Clinical Applications of Bee Venom Acupoint Injection

Bee venom is a complex natural mixture with various pharmaceutical properties. Among these properties, its peptides and enzymes have potential medical therapy for pain relief and inflammation. In clinical settings, this therapy has been used widely to treat diseases by injecting into acupoints. In this article, we have conducted various research from PubMed, Cochrane Library, and Clinical Key from inception of July 2020. The results revealed that bee venom therapy has been reported effective in anti-inflammatory, antiapoptosis, and analgesic effects. Moreover, bee venom acupuncture has been commonly used for clinical disorders such as Parkinson disease, neuropathic pain, Alzheimer disease, intervertebral disc disease, spinal cord injury, musculoskeletal pain, arthritis, multiple sclerosis, skin disease and cancer.


Introduction
Bee venom (BV) is a complex natural mixture produced by Apis mellifera, an European honey bee [1,2]. It is composed of different peptides, proteins, and bioactive components, including melittin, phospholipase A2 (PLA2), and apamin [3]. These components have various pharmaceutical properties, including anti-inflammatory, antinociceptive [4,5], antiapoptosis, antiarthritic [6], and anticancer effects [1,2,7]. BV therapy has been used for a long time [1] in various forms including the administration of live bee stings, the injection of BV, and BV acupuncture (BVA) [8]. Despite its clinical complications, toxicity or anaphylaxis, it is a safe form of acupuncture [6,[9][10][11] and it has interesting pharmacological properties from a biotechnological point of view. Due to the bioactive compounds isolated from BV and its pharmacological actions, BVA is an effective treatment that is commonly used. Numerous studies of BVA have emerged in the last 20 years [12,13], and it has gradually become a popular alternative treatment [14][15][16]. BVA has mostly been used to treat intervertebral disc disease [14,17,18], neuropathic pain [19], arthritis [20,21], and skin diseases [22,23].

Apamin
Apamin is an 18-amino acid peptide and also the smallest neurotoxin in BV [2]. However, it makes up 37%-44% of dry BV and is essential to its biological function [3]. Alvarez-Fischer et al. [39] proved that apamin has anti-inflammatory and antinociceptive functions. Apamin, a blocker of Ca 2+activated K + channels, influenced the permeability of cell membranes toward potassium ions [40]. Moreover, apamin is also able to transport across blood-brain barrier and affect the function of the central nervous system.

Parkinson Disease Preclinical Studies
Parkinson disease is a typical neurodegenerative problem [41] caused by the selective loss of dopaminergic neurons of the substantia nigra pars compacta [42,43]. Its symptoms are characterized by resting tremors, rigidity, bradykinesia, and postural instability [44]. Recently, alternative therapies such as BV are increasing in number. From analyzing an acute 1-methyl-4-phenyl-1,2,3,6tetrahydropyridine (MPTP)-induced mouse model, an animal model of Parkinson disease, Doo et al. [45] suggested that BVA could effectively protect dopaminergic neurons against MPTP toxicity, possibly through the inhibition of Jun activation [45] and the phosphoinositide 3-kinases/protein

Apamin
Apamin is an 18-amino acid peptide and also the smallest neurotoxin in BV [2]. However, it makes up 37%-44% of dry BV and is essential to its biological function [3]. Alvarez-Fischer et al. [39] proved that apamin has anti-inflammatory and antinociceptive functions. Apamin, a blocker of Ca 2+ -activated K + channels, influenced the permeability of cell membranes toward potassium ions [40]. Moreover, apamin is also able to transport across blood-brain barrier and affect the function of the central nervous system.

Parkinson Disease Preclinical Studies
Parkinson disease is a typical neurodegenerative problem [41] caused by the selective loss of dopaminergic neurons of the substantia nigra pars compacta [42,43]. Its symptoms are characterized by resting tremors, rigidity, bradykinesia, and postural instability [44]. Recently, alternative therapies such as BV are increasing in number. From analyzing an acute 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced mouse model, an animal model of Parkinson disease, Doo et al. [45] suggested that BVA could effectively protect dopaminergic neurons against MPTP toxicity, possibly through the inhibition of Jun activation [45] and the phosphoinositide 3-kinases/protein kinase B (PKB) pathway (also known as Akt [PI3K/Akt]) [46]. Another study investigated the anti-inflammatory effects of BV in Parkinson disease. After MPTP injection, it revealed a remarkable decrease in T helper cells (TH+). However, TH+ cells demonstrated a notable survival rate in animals when treated with BVA at ST36 [47]. The potential mechanism of BVA in Parkinson's disease works by reinstating both brain TNF-α and IL-1β in brain tissues, inducing a significant elevation in caspase-3 activity and elevating the expression of apoptosis-related genes such as caspase-3 and Bax [46,48]. In another study, it examined the compounds that play the most effective roles in Parkinson disease and revealed that BV and apamin could protect against MPTP-induced dopaminergic cell loss by increasing striatal dopamine levels. Treatment with BV instead of apamin could increase TNF-α levels in MPTP-induced animal models [39]. Other studies have suggested that bvPLA2 may enhance the motor dysfunction and also pathological functions associated with Parkinson disease in human A53T α-Syn mutant transgenic (A53T Tg) mice by reducing the appearance of α-Syn, the activation of microglia, and the ratio of M1/M2 (M1 microglia produce toxic substances to neurons; M2 microglia generate anti-inflammatory as well as tissue repair elements to enhance survival and repairment) [49].

