Effect of Traditional Chinese Medicine on Long-Term Outcomes of Snakebite in Taiwan

Herein, we review the characteristics of the six predominant venomous snakes in Taiwan and the effects of traditional Chinese medicine on the long-term outcomes of snakebite venom. We electronically searched databases, including PubMed, ClinicalKey, China National Knowledge Infrastructure, National Digital Library of Theses and Dissertations in Taiwan, and Airiti Library, from their inception to November 2019 by using the following Medical Subject Headings’ keywords: snakebite, long-term, chronic, Chinese medicine, CAM, herb, and Taiwan. The most common long-term effects of snakebite envenomation include “migraine-like syndrome”, brain injuries caused by hypoxia or intracranial hemorrhage, and chronic kidney disease. In addition, hypopituitarism is also worth mentioning. Traditional Chinese medicine can potentially be used in a complementary or alternative treatment for these effects, but additional studies are needed.


Introduction
Snakebite envenomation is a rare medical issue, with an incidence rate lower than that of other diseases. However, it can lead to serious complications or death. In 2009, the World Health Organization included snakebites in its list of "neglected tropical diseases" [1,2]. Annually, 1.8-2.7 million cases of snakebite envenomation occur and 94,000 people are killed worldwide. The incidence of snakebite envenomation is particularly high in South and South-East Asia [3].
Taiwan is located in a subtropical region near South-East Asia. The climate in Taiwan is warm and humid and the topography is diverse, which is suitable for the survival of various snake species [4]. In Taiwan, more than 20 snake species are venomous, six of which are particularly prevalent and can be classified into two major venomous snake families: Bungarus multicinctus and Naja atra, which are elapids; and Trimeresurus stejnegeri, Deinagkistrodon acutus, T. mucrosquamatus, and Daboia siamensis, which are vipers [5].
T. stejnegeri (red tail Bamboo Viper) is widely distributed and is the only major venomous snake in Taiwan that is not a protected species [5]. It is arboreal and usually active at low altitudes in mountains, bamboo groves, or orchards [6]. De. acutus (Hundred-Pace snake) is mostly distributed in

Clinical Manifestations of Snake Envenomation in Taiwan
Despite the fact that the six predominant venomous snake species in Taiwan can be divided into hemotoxic, neurotoxic, and mixed-type subgroups, symptoms following snakebite envenomation from different species share much in common. Local effects include local pain, petechiae, ecchymosis, swelling, blistering, infection (cellulitis or abscess), and cutaneous or muscle necrosis. Local effects induced by envenomation of De. acutus, T. mucrosquamatus, and N. atra are especially severe and may progress to compartment syndrome [5,16,30]. A report described the case of a 41-year-old woman who developed necrotizing fasciitis following De. acutus envenomation in Taiwan [14]. A 60-year-old man was reported to develop respiratory failure after a T. mucrosquamatus bite; the bite site, on the occipital lobe, demonstrated severe edema, which was followed by acute airway obstruction and respiratory failure [31].
Systemic effects include hypotension and hemostatic alterations that may result in ischemic stroke, intracranial hemorrhage, or disseminated intravascular coagulation (DIC).
Other symptoms highly associated with envenomation by particular species include acute kidney injury and acute pituitary failure, which are the most relevant to Da. siamensis envenomation [32,33]; ptosis and respiratory failure, which are the most relevant to B. multicinctus envenomation; and ophthalmia, which is the most relevant to N. atra envenomation. The main toxins and clinical manifestations of snake envenomation in Taiwan are summarized in Table 1.

