Rheumatoid arthritis (RA) is one of the representative diseases of paralysis, characterized by chronic inflammation of joint tissue, and the clinical manifestations are joint pain, swelling, deformation, and unfavorable flexion and extension. At present, there is no specific treatment method, and with the development of the disease, the disability rate is high, the prognosis is poor, and it is easy to recur, and in severe cases, it may lead to disability and bring a large burden to society [1-2]. According to Tibetan medical theory, RA belongs to the category of "Zhenbu disease", due to the imbalance of the three energy substances that make up the human body, "Long", "Chiba", and "Bacon", resulting in the yellow water in the body spreading around the body and accumulating in the joint cavity, eventually causing joint swelling, pain, fever, and stiffness [3-4]. Nonsteroidal anti-inflammatory drugs (NSAIDs) are the main clinical treatment for RA, but long-term treatment can lead to drug resistance and limited therapeutic efficacy [5]. Therefore, it is of great significance to find drugs with few adverse reactions and can effectively treat RA.
Notopterygium incisum Ting ex H. T. Chang or N. Broad-leaved Notopterygium incisum Ting ex H. T. Chang The dried roots and rhizomes of franchetii H. de Boiss. are mainly distributed in Aba, Sichuan, Tibet, Qinghai and other places. According to the 2020 edition of the Chinese Pharmacopoeia and the Dictionary of Chinese Ethnic Medicine, it is a commonly used medicine in Chinese, Tibetan, Qiang, and Mongolian medicines, with the effects of dispersing cold, dispelling rheumatism, relieving pain, and benefiting joints, and is mainly used for the treatment of wind-cold, dampness, and limb pain, and has a long history of medicinal use [6-8]. In Tibetan medicine, it is a clinical medicine for the treatment of "Chenbu disease" [9]. According to research reports, Qianghuo has anti-inflammatory, analgesic, and antipyretic effects, and is widely used in RA diseases, but there are few inductive reports on it in the literature. This article reviews the research progress and mechanism of action of Qianghuo anti-arthritis active ingredients, and provides a theoretical basis for its pharmacodynamic material basis, development and utilization.
1 Active ingredient of Qianghuo anti-RA action
In recent years, researchers have conducted a lot of research on the chemical composition of Qianghuo, which mainly includes compounds such as coumarins, polyennes, terpenoids, alkaloids, flavonoids, organic acids, and organic esters [10]. Peng Ren [11] found that there were 89 coumarins, which could be divided into 19 simple coumarins, 12 angular furans, 8 anglatins, 49 linear coumarins, and 1 dihydroisocoumarin according to the parent nuclear classification. There are 46 polyalnes, 28 organic acids and esters, 17 sesquiterpenoids, and 3 flavonoids. Among them, coumarins (1~12, 22~46), polyenynes (13), flavonoids (47, 48) and phenolic acids (14~21) have played a great role in anti-inflammatory and analgesic, inhibition of abnormal proliferation of synovial fibroblasts, osteocyte protection and antioxidation, which are closely related to the pathogenesis of RA, as shown in Table 1. Therefore, from the perspective of pathogenesis, the active ingredients that may work on Qianghuo anti-RA were discovered, as shown in Figure 1.
Coumarinoids are the earliest and most studied components in Qianghuo [24]. Among these components, Qianghuol (24) and Isoimperatorin (25) have a high content, and the 2020 edition of the Chinese Pharmacopoeia records that the total amount of Qiangol should not be less than 0.40%, which is an important index component for the quality evaluation of Qianghuol [25]. Twenty-four coumarins were isolated from Qianghuo extract, most of which had good anti-inflammatory effects and could effectively inhibit the generation of nitric oxide in the inflammatory response model [16]. In addition, coumarins have been shown to be widely used in the treatment of bone and joint diseases [26]. Fusarium celeryl glycol is the most representative polyalkyne compound, and it is also a characteristic compound in Qianghuo, and most of the other components of this class are derivatives of Fuvalryl glycol.
