Classic mechanisms and experimental models for the anti‐inflammatory effect of traditional Chinese medicine

Abstract Inflammation is a common disease involved in the pathogenesis, complications, and sequelae of a large number of related diseases, and therefore considerable research has been directed toward developing anti‐inflammatory drugs for the prevention and treatment of these diseases. Traditional Chinese medicine (TCM) has been used to treat inflammatory and related diseases since ancient times. According to the review of abundant modern scientific researches, it is suggested that TCM exhibit anti‐inflammatory effects at different levels, and via multiple pathways with various targets, and recently a series of in vitro and in vivo anti‐inflammatory models have been developed for anti‐inflammation research in TCM. Currently, the reported classic mechanisms of TCM and experimental models of its anti‐inflammatory effects provide reference points and guidance for further research and development of TCM. Importantly, the research clearly confirms that TCM is now and will continue to be an effective form of treatment for many types of inflammation and inflammation‐related diseases.


| INTRODUC TI ON
Inflammation was one of the first diseases to be identified and diagnosed. More than 2000 years ago, the Roman physician Aulus Cornelius Celsus first described the classic symptoms of inflammation. 1 Within the medical profession, inflammation is described as a complex defense response to injury in various cells, living tissues and the vascular system. Common inflammatory stimulants include physical factors (high heat, low temperature, UV, etc.), chemical factors (strong acids, strong bases, irritant solvents, etc.), mechanical factors (cutting, striking, squeezing, etc.), biological factors (parasites, bacteria, viruses, etc.) and immune factors (allergies and autoimmune diseases). 2 In other words, a huge number of factors in our lives may contribute to inflammation, with the result that inflammation is the most common disease or pathogenesis during our lifetime.
An inflammatory response is initially a beneficial protective behavior of the human body. However, uncontrolled inflammation may lead to discomfort and damage to the body, and may even endanger life.
For example, as is well known, when COVID-19 is induced by SARS-CoV-2, the body produces an immediate inflammatory response. If not controlled in time, the coronavirus will ultimately cause overproduction of cytokines (known as a cytokine storm), damaging the tissues and organs seriously and even threating life. 2,3 Therefore, considerable research effort has been devoted to finding antiinflammatory drugs for the prevention and treatment of a variety of inflammatory diseases.
In 1880, aspirin may have been the first drug shown to be effective against inflammation, and subsequently hundreds of drugs have been approved to treat various inflammatory diseases. Generally, modern chemical and biological agents can be classically divided into non-steroidal anti-inflammatory drugs (NSAIDs) and steroidal antiinflammatory drugs (SAIDs). NSAIDs are one of the most common drugs used in daily life as treatments for colds, fever, headaches, etc.
However, NSAIDs only relieve symptoms, and cannot eliminate the basic inflammatory factors and prevent the continued development of the disease. 4 Thus, NSAIDs are generally used for mild inflammatory symptoms, while critical or severe diseases should be treated with SAIDs. SAIDs are mainly composed by glucocorticoids, which have a strong anti-inflammatory effect. Unfortunately, SAIDs are a double-edged sword, very easily causing adverse reactions that harm the human body and leave sequela. 5 Because of the inevitable shortcomings of these traditional anti-inflammatory drugs, researchers are exploring novel strategies with higher efficacies and lower toxicity to control inflammation. With the gradual modernization and international recognition of TCM, increasingly people acknowledge and accept Chinese medicine. Many TCM remedies, for example, Tripterygium wilfordii Hook.f., 6 Andrographis paniculata (Burm. f.) Nees, 7 Coptis chinensis Franch, 8 etc., have proved to have good anti-inflammatory activity. Therefore, TCM is considered to be an effective anti-inflammatory strategy.
In fact, TCM has been used to treat inflammatory diseases for thousands of years. Within the medical community it is widely believed that inflammation is involved in the pathogenesis, complications, and sequelae of many diseases, 9,10 and there is a great deal of evidence that the therapeutic effects of many TCMs are mediated by their anti-inflammatory activities. 11,12 Thus, scientists around the world are developing drugs based on the anti-inflammatory properties of TCMs. In 2015, Phynova Joint and Muscle Relief Tablets (Siegesbeckiae Herba extract) became the first Chinese medicine product to be approved by the MHRA (Medicines and Healthcare products Regulatory Agency) for marketing in the UK. 13 And its antiinflammatory effect is the mechanism by which Phynova relieves joint and muscle pain. In summary, a crucial mechanism of TCM is its anti-inflammatory action and TCM will prove to be an important effective treatment for inflammatory disease.