Migraine-Like Syndrome
Migraine-like syndrome following snakebite envenomation was reported in a study from Sri Lanka. It revealed that 112 of 816 snakebite victims presented with long-term complications, and 46 patients (5.6%) developed "migraine-like-syndrome", which presented as headaches, dizziness, and sensitivity to light [36]. Hypercoagulability is suspected to be related to migraine pathophysiology, particularly migraine aura [35]. The pathogenesis of snake venom-induced migraine-like syndrome remains unclear. It has been hypothesized that it is caused by hemotoxins, which have procoagulant properties and may affect cranial blood vessels. T. stejnegeri, T. mucrosquamatus, and Da. siamensis venoms contain an abundance of procoagulant proteins, such as Factor V activator, Factor IX activator, Factor X activator, thrombin-like enzymes, and prothrombin activator [20,37,39].
In traditional Chinese medicine, the pathogenic factor of migraine is considered to be blood stasis, which could correspond to hypercoagulability. Therefore, to treat migraines, traditional Chinese medicine practitioners prescribe herbs, such as Angelica sinensis (Dang gui) and Ligusticum chuanxiong (Chuan xiong), that promote blood flow and dispel blood stasis [40]. The pharmacological activities of A. sinensis and L. chuanxiong include antioxidant, anti-inflammatory and antinociceptive effects [41,42]. Ligustilide, one of the main active components of A. sinensis and L. chuanxiong, is capable of regulating the release of calcitonin gene-related protein (CGRP): a highly potent vasoactive peptide that can cause vasodilatation [42]. In addition, Xiang Fu Chuan Xiong Cha, an ancient formula, composed of Cyperi Rhizoma and Chuanxiong Rhizoma at a 2:1 weight ratio, was used for curing migraines and headaches. A recent animal study demonstrated that Cyperi Rhizoma and Chuanxiong Rhizoma (CRCR) significantly increased cerebral blood flow and decreased several substances involved in neurogenic inflammation, thereby relieving migraines [43]. Fructus Viticis (Man jing zi) is another traditional Chinese medicine that is commonly used for curing migraines and headaches. However, Fructus Viticis does not increase cerebral blood flow but regulates neuropeptides such as 5-hydroxytryptamine (5-HT), γ-aminobutyric acid (GABA), calcitonin gene-related peptide, and substance P [44].

Brain Injuries
Snakebite envenomation may lead to serious neurological complications, including acute respiratory failure (due to muscle paralysis) [19], ischemic stroke [45], and intracranial hemorrhage [46], which can result in brain injuries.

•
Respiratory failure Respiratory failure is caused by neurotoxic envenomation in most cases. Neurotoxins are a major component of the venoms of most elapid snakes such as B. multicinctus and N. atra. These toxins can be divided into two groups: α-neurotoxins and β-neurotoxins. α-neurotoxins act on postsynaptic terminals, and belong to the family of three-finger toxins that block acetylcholine receptors; β-neurotoxins act on presynaptic terminals, and belong to the family of heterodimeric PLA2s that inhibit the release of acetylcholine from presynaptic terminals [15,38,47]. The combined effects of α-neurotoxins and β-neurotoxins block neuromuscular transmission and result in muscle paralysis. In respiratory muscles, they induce acute respiratory failure [31,48] and result in hypoxic brain injuries. •

Cerebral infarction and intracranial hemorrhage
Hemotoxins are responsible for cerebral infarction and intracranial hemorrhage following snakebite envenomation. Hemotoxins are major components of Viperidae venoms, such as from T. stejnegeri, T. mucrosquamatus, De. acutus, and Da. siamensis. These hemotoxins can be categorized into two groups: hemorrhagic or nonhemorrhagic.
Nonhemorrhagic toxins include Factor V activator, Factor IX activator, Factor X activator, prothrombin activator, thrombin-like enzymes, and aggregoserpentin. These toxins cause thrombosis as a result of their procoagulant properties and activation of platelet aggregation [13,37,47]. A case report described a 49-year-old woman who developed acute cerebral infarction following a T. stejnegeri snakebite in Taiwan [45]. Both cerebral infarction and intracranial hemorrhage can result in brain injuries that cause long-term sequelae such as hemiplegia or cerebellar ataxia [36,49].