In addition, it has been reported that fuvalryl is also the main active ingredient in the inhibition of 5-lipoxygenase and cyclooxygenase, and has significant anti-inflammatory effects [27]. Flavonoids have been reported in Qianghuo, and only three such compounds have been found, among which geranidins (47) and geranidins (48) have strong antioxidant and free radical scavenging abilities, and have anti-inflammatory and anti-bone cell deformation effects [22-23]. Phenolic acids are widely found in nature, and their biological activities are diverse, with anti-inflammatory, antioxidant and other abilities. Ferulic acid (21), as an active ingredient extracted from Qianghuo, has shown good anti-inflammatory properties in a variety of animal and cell models, inhibiting the expression of inflammatory factors and inflammatory cell infiltration, and can inhibit synovial cell proliferation and angiogenesis, reduce osteoclast formation and bone resorption capacity, and protect joint tissue [28].
2 Mechanism of action of Qianghuo active ingredient anti-RA
2.1 Anti-inflammatory action
2.1.1 Inhibition of pro-inflammatory factors
Anti-inflammatory disease is the key to the treatment of RA as an autoimmune disease characterized by synovitis and vasculitis that eventually causes destruction of articular cartilage and bone, and inflammation is present throughout the development of RA [29]. Cytokines such as tumor necrosis factor (TNF), interleukin (IL), interferon, and chemokine are involved in the innate immunity and adaptive immunity of RA, and affect the occurrence and development of RA, among which pro-inflammatory factors such as TNF-α, IL-1, IL-6, and IL-17 increase the permeability of microvessels, resulting in redness, swelling, heat and pain in RA joints [30].
因此，抑制促炎因子的表达对RA起到很好的治疗作用。 此外，通过抑制Janus激酶（Janus kinase，JAK）/信号转导和转录激活因子（signal transduction and activators of transcription，STAT)、丝裂原活化蛋白激酶（Mitogen-activated protein kinase，MAPK）和核因子-κB（nuclear factor-κB，NF-κB）信号通路等可以直接或间接下调通路相关促炎因子的表达，也可以达到抑制炎症、减轻RA疼痛的作用[31]。
Coumarin compounds are a major component of Qianghuo and have obvious anti-inflammatory effects. Bergamot lactones (23), uncaulins (43), colevanol, and isoimpagenin are all coumarin compounds, and they have been found to be the main active substances with anti-inflammatory effects through the mining and resource evaluation of anti-inflammatory quality markers of Qianghuo [32]. Among them, procaulin can prevent the infiltration of inflammatory cells, reduce the level of pro-inflammatory factors, and significantly inhibit the expression of pro-inflammatory factors such as IL-1β, IL-6, and TNF-α [18]; Bergamot lactones can significantly improve the expression of IL-1β-induced inflammatory factors and mediators, and inhibit lipopolysaccharide-stimulated TNF-α, IL-1β, IL-6, and nitric oxide production through the JAK/STAT signaling pathway, while inhibiting the release of lipopolysaccharide-induced inflammatory factors in RAW264.7 cells in a dose-related manner, exerting anti-inflammatory effects [33-34].
As an indicator component of Qianghuol, it can improve RA by targeting JAK/STAT signaling, and it was found that it could significantly reduce arthritis scores and synovial tissue inflammation in collagen-induced arthritis (CIA) mice. In vitro experiments have found that it can inhibit the production of a variety of pro-inflammatory and chemokine in lipopolysaccharide/γ interferon and TNF-α-treated macrophages [35]. In lipopolysaccharide-induced RAW264.74 cells, isoimpahuin 20 and 100 μg/mL significantly inhibited TNF-α, IL-6, and nitric oxide [36].
In addition, 5 μg/mL of quinocoside (3) has anti-inflammatory and antioxidant effects on lipopolysaccharide-induced chondrocyte inflammation model, and can significantly down-regulate the expression of inflammation-related genes IL-6, matrix metalloproteinases 3 (MMP3), MMP13, and inducible nitric oxide synthase [37]. Psoralen derivatives can also exert anti-inflammatory effects on RAW264.7 cells by regulating NF-κB and MAPK signaling pathways, and Wu et al. [13] found that psoralen (38) combined with bergamot, licohotol, and purple uncortop can significantly improve RA and reduce the number of inflammatory cells in CIA rats.
In addition to coumarins, flavonoids and phenolic compounds are important material bases for anti-inflammatory activity. As a natural flavonoid glycoside, geranidinin has significant anti-inflammatory effects, and 20 mg/kg ig was used in male Wistar rats with arthritis induced by complete Freund's adjuvant for 2 weeks, and rheumatoid factor (RF), anti-citrullinated peptide antibodies (ACPA), TNF-α, IL-13 and IL-17 levels, geranidinin was found to significantly reduce the levels of RF, ACPA, TNF-α, IL-17, and other pro-inflammatory factors [38]. Ferulic acid is the most common anti-inflammatory and analgesic phenolic component, which has a good inhibitory effect on arthritis symptoms, and Zhu et al. [39] found that ferulic acid 25 and 50 mg/kg can significantly reduce the primary (foot edema volume) and secondary lesions of arthritic rats induced by complete Freund's adjuvant, and can reverse changes in biochemical parameters and inflammatory markers, such as C-reactive protein and RF.