Clinical Studies
Furthermore, in terms of clinical trials, researchers of another study recruited 43 grownups with idiopathic Parkinson disease that were randomly separated into 3 groups: acupuncture, BVA, and without-treatment groups. The results revealed that the BVA and acupuncture groups both had significant improvements in the Unified Parkinson's Disease Rating Scale, the Berg Balance Scale, and 30-m walking time [42,50]. Thus, BVA may play an supporting role in preventing the development of Parkinson's disease [51].

Neuropathic Pain
Preclinical Studies BVA has another role in alleviating neuropsychiatric disorders [20]. Cold allodynia is an important symptom of neuropathic pain. Some research have studied whether treatment with BV injection could reduce cold allodynia in rats with chronic constriction injuries (CCIs) of the sciatic nerve. In one study, diluted BV (DBV) was injected (low-dose group: 0.25 mg/kg, high-dose group: 2.5 mg/kg) into the ST36 acupoint 2 weeks after sciatic nerve CCI for each group; and 2 weeks of low-dose DBV (0.25 mg/kg) injection for both groups. High dose of DBV (2.5 mg/kg) notably decreased cold allodynia since it can completely block an intrathecal pretreatment of idazoxan (α2-adrenoceptorantagonist) [52]. Other research has revealed that BVA attenuated pain through α 2 -adrenergic receptors, instead of α 1 -adrenergic receptors [52][53][54][55][56][57]. Combined with the inhibition of peripheral β-adrenoceptors, BVA could complement the strategies for decreasing inflammatory pain [58]. Other receptors, such as α4β2 nicotinic acetylcholine receptors, may also be influenced by BVA, and alleviates cold allodynia in oxaliplatin-injected rats [59]. Moreover, numerous articles have proved that BVA increases Fos expression and reduces nociceptive behavior [60][61][62]. In addition, intrathecal clonidine-induced analgesia was notably improved in efficacy when it combined with BVA [19].

Peripheral Neuropathy Preclinical Studies
Some chemotherapeutic agents induce peripheral neuropathy [63] because of the damage to sensory and motor nerve of the peripheral nervous system [64]. Oxaliplatin is an anticancer drug that leads to neuropathic cold allodynia [65][66][67][68]. Some articles have discussed whether BVA could decrease mechanical allodynia. According to the study by Yoon et al., after mice were injected with DBV (0.1 mg/kg) at ST36 of the right hind limb for 2 weeks and followed by oxaliplatin (10 mg/kg) injection, the mice had notably reduced ipsilateral mechanical allodynia. In the study, pre-injecting 2% lidocaine (40 mg/kg) into ST36, or yohimbine (25 µg/mouse), an alpha-2 adrenoceptor antagonist, into the spinal cord will both completely blocked the antiallodynic effect of DBV. However, the effect of DBV did not change with the pretreatment of naloxone (20 µg/mouse), an opioid receptor antagonist [66]. Similarly, another study revealed that the antiallodynic effect of BVA was related to the serotonin system and could be blocked by methysergide (mixed 5-HT1/5-HT2 receptor antagonist, 1 mg/kg i.p.) or MDL-72222 (5-HT3 receptor antagonist, 1 mg/kg i.p). That is, serotonin levels in the spinal cord could be elevated using BVA, and it was suggested that BVA alleviated cold allodynia in oxaliplatin-injected rats mainly through the activation of spinal 5-HT3 receptors [67,68]. Paclitaxel, a chemotherapy drug used against various tumors, also induced painful peripheral neuropathy. In this study, BV decreased the side effects of paclitaxel. In addition, the antihyperalgesic effect of BVA treatment at acupoint ST36 was more pronounced than at L11. Moreover, to investigate which of these compounds, BVA (1 mg/kg), melittin (0.5 mg/kg), and PLA2 (0.12 mg/kg), performed best analgesic effect when injected into ST36; the results demonstrated that melittin was the strongest analgesic among all [53].