| THE CL A SS I C MECHANIS MS OF THE ANTI -INFL AMMATORY EFFEC TS OF TR AD ITI ONAL CHINE S E MED I CINE
The pathological mechanism of inflammation is a complex defense response involving diverse cells and various factors. The cells involved in the inflammatory response are phagocytes (mononuclear macrophages, neutrophils and eosinophils), platelets and endothelial cells, which are activated in response to inflammatory stimuli. After activation, the cells produce inflammatory mediators that initially protect the body by removing irritants but eventually develop into inflammatory diseases. Inflammation is usually divided into three distinct phases: it starts as increased vascular permeability, followed by infiltration of leukocytes, which eventually cause granuloma formation and tissue repair. Due to the complexity of its constituents, TCM exhibits anti-inflammatory effects at different levels, via multiple pathways with various targets (Figure 1). Firstly, TCM may regulate the hypothalamic-pituitary-adrenal (HPA) axis via endogenous hormones to relieve inflammation. Then TCM could subsequently inhibit the production and release of inflammatory mediators and interfere with binding to receptors. At the same time, TCM could also counter oxidative stress and interact with multiple signaling pathways. In addition, TCM could activate the immune system to alleviate inflammation. In summary, the classic mechanisms of antiinflammatory effects of TCM are various and effective.

| The hypothalamic-pituitary-adrenal (HPA) axis
A lot of TCMs exhibit glucocorticoid-like pharmacological activity or regulate the function of HPA axis, 14 ultimately leading to increased endogenous cortisol secretion, which exerts antiinflammatory effects. Thus, they are called HPA-dependent anti-inflammatory drugs. For instance, Gastrodiae Rhizoma water extract 15 ameliorates inflammation via increased plasma corticosterone (CORT), adrenocorticotrophic hormone (ACTH),

F I G U R E 1
The classic mechanisms of the anti-inflammatory effects of Traditional Chinese Medicine hypothalamic corticotropin-releasing factor (CRF), and glucocorticoid receptor (GR) concentrations. CRF is a neuroregulatory factor found in the brain that regulates the transmission of serotonin (5-HT), which is a key molecule in the occurrence and progression of inflammation and mainly regulates vascular permeability. Chinese herbal compound prescriptions could also exert anti-inflammatory effects through the HPA axis. Bu-Shen-Yi-Qi-Tang relieves respiratory inflammation and inhibits hypothalamic-pituitary-adrenal axis activity in asthmatic mice. 16 In addition, like SAIDs, TCM can exhibit anti-inflammatory effects via increased endogenous cortisol secretion, but lacking the side effects of glucocorticoids, they can be more safely applied in clinical situations. Thus, its action on the HPA axis is an effective and significant mechanism by which TCM counteracts inflammation.

| The metabolism of arachidonic acid (AA)
Arachidonic acid (AA) is an unsaturated arachidic acid catalyzed by phospholipase A2 (PLA2) after activation by inflammatory stimuli and inflammatory mediators. 17 AA is then metabolized by cyclooxygenase (COX) and 5-lipoxygenase (5-LOX) pathways to produce various metabolites. Arachidonic acid metabolites -prostaglandins (PGs), leukotrienes (LTs), and 12-hydroxyeicosatetraenoic acid (12-HETE) -are actively involved in the development of various inflammatory diseases, like cancer, pneumonia and arthritis. Therefore, many of the key enzymes involved become important drug targets. 18 Substantial evidence exists that TCM could target these enzymes to exert anti-inflammatory effects.

| Phospholipase A2 (PLA2)
PLA2 is the rate-limiting enzyme mediating the biosynthesis of AA from membrane phospholipids and is an extremely important target for drugs such as SAIDs. Several Chinese medicines also inhibit PLA2 to relieve inflammation. Scutellarin, a bioactive constituent in Scutellaria baicalensis Georgi, has been confirmed as a potent inhibitor of PLA2. 19 Similarly, the world-famous Chinese medicine Yunnan Baiyao exhibits anti-inflammatory effects via regulation of the PLA2/AA metabolic pathway in an acute inflammation rat model. 20 Thus, like steroidal anti-inflammatory drugs, TCMs can exert antiinflammatory effects via PLA2, confirming the scientific explanation of their application in critical inflammatory diseases.
COX is a key rate-limiting enzyme in PG synthesis, which can induce AA to produce various PGs and thromboxane A2 (TXA2), leading to various physiological and pathological effects. Thus, COX is an extremely important target for drugs such as NSAIDs. The classical NSAID aspirin is a potent inhibitor against COX. Aspirin was developed from willow bark as an anti-inflammatory and analgesic drug. 21 Similarly, TCM also exerts anti-inflammatory effects through COX. As recently reported, a series of bioactive constituents in the Chinese medicine Huo-Luo-Xiao-Ling Dan have been shown to be inhibitors of COX. 22 Acetyl-11-ketoβ-boswellic acid, β-boswellic acid, acetylα-boswellic acid, acetylβ-boswellic acid, and betulinic acid were COX-1 selective inhibitors. Senkyunolide O and cryptotanshinone were COX-2 selective inhibitors. Roburic acid and phenethyl-trans-ferulate inhibited COX-1 and COX-2 equally. In addition, a large number of TCMs have also been confirmed to exert anti-inflammatory effects via COX. [23][24][25] Thus, COX is clearly a significant target for Traditional Chinese medicines.