2.1.2 Regulation of immune cells
Cytokines produced by immune cells can induce the formation and aggregation of immune cells, leading to persistent inflammation [30]. CD4+ T cells can be stimulated by cytokines to differentiate into different helper T cells (Th), such as Th1, Th2, Th17 and regulatory T cells (Treg), among which Th1 secretes pro-inflammatory factors, causing synovial inflammation and then cartilage damage. Th2 can secrete anti-inflammatory factors to alleviate the inflammatory response of RA. Th17 can also secrete pro-inflammatory factors and induce inflammation; Treg can secrete immunosuppressive factors, which exert an effective immunosuppressive effect to inhibit the inflammatory response of RA [40-41]. Therefore, regulating the balance of Th1/Th2 and Th17/Treg cells is of great significance to improve the joint swelling caused by RA and the synovial inflammation of the joint. Studies have shown that psoralen can regulate Th1/Th2 lymphocyte balance in mouse models of rheumatoid arthritis, and the intervention of ig psoralen 40 mg/kg can effectively reduce the differentiation of CD4+ cells into Th1 cells in mouse spleen lymphocytes and regulate Th1/Th2 balance [42].
2.2 Inhibit abnormal proliferation of synovial fibroblasts
Dysplasia synovial fibroblasts are another pathological feature of RA, which may lead to tumor-like proliferation and aggressive migration, resulting in joint destruction, deformity, and even loss of function [43]. In recent years, studies have shown that the active ingredient of Qianghuo has the function of inhibiting synovial fibroblast proliferation and reducing synovial tissue proliferation and joint destruction. Isopsoralen (45) is an effective active ingredient in the treatment of RA in Qianghuo, which can inhibit the inflammatory phenotype of RA fibroblast-like synoviocyte (FLS) and prevent the occurrence and progression of RA-like manifestations in the CIA model, and improve the inflammatory phenotype of RA fibroblastic synovial cells by inhibiting the cytokine production, migration, invasion, and pro-angiogenic ability of RA fibroblastic synovial cells [20].
Imperatorin, a naturally occurring furanocoumarin, not only significantly reduces collagen-induced arthritis by reducing synovial hyperplasia, but also enhances the apoptosis index of ankle cells. In addition, imperatorin can inhibit cell proliferation and induce apoptosis of RA-FLS, which may be related to the mitochondria/cysteine aspartate protease-mediated signaling pathway, which accelerates apoptosis by significantly reducing IL-1β-induced RA-FLS cell viability [19]. In addition, it was found that the inhibition of CIA-FLS cells by ambirol and isoimperatorin was dose-related at the same time [15]. Chen et al. [44] measured the proliferation and apoptosis of human RA fibroblast MH7A cells by using CCK8 kit and flow cytometry, and found that geranithin could inhibit the proliferation of MH7A cells induced by TNF-α in a dose-related manner, and could increase the apoptosis rate of MH7A cells.
2.3 Anti-bone destructive effect
RA is characterized by systemic bone loss and joint destruction, in which the balance of osteoclasts and osteoblasts plays an important role in the treatment of RA, and osteoclasts play an important role in the growth, development, repair, and reorganization of bone, and their hyperfunction can lead to bone degenerative changes, such as osteoporosis [45]. In RA, bone tissue homeostasis is disrupted, and osteoclast production is greater than apoptosis, which leads to abnormal bone remodeling in RA. At the same time, due to the increase in the expression of synovitis and others, the differentiation and maturation of osteoblasts are blocked, which inhibits bone resorption and cannot compensate for pathological bone resorption, resulting in bone destruction [46]. In addition to osteoclasts and osteoblasts, chondrocytes are also very important in inhibiting cartilage destruction in RA. Chondrocytes are the only cells present in cartilage, so apoptosis of chondrocytes may cause bone destruction and affect the patient's quality of life [47].