Chronic Kidney Disease
Among the six predominant venomous snakes in Taiwan, Da. siamensis envenomation is the most relevant to kidney injury. Da. siamensis venom primarily demonstrates hemotoxic effects with some neurotoxic effects [5]. The causes of kidney injury are multifactorial and include bleeding, hypotension, intravascular hemolysis, disseminated intravascular coagulation, and nephrotoxicity [5,55].
In most cases, acute kidney injuries caused by snakebite envenomation are healed within days or months after treatment [55]. However, in some cases, acute kidney injuries develop into chronic renal disease. A descriptive study in Sri Lanka revealed that 20 (37%) of 54 patients who had acute kidney injury after a snakebite developed chronic renal disease. Furthermore, the renal histology of six patients demonstrated predominant glomerular sclerosis and interstitial nephritis [56].
A large retrospective study in Taiwan reported that traditional Chinese medicine reduced end-stage renal disease risk by 60% in patients with chronic kidney disease [57]. Some herbs such as As. membranaceus, Ophiocordyceps sinensis, Rheum palmatum, and Cortex Moutan (root bark of P. suffruticosa), have displayed benefits in treating kidney injuries [58]. Furthermore, As. membranaceus, with strong anti-inflammatory effects, can reduce proteinuria and attenuate kidney injury in several animal models [59].

Hypopituitarism
Hypopituitarism is not a common complication following snakebite envenomation. Nevertheless, there were several case reports that revealed that victims suffered from hypoglycemia, fatigue, loss of libido (male), and amenorrhea (female) after a Russell's viper bite [27,60]. A retrospective study in Burma reported that seven of 24 individuals had clinical features of hypopituitarism, while four of 24 individuals had pituitary hormonal abnormalities without evident manifestations [61]. In India, a 45-year-old male was reported to develop delayed hypopituitarism after a Russell's viper bite. The patient complained about lethargy and loss of appetite for six months, and laboratory data showed a decrease of T4, LH and cortisol. As a result of adrenal and thyroid deficiency, a brain MRI was arranged. The image demonstrated an empty sella with a thinned out pituitary gland [62]. The imaging of pituitary damage caused by snakebite is different from that of postpartum necrosis (Sheehan's syndrome), which usually showed hemorrhage into a normal size pituitary. Venoms of other snakes reveal similar hemotoxic effects on intravascular coagulation to Russell's viper venom. However, Russell's viper envenomation is particularly relevant to pituitary damage [63].
Hypopituitarism following snakebite envenomation is considered to be related to acute kidney injury and chronic kidney disease. A prospective observational study in India showed that nine of 96 patients developed hypopituitarism after a Russell's viper bite. All of the nine patients suffered from acute kidney injury and had dialysis, while five (55.56%) of the nine developed varying degrees of chronic kidney disease on long-term follow up [64].
Therapeutic agents currently used for hypopituitarism include hypophysis hormones and steroids [62]. In traditional Chinese medicine, the pathogenic factor of hypopituitarism is considered Toxins 2020, 12, 132 6 of 11 to be "kidney deficiency". Hence, to treat hypopituitarism, practitioners prescribe herbs or a formula to nourish both kidney-yang and kidney-yin, such as Er-Xian Decoction (EXD). EXD is a traditional Chinese medicine formula composed of Herba Epimedii (Yinyanghuo), Radix morindae Officinalis (Bajitian), A. Sinensis, R. Anemarrhenae (Zhimu), Phellodendri Chinensis Cortex (Huangbo), and R. Curculiginis (Xianmao). It was usually used to treat climacteric syndrome and was proved to be effective to trigger the hypothalamic-pituitary-testicular axis and enhance levels of GnRH and LH in an animal model [65]. A study in China showed that Tiannianyin (TNY), a Chinese medicinal formula, can elevate PCNA expression in the anterior pituitary cells of aging rats, and enhance levels of testosterone [66]. These findings are summarized in Table 2. Table 2. Effects of traditional Chinese medicine (TCM) on long-term outcomes of snake envenomation.

