Autophagic effects of Chaihu (dried roots of Bupleurum Chinense DC or Bupleurum scorzoneraefolium WILD)

Chaihu, prepared from the dried roots of Bupleurum Chinense DC (also known as bei Chaihu in Chinese) or Bupleurum scorzoneraefolium WILD (also known as nan Chaihu in Chinese), is a herbal medicine for harmonizing and soothing gan (liver) qi stagnation. Substantial pharmacological studies have been conducted on Chaihu and its active components (saikosaponins). One of the active components of Chaihu, saikosaponin-d, exhibited anticancer effects via autophagy induction. This article reviews the pharmacological findings for the roles of autophagy in the pharmacological actions of Chaihu and saikosaponins.


Chaihu regulates qi stagnation in Chinese Medicine (CM) theory
The CM approach to relieving symptoms (e.g., physical discomfort and emotional instability) is to soothe stagnation of gan qi [17]. Gan qi stagnation can lead to (1) distention and pain in the chest and flank, and menstrual dysregulation, (2) impaired digestive functions such as loss of appetite, dyspepsia, flatulence, and regurgitation, and (3) emotional instabilities such as depression, anxiety, and insomnia [18]. Chaihu is often prescribed to relieve the symptoms of qi stagnation in CM [5].

Autophagy in health and diseases
Autophagy has been highlighted for its protective roles in various physiological and pathological conditions including (1) cellular homeostasis and genome stability maintenance, (2) immunomodulation, (3) hepatoprotection and aggregate removal, (4) cancers, and (5) emotional instability conditions [33][34][35]. Autophagic regulation is mainly responsible for maintenance of normal cellular and hormonal homeostasis, defense against pathogen invasion, and protection against toxic protein aggregate accumulation, and beneficial improvements in all of these at the cellular level are related to improved qi stagnation (Table 1).
Newborn mice under starvation showed immediate increases of autophagy in various tissues, which returned to the basal levels after nutrient supply restoration [36][37][38]. Mice deficient in autophagy-related gene (Atg) 5 showed a substantial increase in nutrition deprivationinduced death, suggesting an essential role of autophagy in energy maintenance [39]. Autophagy is a protective mechanism that eliminates abnormal proteins and defective organelles such as mitochondria, peroxisomes, or ER membranes. For example, hepatocytes from Atg7knockout mice exhibited accumulation of abnormal mitochondria and ER structures [40], and associated cellular degeneration [39]. A recent study further revealed essential roles of autophagy in limiting DNA damage and chromosome instability, and failure of the autophagy process can result in carcinogenesis or cell death [41].