The coumarin compounds bergamot lactone and bergamot (42) in Qianghuo had obvious regulatory effects on osteoclasts and osteoblasts. It has been found that the number of osteoblasts in rat osteoblasts treated with bergamot lactones for 24, 48, and 72 h showed that the number of osteoblasts increased significantly, and the expression levels of osteocalcin and alkaline phosphatase were significantly increased [48]. Zheng et al. [14] found that bergamot lactone could significantly inhibit osteoclast cell production and bone resorption under lipopolysaccharide stimulation conditions. As a regulator of osteoblasts, bergamot promotes the nuclear translocation of β-catenin by activating the Wnt/β-catenin signaling pathway, significantly promoting osteoblast differentiation and bone formation [17].
In addition, dihydrocarvirysol (46) can activate autophagy by inhibiting serum and glucocorticoid-induced protein kinase 1 expression, thereby attenuating lipopolysaccharide-induced chondrocyte inflammation and apoptosis[21]. Flavonoids also have a good resistance to the destruction of bone cells, and geranithin can improve IL-1β-induced deformation and decrease in the number of chondrocytes in neonatal rat osteoarthritis, and can increase the phosphorylation level of chondrocytes p65, weaken the transport of NF-κB p65 from the cytoplasm to the nucleus, and inhibit the activation of the NF-κB pathway[23].
2.4 Antioxidant effect
Oxidative stress is an important cause of RA and one of the pathological results of RA. Oxidative stress refers to an increase in cytokine secretion and aggravation of joint inflammation by increasing the body's lipid peroxidation response [49]. The process of reactive oxygen species generation has been shown to be positively correlated with the degree of inflammation and the rate of joint destruction [50]. In RA patients, reactive oxygen species increase in the body, weakening their antioxidant capacity, aggravating inflammatory tissue damage, and prompting the synovial membrane of RA to secrete metalloproteinases, thereby destroying cartilage.
Superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px) are antioxidant enzymes that can remove reactive oxygen species from organisms and protect cells from oxidative stress. Therefore, increasing the activity of SOD, CAT, GSH-Px can reduce the level of oxidative stress in the body and protect the body's cells from damage. In addition, nitric oxide is a type of reactive nitrogen radical. In the pathogenesis of RA, high levels of nitric oxide can stimulate reactive oxygen species, induce lipid peroxidation, and affect cartilage damage in inflammatory joints [51]. Therefore, inhibiting nitric oxide production can also help alleviate inflammation and oxidative stress damage.
Purple procamidin can inhibit lipopolysaccharide stimulation of mitochondrial fission-dependent dynamic protein-associated protein 1 phosphorylation and large reactive oxygen species production in chondrocytes [52]. Oxidized imperatorin (7) exhibits potent antioxidant activity in in vitro free radical scavenging experiments [53]. Bergamot lactones act as antioxidants to prevent the accumulation of reactive oxygen species, inhibit oxidative stress caused by isoprenaline-induced free radical production in rats, and even regulate the levels of GSH-Px, CAT, and SOD [54].
In addition, flavonoids have obvious antioxidant effects in Qianghuo. Geranidin, geranidin, are flavonoids in Qianghuo. Studies have shown that they can improve hydrogen peroxide-induced oxidative stress in a dose-related manner, restore cellular antioxidant enzyme activity, reduce malondialdehyde levels induced by hydrogen peroxide, and exert a protective effect on endothelial cells that induce oxidative stress by protecting cells from the harmful effects of hydrogen peroxide and enhancing the activity of cellular antioxidant systems [22]. In rat adrenal pheochromocytoma PC12 cells, geranithin was able to increase cell viability, reduce lactate dehydrogenase release and reactive oxygen species levels, and inhibit oxidative stress [55].
The mechanism of Qianghuo and its active ingredients in the treatment of RA is shown in Figure 2.
3 The anti-RA effect of Qianghuo compatibility and its preparations
The compatibility of traditional Chinese medicine is the main form of clinical medication, and reasonable compatibility can play a synergistic role and reduce toxicity. Studies have shown that the two drugs must help each other to enhance the potency, and the compatibility effect is significantly higher than that of the drug alone, and the combination of drugs can significantly reduce the swelling of the toes in arthritic rats to a certain extent, and increase the pain threshold of mechanical stress stimulation [56]. In addition, the compatibility of TCM compounds is the main way of TCM treatment, with the Jun medicine in the compound as the main material base to exert the main effect, and the Chenzuo medicine to assist and reconcile to achieve the best efficacy and therapeutic purpose [57]. Sun Hongbing et al. [58] found that Qianghuo is often compatible with drugs such as fengfeng, Chuanxiong, and angelica. Wind prevention is the minister, which can relieve the surface of wind, sweating and pain relief; Chuanxiong is used as a support, which can be used to invigorate blood and relieve pain; Angelica is used as an envoy, which has the effect of relieving the fear of sweating too much and hurting the blood.