Clinical Studies
Yoon et al. [64] report BV pharmacopuncture can reduce the scores of World Health Organization Common Toxicity Criteria for Peripheral neuropathy, Patient Neurotoxicity Questionnaire and Visual Analogue System, and also can improve the life quality in 11 Chemotherapy-induced peripheral neuropathy patients with weeks follow-up.

Alzheimer Disease Preclinical Studies
Alzheimer disease is a neuroinflammatory disease caused by the accumulation of 2 hallmark proteins, amyloid-β peptides and neurofibrillary tangles [69]. However, CD4+Foxp3+ Treg can modulate the neuroinflammation by suppressing the Th effector cell activation [38]. Treatment with bvPLA2 remarkably increased the numbers of Treg cells. Ye et al. suggested that bvPLA2 treatment may inhibit the progression of Alzheimer disease in 3xTg mice through increased glucose metabolism in the brain, decreased amyloid beta deposits in the hippocampus, reduced neuroinflammatory responses, and lower the percentage of CD4+ T cells infiltrating in the hippocampus [38]. These results indicate that BV may protect the brain tissue by reducing neuroinflammation.

Intervertebral Disc Disease Preclinical Studies
Intervertebral disc disease is a spinal disease that often causes neurological pain and dysfunction. Typically, anti-inflammatory and analgesic medicines are used to decrease nociceptive signals and ameliorate suffering. However, Tsai et al. revealed that BVA effectively alleviated pain symptoms [17]. Forty dogs with neurological disorder triggered by intervertebral disc disease underwent separate treatment for 6 weeks one treated by BV the usual therapy (oral prednisone and the nonsteroidal anti-inflammatory drug [NSAID] carprofen) another by usual therapy alone. BVA considerably improved grades recorded with the myelopathy scoring system as well as functional numeric scale [17].

Spinal Cord Injury Preclinical Studies
Spine injury is an inflammatory response that stimulated the secretion of proinflammatory cytokines, consisting of IL-1β and IL-6 [70][71][72]. Furthermore, spinal cord injuries lead to the increasing level of anti-inflammatory cytokines such as interleukin-10 (IL-10) [73]. One model of spinal cord compression showed that BVA at acupoints GV3 and ST36 notably increased the secretion of IL-10 in the first 6 h and reduced the expression of IL-6 in 24 h after treatment. However, there is no influence on the level of IL-1β and interleukin-4 (IL-4) cytokines. [74]. In another experiment on below-level neuropathic pain in rats with spinal cord injuries, DBV injection at acupoints can not only decreased glia expression and neuropathic pain but also enhanced the speed of recovery [75].

Central Poststroke Pain
Clinical Studies Following cerebrovascular incidents, central poststroke pain often manifests as sensory disturbances and neuropathic pain [76]. Pharmacopuncture is a widely used intervention for poststroke pain [77]. In a single-blind randomized controlled clinical trial, they recruited 20 patients who were suffering from central poststroke pain. These patients were injected with either DBV or saline at acupoints twice a week for 3 weeks. After treatment, the BV injection group showed more pronounced decreases in visual analog scale (VAS) scores than the saline injection group did [78]. A systematic review surveyed 138 potential articles, in which only four were randomized controlled trials that meeting the researchers' criteria. The review demonstrated that injection with BV was more effective than injection with saline in decreasing poststroke shoulder pain [79].

Musculoskeletal Pain
Clinical Studies More and more people are affected by chronic neck pain or lower back pain. These patients are often prescribed NSAIDs [80] or alternative treatments such as BVA [81,82]. In one recent blinded human trial, 54 people suffering from chronic lower back pain were assigned to either BVA or sham acupuncture groups. Each patient received six treatments over 3 weeks. Participants in the BV group had a notable reduction in pain (26%) after 3 weeks, as recorded using the VAS [83,84]. In another study, 60 participants were randomly treated with either normal saline or BV injection. Both groups were treated twice a week for 4 weeks with injections at six acupoints (BL23, BL24, and BL25 on bilateral sides). The results revealed that BVA was more effective in treating chronic lower back pain.
BV injection at acupoints was more effective than normal saline aqua-acupuncture (used as a placebo) in the treatment of 30 patients suffering from acute ankle sprains after daily treatments for 1 week [25].