Chaihu-mediated autophagy induction
Maintenance of normal homeostasis by defense against pathogen infections is critical. Fever is an immune response initiated by inflammatory mediators such as interleukin (IL)-1, IL-6, tumor necrosis factor (TNF)-α, macrophage inflammatory protein 1, and interferon (IFN) for heat production, and depends on antipyretics (IL-10, glucocorticoids, and neuropeptides) for heat dissipation [42,43]. Chaihu is prescribed as the major herbal medicine to resolve alternating chills and fever, headache, distention in the chest and flank, or loss of appetite in CM [18,44]. Chaihu was reported to exert its antipyretic effect through the thermoregulatory center in the hypothalamus [45]. Chaihu inhibited increases in cyclic adenosine monophosphate (c-AMP), an endopyrogen, in the hypothalamus and promoted the release of antipyretic substances [46]. Furthermore, total saikosaponins exerted potent anti-endotoxin effects with a simultaneous reduction in body temperature elevation in vivo [47]. All of these beneficial effects can be attributed to the maintenance of cellular homeostasis, a key process regulated by autophagy.
In liver ischemia-reperfusion injury, autophagy induction attenuated the organ damage, and delayed inflammatory or oxidative damage [48]. Furthermore, autophagy suppression was found to be a response to excessive alcohol intake, which might be a reason for the abnormal protein aggregation observed in liver diseases [40]. In vitro studies further showed a dysfunction of autophagy in cells with hepatitis C virus infection [48,49]. Autophagy was also found to regulate the immunological responses to invading microorganisms [50]. Another study showed that plasmacytoid dendritic cells recognized viruses via Toll-like receptors (TLRs) with a requirement for autophagy [51]. In addition, defective autophagy was involved in inflammatory diseases such as systemic lupus erythematosis and Crohn's disease [52,53]. Emerging evidence has suggested roles for autophagy in immunological responses including antimicrobial activity, antigen presentation, cytokine production, and regulation of lymphocytes [50,54]. For example, disruption of the virulence factor from the HSV-1 virus, which inhibited the host autophagy proteins, could prevent fatal encephalitis [55]. In addition, autophagy exhibited protective functions in the spleen, bone marrow, or liver through activation of immune responses such as detoxification and degradation of toxins and inflammatory proteins [56][57][58]. Table 1 Comparisons of CM applications, pharmacological actions, and autophagy effects of Chaihu Chaihu regulated the immune responses against invading pathogens by stimulating the secretion of glucocorticoids and inhibiting inflammation and anaphylaxis [59,60], and was involved in inflammatory processes such as infiltration, capillary permeability, and release of cytokines [46]. Chaihu or its component saikosaponins eliminated exogenous pyrogens through their antibacterial properties [61], and possessed antiviral activities toward hepatitis B [62], human coronavirus 229E [9], influenza virus [11], and respiratory syncytial virus [63]. Ssd reduced the levels of cyclooxygenase and lipoxygenase in vitro, promoted IL-2 and IL-4 production, and inhibited IL-6, TNF-α, and IFN-γ expression in mouse T lymphocytes [64,65]. The prominent anti-inflammatory effects of Chaihu could be mediated through autophagy induction, a key process for pathogen elimination and immunity regulation. Our group was the first to report the autophagic activities of Chaihu and Ssd [16]. We hypothesized that Chaihu harmonizes the exterior and interior of the human body and soothes gan qi stagnation through autophagy induction.

Chaihu-induced autophagy alleviates gan qi stagnation
In CM theory, Chaihu soothes stagnation of gan qi and promotes circulation of qi, and thus alleviates distention and pain in the chest and flank, menstrual dysregulation, impaired digestive functions such as loss of appetite, dyspepsia, flatulence, and regurgitation, and emotional instabilities such as depression, anxiety, and insomnia [18]. Chaihu is used to treat diseases related to the digestive system, e.g., hepatitis, liver cirrhosis, cholecystitis, pancreatitis, gynecological diseases, and hyperlipidemia [5].
Saikosaponins alleviated hepatocytes from oxidative and inflammatory stresses, and inhibited liver fibrosis [66]. Further studies demonstrated the protective effects of saikosaponins in reducing lipid peroxidation in hepatocytes [67], regulating intracellular calcium levels to prevent hepatocyte injury [68], suppressing activation of hepatic stellate cells as the major matrix-producing cells in liver fibrosis [69,70], and reducing collagen I deposition in the rat liver [71]. Saikosaponins exhibited regulatory effects on cytokines such as ILs, TNF, and IFN [64,65], inhibitory effects on infiltration of macrophages and T lymphocytes [72], and bidirectional modulation of splenic T lymphocyte proliferation [64]. These findings suggest that the hepatoprotective effects of Chaihu and saikosaponins are related to improvement of gan qi stagnation. In addition to liver diseases, Chaihu is commonly used for chronic pancreatitis [73]. Saikosaponins exhibited potent stimulatory effects on pancreatic enzyme secretion in rats [74]. Chai-hu-shu-gan powder inhibited the expression of nuclear factor-κB (NF-κB) and TNF-α mRNA in the pancreas to achieve anti-inflammatory and antifibrotic effects [75]. Moreover, the same prescription reduced the abnormally high plasma level of cholecystokinin in chronic pancreatitis, improved the gastric movement, and avoided nausea and flatulence [76,77].
In liver ischemia-reperfusion injury, autophagy induction attenuated the ischemic and reperfusion damage to the organ, probably because a decrease in autophagy would lead to accumulation of dysfunctional mitochondria, resulting in cellular damage and failure in energy production, and eventually cell death [48]. In liver disease, suppression of autophagy caused abnormal protein aggregation [40]. In liver fibrosis, autophagy activation might be beneficial to the recovery of the liver function [78]. All of these findings indicate that Chaihu-induced autophagy might relieve liver disease-related symptoms through anti-inflammatory, organ-protective, and aggregate removal functions, which are related to alleviation of gan qi stagnation.