Long-Term Sequelae
Potential TCM Treatment Possible Mechanism References "Migraine-like syndrome" A. sinensis, L. chuanxiong Regulate the release of CGRP [41,42] CRCR Increases cerebral blood flow and decreased several substances involved in neurogenic inflammation [43] F. Viticis Regulates neuropeptides [44] Brain injuries secondary to hypoxia, ischemic stroke or ICH

Conclusions
Envenomation by the six prevalent venomous snakes in Taiwan can lead to long-term effects such as headaches, dizziness, brain injuries, chronic kidney disease, and hypopituitarism. TCM can relieve the symptoms mentioned above, which is suggested to result from its antioxidative, anti-inflammatory and antinociceptive properties. In addition, since TCM treatments use acupuncture or natural products in general, the cost is lower than of Western medicine. TCM is symptomatic medicine; its treatment mainly focuses on the symptoms of the diseases. The characterization of TCM is pattern identification and treatment, which is different from disease identification and treatment in the Western medicine, which emphasizes the etiology of the disease. Therefore, it is suggested that in the acute stage of snakebite envenomation, Western medicine such as antivenoms and supportive treatment must be used. In addressing the long-term effects of snakebite envenomation, such as "migraine-like syndrome", TCM could be a complementary treatment. Therefore, how to combine Western medicine and TCM to produce a more beneficial treatment for the patients with snakebite envenomation could be an issue in the future.

Materials and Methods
We searched the PubMed, ClinicalKey, China National Knowledge Infrastructure, National Digital Library of Theses and Dissertations in Taiwan, and Airiti Library database from their inception to November 2019. Medical Subject Headings' keywords included "snakebite", "long-term", "chronic", "Chinese medicine", "CAM", "herb", and "Taiwan". Languages were limited to English and Chinese. The filter process was first carried through the website search engine, which yielded 250 articles. We manually screened the studies and excluded 145 articles either for lack of abstract, or the abstract not being related to snakebite envenomation. Another 69 articles were excluded because the full text was not available or it was not related to snakebite envenomation. In total, 36 articles were included in the study, including prospective and retrospective studies related to snakebite envenomation. A flowchart of the search process is displayed in Figure 1. For detailed information of the articles we yielded and excluded, please check the Supplementary Materials Table S1. Regarding the images of the six predominant venomous snakes in Taiwan, the information source for this study has been shown in reference [12].
identification and treatment in the Western medicine, which emphasizes the etiology of the disease. Therefore, it is suggested that in the acute stage of snakebite envenomation, Western medicine such as antivenoms and supportive treatment must be used. In addressing the long-term effects of snakebite envenomation, such as "migraine-like syndrome", TCM could be a complementary treatment. Therefore, how to combine Western medicine and TCM to produce a more beneficial treatment for the patients with snakebite envenomation could be an issue in the future.

Materials and Methods
We searched the PubMed, ClinicalKey, China National Knowledge Infrastructure, National Digital Library of Theses and Dissertations in Taiwan, and Airiti Library database from their inception to November 2019. Medical Subject Headings' keywords included "snakebite", "longterm", "chronic", "Chinese medicine", "CAM", "herb", and "Taiwan". Languages were limited to English and Chinese. The filter process was first carried through the website search engine, which yielded 250 articles. We manually screened the studies and excluded 145 articles either for lack of abstract, or the abstract not being related to snakebite envenomation. Another 69 articles were excluded because the full text was not available or it was not related to snakebite envenomation. In total, 36 articles were included in the study, including prospective and retrospective studies related to snakebite envenomation. A flowchart of the search process is displayed in Figure 1. For detailed information of the articles we yielded and excluded, please check the Supplementary Materials- Table S1. Regarding the images of the six predominant venomous snakes in Taiwan, the information source for this study has been shown in reference [12].

Supplementary Materials:
The following are available online at www.mdpi.com/xxx/s1, Table S1: Review Matrix on the Use of Effect of traditional Chinese medicine on long-term outcomes of snakebite in Taiwan.