Clinical Studies
Osteoarthritis is a type of arthritis that often affects the knee joints. BV can relieve the pain associated with knee osteoarthritis. BVA strongly stimulated aromatase activation in human leukemic cell line FLG19.1 and human osteoblast cells, enabling estrogen production by bone-derived cells to inhibit the development of osteoarthritis [85]. In one study, 60 patients with knee osteoarthritis received BVA twice a week continued for 4 weeks, and BVA was proven to be more effective than traditional acupuncture for pain relief [86]. In another study, 538 participants received weekly dermal injections of BV or histamine. After 12 weeks, the BV biotherapy group showed a remarkable improvement over the control group in Western Ontario and McMaster Universities Arthritis Index (WOMAC) pain scores [87]. Another comparative study revealed that a combination of intra-articular injection with intra-acupoint injection was more effective than conservative treatments for osteoarthritis of the knee [88].

Rheumatoid Arthritis Preclinical Studies
Rheumatoid arthritis is an autoimmune disorder characterized the proliferation of synovium and cellular infiltration that can lead to progressive joint destruction, deformity, disability, swelling, and pain in multiple joints [89,90]. Low-dose methotrexate is commonest treatment for rheumatoid arthritis. However, one side effect of methotrexate is hepatotoxicity, which can result in poor medication compliance [91]. Therefore, patients are likely to seek alternative treatments. Previous studies have indicated that BV injection may have anti-inflammatory and antinociceptive effects on rheumatoid arthritis in rats [92]. Li et al. [29] used Freund's adjuvant-induced animal model to induced arthritis and found that treatment with BV at ST-36 notably suppressed Fos expression in the superficial layer of the lumbar spinal cord and thus reduced paw edema and nociceptive behaviors in the injected side of the paw. On the other hand, in a type-II collagen-induced arthritis (CIA) model, Lee et al. [93] found that BV injection therapy will inhibit immune responses. TNF-α production was notably lower in the BV group than in the control group, while IL-1β remained the same level [93]. Another study revealed that free radical levels and protease activity were significantly increased in rats with CIA compared with normal rats. BV injection (0.25 mg/kg) was an effective modulator of rheumatoid arthritis, inhibiting protease activities and removing reactive oxygen species (ROS) [94]. In addition, in combination use with methotrexate, BV may have increased bioavailability and antiarthritic effects [95]. Reduced hepatotoxicity was mostly due to the decreased expression of TNF-α and NF-kB (p65) in the synovial membrane of the hind paw [91]. These studies indicated that BV injection had antiarthritic and antinociceptive effects on rats with arthritis and can be an alternative therapy for rheumatoid arthritis [1].

Multiple Sclerosis Preclinical Studies
Multiple sclerosis is an autoimmune disease potentially leads to brain and spinal cord disabling diseases due to inflammation of the central nervous system. The etiology of multiple sclerosis is unknown. It may be induced by viral infection, environmental factors, or genetic factors [96]. Since 2000, several alternative medications have been used to treat multiple sclerosis, such as BV, cannabis, cannabinoids, acupuncture, and acupressure [97]. Some experiments have proven that BVA had effects on patients with multiple sclerosis. Pretreatment with BVA suppressed demyelination, glial activation, expression of cytokines such as interferon (IFN)-γ, interleukin-17 (IL-17), interleukin-17 A (IL-17A), TNF-α, IL-1β, and chemokines (monocyte chemotactic protein-1 (MCP-1) and macrophage inflammatory protein (MIP)-1α). In an animal model of multiple sclerosis, BVA will induce nitric oxide synthase (iNOS) activity, activate p38 mitogen-activated protein kinase (MAPK) and promote NF-κB signaling pathways in the spinal cords of rats with acute myelin basic protein (MBP)(68-82)-induced experimental autoimmune encephalomyelitis (EAE). BVA treatment may decrease the number of CD4 + , CD4 + /IFN-γ + , and CD4 + /IL-17 + T cells and increase the number of CD4 + /Foxp3 + T cells in the spinal cords and lymph nodes of rats with acute EAE [96].