Chaihu-mediated autophagy intervenes in carcinogenesis
In CM theory, tumor formation is the result of stasis of xue (blood), retention of jin ye (fluid), and accumulation of tan (phlegm) [79]. A recent study demonstrated the anticancer effects of Ssa and Ssd via autophagy induction and autophagic cell death [16]. In addition, Chaihu is a commonly prescribed herb in contemporary formulations (Table 2) with preventive or therapeutic effects on cancer [80]. Patients treated with "xiao Chaihu" decoction exhibited a significantly lower incidence of hepatocellular carcinoma [81], reductions in cancer pain and tumor size [82,83], and prevention of liver cancer relapses [84]. The decoction had multiple functions in immunomodulation, apoptosis, and anti-angiogenesis [85][86][87].
The signaling pathway of autophagy is associated with the key regulatory proteins of carcinogenesis, such as tumor suppressor gene p53, phosphatase and tensin homolog (PTEN), death-associated protein kinase, and proto-oncoprotein B-cell CLL/lymphoma 2 (Bcl-2) [39,88]. Autophagy was responsible for massive cancer cell death in vitro and in vivo [89][90][91]. Autophagic inducers also promoted autophagic cell death in tumors or augmented the efficacy of chemotherapeutic agents when used in combination during cancer therapy [92,93]. By eliminating genomic mutations, damaged proteins, and cytotoxic substances, autophagy protected cells against cancers [94]. However, the roles of autophagy in cancers remain controversial, because autophagy might promote tumor growth by providing energy to poorly-vascularized tumor cells [95].
Despite its adaptive and pro-survival roles, autophagy can lead to type II programmed cell death [96]. Autophagy promoted autophagic cell death in cells [97], and killed apoptosis-resistant cancer cells under chemotherapy [98]. Moreover, autophagy was associated with massive cancer cell death in cancerous tissues derived from different organs [99,100]. Ssd was able to induce autophagic cell death in a panel of apoptosis-resistant cells via direct inhibition of SERCA [16]. The anticancer effects of Chaihu can be attributed to its autophagyinducing ability.

Chaihu-mediated autophagy modulates stress hormone-regulated metabolism
Chaihu could mediate its protective effects on gan qi stagnation-induced emotional instability through lipid metabolism and hormonal regulation [101]. In fact, analyses of plasma metabolites in a rat model of gan qi stagnation stimulated by chronic immobilization stress revealed elevated levels of lactic acid, saturated fatty acid, and blood sugar, and reduced levels of unsaturated fatty acid and high density lipoprotein [102]. Another study applied stress to a macaque model with premenstrual syndrome, and demonstrated increased plasma levels of serotonin (5-HT), noradrenalin, and prolactin [103].
As a regulator of lipid and glucose metabolism [104], loss of autophagy caused abnormal accumulation of lipids in mouse hepatocytes and a significant increase in plasma triglycerides, with reductions in fatty acid betaoxidation [105] and pancreatic β-cell mass [106]. Coincidently, saikosaponins increased hepatic uptake of cholesterol and decreased plasma levels of cholesterol and triglycerides [107]. Furthermore, a study on depressive patients revealed correlations between the plasma levels of cholesterol, triglycerides, and serum neurotransmitters, and depression [108]. As saikosaponins were able to reduce the plasma levels of cholesterol, triglycerides, and phospholipids [107], Chaihu might attenuate depressive symptoms by regulating metabolite, hormone, and neurotransmitter levels via autophagy-mediated lipid metabolism in the human body.

Conclusions
The function of Chaihu in harmonizing the exterior and interior of the body is related to its pathogen control and immunomodulation properties. Furthermore, Chaihu's function in resolving gan qi stagnation might arise through its supportive roles in protecting organs, preventing damage to cells and organs, and restoring visceral and cellular metabolic conditions. All of these protective pharmacological effects of Chaihu might be attributed to its autophagy induction.

Competing interests
The authors declare that they have no competing interests.
Authors' contributions VKWW conceived and planned the review. BYKL and JFO carried out the review plan and wrote the manuscript. All authors read and approved the final manuscript.