radiation equipment (111). NIR lasers have been developed and utilized in the medical field for decades, with their safety and theoretical foundations well-established (112). Cytochrome c oxidase (CCO) in mitochondria is one of the main photoreceptors when NIR is irradiated to biological tissues. CCO absorbs photons and its activity is enhanced, optimizing the function of the electron transport chain, increasing ATP production and improving cellular energy metabolism. This process not only enhance cellular respiratory efficiency but also mitigates the formation of free radicals and alleviates oxidative stress by upregulating antioxidant enzymes, such as superoxide dismutase (SOD).

NIR is able to down-regulate the expression of inflammatory cytokines including TNF-α, IL-1β, and IL-6, while up-regulating the levels of anti-inflammatory mediators like IL-10, thereby reducing the inflammatory response and maintaining an appropriate immune response. This modulatory effect makes NIR excellent in treating many types of pain, such as arthritis, musculoskeletal pain, and post-operative pain, by reducing inflammation and promoting repair of damaged tissue to achieve pain relief. For chronic wounds such as diabetic foot ulcers and burns, NIR can accelerate the healing process and reduce the risk of infection.

Mechanisms of activation of the immune system by NIR include enhancement of immune cell function. By improving cellular energy metabolism, NIR contributes to maintaining the physiological activities of immune-related cellular components, including macrophages and lymphocytes, by regulating their functional integrity. In addition, NIR promotes macrophage polarization towards the M2 type, which exhibits enhanced anti-inflammatory and reparative properties. For T cells, NIR may enhance the proliferation and differentiation of CD4+ T cells, support Th1/Th2 balance, and stimulate the production of regulatory T cells (Treg), which play a critical role in suppressing autoimmune responses and maintaining immune tolerance. NIR also affects the immune system indirectly through neuroendocrine pathways, for example, it regulates the hypothalamic-pituitary-adrenal axis (HPA axis), which in turn modulates the secretion of immunomodulatory hormones like glucocorticoids.

Although the concept of using lasers as vaccine adjuvants emerged in the 1960s, recent studies have identified mast cells as key mediators of their immunostimulatory effects; however, the precise cellular mechanisms, and in particular how light triggers mast cell activation, remain incompletely elucidated (113). Physiological and biological activities of 1270 nm near-infrared light as a vaccine adjuvant in a mouse model of influenza studied by Yohei Maki et al. The near-infrared photoadjuvant effect is wavelength specific. 1270 nm band is preferred based on the following molecular absorption properties (1): Oxygen molecule absorption matching: Under the partial pressure of oxygen in biological tissues, this band covers both the discrete absorption line of O₂ monomer (1268 nm) and the continuous absorption band of O₂-O₂/O₂-N₂ collision complex (1264 nm) (114). Measured absorption cross sections showed that the peak absorption cross section of 21% O₂ mixture at 294 K body temperature environment amounted to 2.52 × 10^-²⁶ cm²/molecule (monomer) versus 4.87 × 10^-¹⁹ cm⁵/molecule² (complex) (114). (2) Tissue penetration optimization: Near 1450 nm (one of the main absorption peaks of water), water molecules in tissues strongly absorb photons, resulting in a significant increase in light attenuation and a significant shortening of the effective attenuation depth (penetration depth) (115). In contrast, the linear absorption coefficient of water at 1270 nm (0.122 cm-¹) is much lower than that at 1450 nm (3.05 cm-¹), so the penetration depth can theoretically be increased by 25 times (116). (3) Mitochondrial energy matching: 1270 nm laser irradiation can directly generate single-linear oxygen (without photosensitizer), and its characteristic phosphorescence peak is located at 1270 nm, which can be detected by near-infrared spectroscopy (117); in neurons and astrocytes, 1270 nm laser-induced single-linear oxygen can elevate the mitochondrial membrane potential (ΔΨm), activate NADH/FADH₂-dependent respiration, and increase the maximal respiration rate, which ultimately promotes ATP production (117); 1270 nm light is highly selective for the generation of single-linear oxygen species and does not induce the accumulation of harmful ROS such as superoxide anion or hydrogen peroxide (118).

Using the P-815 mouse mast cell tumor cell line, mastocytes play a critical role in modulating immunological processes (119)., ROS were detected with H2DCFDA (2’,7’-dichlorodihydrofluorescein diacetate), and the intensity of cellular fluorescence was analyzed following laser irradiation. Continuous-wave 1270 nm near-infrared laser irradiation at 300 m W/cm² markedly elevated DCF-positive cell populations associated with reactive oxygen species in in vitro cultured mast cells. Quantitative analysis revealed that cellular fluorescence parameters, particularly the median fluorescence intensity (MFI), exhibited a radiation intensity-dependent enhancement pattern when contrasted with untreated experimental controls. This systematic investigation provides evidence that continuous-wave near-infrared illumination within the 1270 nm spectrum stimulates intracellular reactive oxygen species generation in mast cell populations under specified irradiation conditions.

In eukaryotic organisms, ROS generation primarily occurs within the mitochondrial as a natural consequence of oxidative phosphorylation during aerobic respiration. Notably, there exists a significant correlation between the mitochondrial ATP synthesis machinery and the concomitant synthesis of reactive oxygen species within cellular systems (120). The concentration of ATP in both intact P-815 cellular models and purified mitochondrial fractions was determined using an enzymatic bioluminescence assay with luciferin-luciferase detection systems. Experimental data revealed that photonic stimulation at 1270 nm wavelength in complete cellular environments significantly augmented bioenergetic molecule biosynthesis, with observed metabolic enhancement demonstrating a direct proportionality to incident radiant flux density. From mitochondria isolated individually, ATP is produced in large quantities at both 1270 nm 200 m W/cm2 and 300 m W/cm2 wavelengths as it is in the whole cell. Experimental findings demonstrate that the mechanism by which laser adjuvants interact with mast cells under continuous-wave 1270 nm near-infrared light exposure originates from the mitochondrial generation of ATP and ROS. These organelle-derived biomolecules act as photosensitive signaling mediators initiating cellular responses to photonic stimulation. Six hours after laser irradiation of mouse skin, RNA was extracted and analyzed using real-time RT-PCR to detect chemokine expression. The relative expression levels of CCL2 and CCL20 chemokine mRNA in mouse skin increased by approximately 2-fold and 4-fold, respectively, six hours after 1270 nm laser irradiation. Treatment of human umbilical vein endothelial cells with near-infrared light irradiation at wavelengths of 1064 nm and 1270 nm, respectively, significantly promoted the synthesis of the endogenous bioactive molecule nitric oxide (NO), which has been shown to be an important member of the reactive oxygen species (ROS) family, in the cells (121). NO exerts regulatory effects on mitochondrial bioenergetics through multiple mechanisms. Experimental evidence indicates that NO interferes with electron transport chain activity, particularly by suppressing electron flux within the mitochondrial respiratory complexes. This inhibition paradoxically correlates with elevated oxygen utilization rates, possibly through a mechanism involving increased mitochondrial membrane polarization (122). Furthermore, nitric oxide exhibits vasodilatory effects on the lymphatic system via specific signaling pathways (123) and may significantly contribute to the structural expansion of lymphatic microvessels situated within the dermal tissue layer.

In summary, NIR at 1270 nm wavelength effectively stimulates mitochondrial biosynthesis of reactive oxygen species and adenosine triphosphate, which enhanced the vaccine adjuvant effect through light reception. This wavelength showed moderate to long-lasting antibody titers making it a promising candidate for clinical applications due to its low energy requirement and unique adjuvant effect (see Table 1 ).