| 5-lipoxygenase (5-LOX)
The enzyme 5-lipoxygenase (5-LOX) is a key enzyme catalyzing AA into leukotrienes (LTs). LTs are recognized mediators of inflammation and play important roles in many diseases, and therefore 5-LOX is considered to be a classic target for anti-inflammatory drugs. Resveratrol, 26

| Leukotrienes (LTs)
Via the catalyst 5-LOX, AA is primarily transformed into 5-hydroperoxyeicosatetraenoic acid (5-HpETE). 5-HpETE is extremely unstable and easily degrades into leukotriene A4 (LTA 4 ). LTA 4 is then catalyzed by leukotriene A4 hydrolase (LTA4H), and eventually modified into the stable isoforms LTB 4 and LTC 4 . Subsequently, LTC 4 can be transformed into LTD 4 , LTE 4 and LTF 4 . Currently, LTB 4 , LTC 4 , LTD 4 , LTE 4 and LTF 4 are recognized inflammation mediators for various diseases. As the rate-limiting enzyme in LT synthesis, LTA4H is the recognized target for drugs. 35 A previous study 36  them. 30 Among these leukotriene metabolites, LTB 4 may be one of the most powerful leukocyte chemokines causing tissue and organ damage, and it has been shown that LTB 4 can also be suppressed by Chinese medicines such as Danggui-Shaoyao-San, 37 Mahuang decoction, 38 Bidens bipinnata L 39 and so on. In summary, leukotrienes are also a key indicator in the evaluation of the anti-inflammatory effects of TCM. it is possible that multiple pathways may be involved in these effects.  As is will known, ROS can induce inflammation, inhibit the activity of various enzymes, and damage endothelial cells, resulting in increased permeability, and causing multicellular and tissue damage. 2,58 There is strong evidence that Chinese medicine can also exert an anti-inflammatory effect through anti-oxidation.

| Inflammatory mediators in body fluids
In body fluids, there are three interrelated systems, namely complement, kinin and clotting, that are important inflammation mediators.
In complement systems, C 3a and C 5a are key inflammation mediators that increase the permeability of vascular tissue, activate the metabolism of AA and promote the release of inflammatory mediators.
In kinin systems, kinins increase the permeability of vascular tissue, contract smooth muscles, and evoke pain in the area of inflammation.
In clotting systems, thrombin and fibrinolytic enzymes are related to the permeability of vascular tissue, leukocyte chemotaxis and vascular inflammation. Many studies 59,60 have shown that complement, kinin and clotting systems play a key role in the anti-inflammatory effect of Chinese medicine.

| Immunological regulation
Inflammation and immune responses are two dominating responses to foreign bodies, two sides of the same problem, but overlapping and inseparable. Recent progress in immunity research has shown the importance of immunity in controlling disease. Since ancient times, TCM has embraced a similar concept of immune regulation and it is accepted that immune regulation is an important mechanism for the pharmacological activity of Chinese medicine. 61 As has been reported, Chinese medicines such as Moutan cortex radicis, 62 Gegen Qinlian decoction, 42 and others can ameliorate inflammatory diseases via immunological regulation. Therefore, immunological regulation is another classical mechanism of the anti-inflammatory effect of TCM.