Acne Preclinical Studies
Acne is a chronic skin problem occurring in the sebaceous units of the skin, including on the face, neck, back, and chest [22]. Propionibacterium acnes is a key factor leading to the inflammatory reaction of acne. Traditionally, antibiotics are the first-line treatment. Nonetheless, antibiotics may result in the emergence of antibiotic resistant pathogens and side effects. Recent research has focused on the anti-inflammatory effects of BV. Heat-killed P. acnes increased levels of TNF-α, interleukin-8 (IL-8) and IFN-γ in HaCaT and THP-1 cells. BV inhibited the Toll-like receptors (TLR2) expression in HaCaT cells [28,98], thus suppressing the secretion of proinflammatory cytokines TNF-α and IL-1β [23,99]. Another study indicated that TLR activation happens during an inflammatory response, and will resulted in the activation of MAPK and transcription factor NF-κB signaling pathways. P. acnes increased the expression of IKK, IκB, and NF-κB in HaCaT cells, and lead to inflammation. Meanwhile, melittin will reduced the phosphorylation of IKK, IκB, and NF-κB [28,98].

Atopic Dermatitis Preclinical Studies
Atopic dermatitis is an inflammatory skin disorder caused by a deficient skin barrier, pruritus, eczema, dry skin, and an abnormal immunoglobulin E (IgE)-mediated allergic response to diverse external antigens [23]. In a mouse model of trimellitic anhydride (TMA)-induced skin impairment, BV (0.3 mg/kg) delivered at the BL40 acupoint reduced levels of both T helper cell type 1 (Th1) and Th2 cytokines and inhibited the synthesis of IL-4 and IgE. That is, BVA relieved inflammatory and allergic responses in the TMA model [100].

Preclinical Studies
Cancer is the second leading cause of death worldwide. More and more new agents, treatment strategies, and biotoxins such as BV are being used to fight against cancer cells. One article investigated whether BV was efficient as an antitumor agent. Moon et al. indicated that through BV injection, Bcl-2, extracellular regulated protein kinases (ERK), and protein kinase B (Akt), will decrease in expression, and lead to apoptosis of cancer cells [101]. BV stimulated the expression of death receptors and inhibition of NF-κB, leading to the apoptosis of lung cancer cells [102]. BV also inhibited Akt and MAPK phosphorylation in Lewis lung carcinoma cells and human umbilical vein endothelial cells. And the expression of VEGF and VEGFR-2 were down-regulated [103]. In addition, BV intervened in calcium fluctuation, ROS generation, and the release of AIFs and EndoG in human melanoma cells [104]. In MDA-MB-231 breast cancer, Raman spectroscopy with multivariate analysis proved the effect of nuclear fragmentation and protein degradation at different concentrations [105]. In summary, with appropriate usage, BV may become a promising antineoplastic drug.

Safety
People concern about the safety and side effects of BVA, since this treatment may lead to sensitization and even fatal anaphylactic reactions. In some cases, after the BV treatment, patients developed acute anaphylactic shock, Guillaume-Barre syndrome or irreversible ulnar nerve injury [20]. Ahn et al. [106] reported administered BVA (0.2 ml) became more redness and itching after few minutes. However, purified essential BV (0.2 ml) presented less allergic than original BV. Both of these pharmacopuncture showed similar anti-inflammatory effects [106]. A study of dose toxicity test in 30 Sprague-Dawley rats treated repeatedly with SBV (0.28 mg/kg) or normal saline (0.2 mL/kg) for 4 weeks showed no significant adverse effect for BV injection after 13 weeks [11]. Comparably, a 13-week repeated research suggested that the dose of BV under approximately 0.07 mg/kg is relatively safe without observed adverse effect [10].

Conclusions
BV injection at acupoints has been reported to be effective for clinical applications including treatment for inflammation, pain, arthritis, Parkinson disease, and cancer, among others. The effectiveness of BV injection at acupoints possibly results from its anti-inflammatory, antinociceptive, and antiapoptosis effects. However, more randomized, double-blinded, controlled clinical trials are required to confirm the scientific evidence of its therapeutic efficacy, and more pharmacological studies should be conducted to understand its therapeutic mechanisms. In the future, we expect BV injection at acupoints to become a new strategy for treating certain diseases.

Materials and Methods
We searched databases consisting of PubMed, Clinical Key, and the Cochrane Library from their inception to May 2020 making use of the following headings and keywords alone or in varied combinations: "Bee Venom (BV)", "Bee Venom Injections", "Acupoints", and "Acupuncture". The results were limited to articles in English only. We included supplementary articles after screening a manual review of the reference lists of reliable studies and review articles. We excluded articles where the full text was not available, articles unrelated to the therapeutics of BV injections at acupoints, articles with limited information, articles duplicated between databases, and case reports. A flowchart of the search procedures is presented in Figure A1, and a summary of the main published applications of BV therapy is shown in Table 1.