Clinically, Qianghuo often uses compound preparations or formulas to resist RA, such as Qianghuo Dihuang decoction, Qianghuo Qingang Fang and Qianghuo Sheng Wet Decoction, which play an important role in the treatment of RA. Modern pharmacological studies have shown that Qianghuo Dihuang decoction has a significant effect in the treatment of adjuvant RA rats, and can effectively improve Th17/Treg imbalance, serum MMP levels, and the expression of vascular endothelial growth factor and NF-κB ligand mRNA in synovial tissue [59].
Qianghuo Qin Gang Fang has a good clinical effect on patients with rheumatism, cold, and dampness, and can reduce the levels of inflammatory factors, MMP2, and MMP9, and alleviate the inflammatory response of patients [60]. Li Wenlong et al. [61] suggested that Qianghuo Sheng Wet Tang may act on the acute stage of RA through related signaling pathways such as inflammation, angiogenesis, synovial hyperplasia, and immune factors. In addition, the understanding and treatment of RA syndrome differentiation and classification is a characteristic of traditional Chinese medicine, and in the Chinese Medical Codex, Qianghuo as a drug for the treatment of RA has been used frequently by doctors throughout the ages, and its basic formula composed of licorice, white peony, parsnip, angelica, cinnamon branch, ephedra, skullcap, myrrh, and frankincense can be used to treat RA from the liver [62], followed by preparations containing Qianghuo can also treat RA rheumatism and stasis, blood stasis obstruction, liver and kidney insufficiency, and damp-heat obstruction syndrome, as shown in Table 2.
4 Conclusion and outlook
As a long-term, recalcitrant and disabling autoimmune disease, RA can be treated with a variety of chemical drugs, but long-term use will produce adverse reactions such as gastrointestinal tract, liver and kidney damage. Qianghuo has the effects of dispersing cold, dispelling rheumatism, relieving pain, and improving joints, and is a common medicine for the treatment of RA. Through modern pharmacological studies, it has been found that the coumarin compounds in Qianghuo, Autrodin, Isoimperatorin, Qianghuol, Bergamot, Psoralen have anti-inflammatory effects, among which Qianghuol, Isoimperatorin and Isopsoralen can inhibit the abnormal proliferation of synovial fibroblasts. In addition, bergamot, bergamot and dihydroceleryl have obvious regulatory effects in bone cells. Alachin, oxidative imperatorin, and bergamot lactones can also act as antioxidants to prevent the accumulation of reactive oxygen species, which may achieve the therapeutic effect of RA. The flavonoids geraniphylin and geranithin also have obvious effects on anti-inflammatory, anti-bone destruction and antioxidant; The phenolic compound ferulic acid mainly acts on anti-inflammatory and antioxidant aspects.
Compared with the current clinical anti-RA drugs, Qianghuo has the advantages of fewer adverse reactions, less easy to form drug tolerance and no addiction. However, due to the fact that the clinical application of Qianghuo is mostly presented in the form of compound preparations, prescriptions, etc., and the chemical composition of Qianghuo itself is complex and diverse, it is still unclear whether there are multiple chemical components that synergize with each other in the treatment of RA in the process of medication. Therefore, in the follow-up experiments, it is necessary to make full use of systems biology, spectral-activity relationship research, multi-omics technology, multi-target high-throughput screening and other active ingredients to further study the pharmacodynamic material basis and mechanism of anti-RA of Qianghuo, so as to provide a basis for in-depth research and enrich the clinical application basis of Qianghuo, and have important guiding significance for promoting the development and utilization of Qianghuo resources, the research and development of new anti-RA drugs and clinical use.
Source: Tang Luhuan, Wu Mengru, Chen Yanyan, Xu Zihang, Yao Yaqi, Li Yubo. Research progress on active ingredients and mechanism of action of Qianghuo anti-rheumatoid arthritis [J]. Chinese Herbal Medicine, 2024, 55(9): 3137-3145.