| Gut microbiota
In recent years, the importance of gut microbiota in disease progression has increasingly been recognized. Gut microbiota are an indispensable part of the human body and their distribution and function are vital for human health. Disturbing the balance of gut microbiota leads to various diseases and is not conducive to controlling disease. Therefore, gut microbiota have become an important target for drug development. On the one hand, Chinese medicines are mostly taken orally into the digestive tract, and thus gut microbes can directly influence the efficacy of these medicines. 63 In the gut, the ingredients in TCM can be metabolized and absorbed. For example, polysaccharides as the main components of many TCM 64

| THE CL A SS I C AL E XPERIMENTAL MODEL OF THE ANTI -INFL AMMATORY EFFEC T OF TR AD ITI ONAL CHINE S E MEDICINE
Inflammation is a common condition related to almost all diseases and thus much effort has been devoted to finding anti-inflammatory drugs for the prevention and treatment of a variety of diseases. Due to the extensive clinical experience of TCM practitioners, Chinese medicine has become a source of new drug development. Although a large number of Chinese medicines with anti-inflammatory effects have been used for thousands of years, it is essential to confirm the mechanisms of actions of TCM using modern pharmacological experiments. As stated above, anti-inflammatory activity is an important aspect of TCM, and therefore a series of in vitro and in vivo anti-inflammatory models are used to study TCM (Figure 2).

| In silico screening
In silico screening consists of artificial intelligence drug screening,

| Molecular and biochemical models
There are a large number of key enzymes involved in the occurrence and development of inflammation. A drug will have high potential as an anti-inflammatory once it shows inhibitory activity on relevant enzymes. Thus, the construction of molecular and biochemical models for enzyme activity is of great importance. Classical molecular and biochemical models of PLA2, COX, 5-LOX, PGE2 synthases, LTA4H and so on have been established for TCM. 19,22,32,69 Models for key targets of signaling pathways have also been established.
Considering the close relationship between oxidative stress and inflammation, molecular and biochemical models of antioxidant activity (DPPH, ABTS and total antioxidant capacity assay) can also be used to evaluate anti-inflammatory effects. 70 At present, these molecular and biochemical models have been widely applied to test the anti-inflammatory effects of TCM. 19,22,71 Additionally, these models are also ideal for drug screening. In conclusion, molecular and biochemical models are more accurate than in silico screening, but the cell-free system is also not very reliable when compared to cell-based models.

| Mouse models
Inflammation usually starts with increased vascular permeability, followed by infiltration of leukocytes, and eventually develops into granuloma and tissue repair. Therefore, the mediator-induced inflammation mouse is one of the most reliable models for antiinflammatory drug research. Mouse models simulate the pathological features of inflammatory diseases better than cell models.
Consistent with clinical practice, animal experiments are divided into acute and chronic inflammatory models. At present, both rat and mouse inflammatory models have been widely applied in the study of TCM.

Chemical-induced paw edema
Carrageenan, histamine, 5-HT, bradykinin, dextran, and lipopolysaccharide (LPS) are commonly applied as chemical inducers in rats. 73,76,78 After stimulation by these chemicals, scientists measure the paw withdrawal thermal latency (PWTL) using the hot plate test, the volume and thickness of edematous paw and the expression of inflammatory mediators, to explore the anti-inflammation effect of TCM. 73,78 The anti-inflammatory activity of Ipomoea stolonifera shown in the mouse ear edema model has been confirmed in the carrageenan-induced rat paw edema model, which also showed the pharmaceutical effects. 73 In the same model, the anti-inflammation effect of Pudilan antiphlogistic oral liquid was revealed, providing scientific support for wide clinical application. 78 Among the chemical-induced paw edema models, the carrageenan induced paw edema model is the most widely used in research on the antiinflammatory effects of Chinese medicine.

Granuloma
As acute models do not adequately reflect the anti-inflammatory effects of TCM, a chronic inflammation model may be more suitable for testing Chinese medicine. Granuloma can accurately represent the pathological progress of chronic inflammation. 73,76 Cotton pellets and glass rods are the classic inducers in rats. 73 After sacrifice, the weight of granuloma, the expression of inflammatory mediators (proinflammatory cytokines, NO and so on) and the level of ROS related indexes are assessed to evaluate anti-inflammatory activity. The anti-inflammatory effect of Taraxacum officinale, 83 Ipomoea stolonifera 73 and Qingdaisan (Formulated Indigo powder) 84

| Other inflammation-related specific disease models
The acute and chronic inflammatory models described above have been widely employed in the early stages of anti-inflammatory drug exploration, but inflammation-related models of specific diseases should be built for further development and in-depth research. For nervous system diseases, more appropriate models for inflammation-related disease research include the intraventricular its anti-inflammation effect had been confirmed in a series of inflammation-related specific disease models. 86 Similarly, pneumonia, bronchitis and influenza are representative inflammationrelated models for the respiratory system. A significant curative effect of Lianhua Qingwen (LHQW) has been shown in multiple inflammation-related specific disease models. 87 In summary, there are also associated inflammatory models for potentially different diseases.

| Other model animal models
Although rats and mice are the most widely used animal models of inflammation, monkeys, rabbits, zebrafish, drosophila and nematodes have also been used to study inflammation. Monkeys, the animal model best able to represent human pathology, have been employed to explore inflammation-related diseases. 88 Rabbits have also been used in anti-inflammatory research on Rhubarb. 89 As reported, tail amputation, LPS stimulation and copper sulfate exposure are the most common zebrafish models for inflammation research, and have been used in studies of schaftoside 90 (the active constituent in Artemisia Annua L. Artemisiae Argyi, Arisaema erubescens [Wall.] Schott. and others) and indolealkylamines 91 (from Veneum Bufonis). Drosophila has also been used in anti-inflammation research on Chinese medicine. 90 In addition, a few studies of the effects of Chinese medicine (Polygonum multiflorum Thunb extract) on inflammation also used Caenorhabditis elegans models. 91 Experimental models of the anti-inflammatory effects of TCM are clearly not limited to rodents, and other model animal models also contribute to inflammatory disease assessment.

| Organoid models
With the continuing progress of science and technology, more and more new technologies have been applied in the study of inflammation. In particular, organoid models are considered to be the closest in vitro model to simulate the in vivo physiological and pathological characteristics of diseases. 92 At present, organoid models of the intestines, lung, liver and stomach have been widely applied in the study of inflammation. As previously reported, organoid models have been used to evaluate the protective effect of TCM on intestinal damage (Glycyrrhetinic acid from Glycyrrhiza uralensis Fisch.), 93 cancer (Gambogic acid from Garcinia hamburgy Hook. f.), 94 regeneration of intestinal epithelia (Trillium tschonoskii) 95 and so on. While research directly utilizing organoids for testing anti-inflammatory effects of TCM is still lacking, we firmly believe that organoids will also become a recognized experimental model for future research on TCM.

| CON CLUS ION
Inflammation is a common condition related to almost all diseases.
TCM has been used to treat inflammatory diseases for thousands of years. A great deal of evidence as proved that its anti-inflammatory effect may be one of the important mechanisms of TCM. 71 The pathological mechanisms of inflammation are complex, involving a variety of cells and factors. TCM can inhibit inflammation at different levels, via multiple pathways with various targets. To date, TCM has been shown to exert an anti-inflammatory effect via well-known mechanisms including the HPA axis, the metabolism of AA, proinflammatory cytokines, signaling pathways, vasoactive mediators, NO, ROS, inflammatory mediators in body fluids, immunological regulation and so on. In other words, the complexity of inflammation, involving multiple targets and pathways, and the holistic treatment theory of TCM, involving multiple components and mechanisms, mean that a specific TCM can exert its effect through multiple mechanisms, and even have an effect on all of these reported mechanisms.
Considering the complexity of the anti-inflammatory mechanisms of TCM, further research requires the use of mature modern medical research strategies. In particular, the application of omics (Transcriptome, Proteome, Metabolome and Microbiome) technology could be used to objectively reveal the overall mechanisms of TCM's anti-inflammation effects, rather than focusing on certain mechanisms. Omics techniques can be used alone or in combination to provide preliminary insights into mechanistic research, while ultimately the mechanisms identified should to be validated by conventional biochemical and molecular assessment. In addition, new technologies such as target fishing technology, network pharmacology and artificial intelligence are also helping us to explore the mechanisms behind the anti-inflammatory effects of TCM. In short, a variety of reliable techniques should be broadly applied to objectively reveal the mechanisms of TCM's anti-inflammatory effects.
For more specific experimental research, the classic experimental models including in silico screening, molecular and biochemical models, cell models, mouse models (acute and chronic inflammatory models) and other animal models can be used to evaluate the antiinflammatory effects of TCM, while organoid models provide a novel potential model. It can be seen that there are abundant models for anti-inflammation research on TCM, and it is important to choose the appropriate model for the research project. First, use of a single model only is not reliable, and multiple model studies are more persuasive.
Second, while in silico screening, molecular and biochemical models, cell models are suitable for drug screening or preliminary evaluation, the final evaluation and mechanistic study should be conducted in vivo. And last, the right animal model must be chosen to accord with the drug indication for specific Chinese medicines. The optimal research strategy is to verify the mechanism in clinical studies. In conclusion, TCM is and will continue to be an effective treatment for a variety of inflammation and inflammation-related diseases. :S102-S109.