Apoptosis Induction of Fibroblast-Like Synoviocytes Is an Important Molecular-Mechanism for Herbal Medicine along with its Active Components in Treating Rheumatoid Arthritis

Rheumatoid arthritis (RA) is a known chronic autoimmune disease can cause joint deformity and even loss of joint function. Fibroblast-like synoviocytes (FLS), one of the main cell types in synovial tissues of RA patients, are key effector cells in the development of RA and are considered as promising therapeutic targets for treating RA. Herbal medicines are precious resources for finding novel agents for treating various diseases including RA. It is reported that induction of apoptosis in FLS is an important mechanism for the herbal medicines to treat RA. Consequently, this paper reviewed the current available references on pro-apoptotic effects of herbal medicines on FLS and summarized the related possible signal pathways. Taken together, the main related signal pathways are concluded as death receptors mediated apoptotic pathway, mitochondrial dependent apoptotic pathway, NF-κB mediated apoptotic pathways, mitogen-activated protein kinase (MAPK) mediated apoptotic pathway, endoplasmic reticulum stress (ERS) mediated apoptotic pathway, PI3K-Akt mediated apoptotic pathway, and other reported pathways such as janus kinase/signal transducers and activators of transcription (JAK-STAT) signal pathway. Understanding the apoptosis induction pathways in FLS of these herbal medicines will not only help clear molecular mechanisms of herbal medicines for treating RA but also be beneficial for finding novel candidate therapeutic drugs from natural herbal medicines. Thus, we expect the present review will highlight the importance of herbal medicines and its components for treating RA via induction of apoptosis in FLS, and provide some directions for the future development of these mentioned herbal medicines as anti-RA drugs in clinical.


Introduction
Rheumatoid arthritis (RA) is a chronic, invasive autoimmune disease that can cause joint deformity, even complete loss of joint function, so it is often referred to as "Deathless cancer" [1]. Pathological characteristic of RA is excessive synovial tissue hyperplasia, pannus formation, and erosion of cartilage, and the typical clinical features is the symmetry of joint swelling, pain, morning stiffness, and deformity, often accompanied by joint organ involvement [2,3]. RA can occur at any age, with the highest the activated Caspase-8 could trigger the intrinsic mitochondrial pathway via the Bid cleavage, following a series of apoptotic events, such as cytchrome c (Cyt-C) release from the mitochondria, apoptosome formation, Caspase cascade reaction, and PARP cleavage, and eventually lead to apoptosis [20,[37][38][39]. Currently, increasing evidences have suggested that induction of apoptosis in FLS via death receptors mediated apoptotic pathway is an important molecular mechanism for herbal medicine extracts and its active components to treatment of rheumatoid arthritis.

Herbal Medicine Extracts
In 2002, a study reported by Liu et al. revealed that a clinical RA drug of Xinfeng capsule (XFC, 1.8 g/kg, p.o.) possessed significant anti-RA effects in rats via reduction of arthritis index, and the potential mechanisms are correlated to up-regulation of Fas and FasL, whereas down-regulation of Bcl-2 in synovia tissues of joints [40]. Furthermore, Liu et al. (2010) investigated the effects of extracts from the roots of Salvia miltiorrhiza (ERSM) on FLS of clinical RA patients (RA-FLS). From the results of this investigation, Liu et al. suggested that ERSM (0.4 mg/mL) has obvious apoptosis inducing activities in RA-FLS via up-regulation of mRNA expressions of Fas [41]. In addition, another research article by Liang et al., in 2017, investigated the anti-RA effects of an interesting traditional Chinese medicine (TCM) clinical prescription, named Fengshi Bitong Prescription (FSBT), and reported that FSBT (9.5, 19, and 38 g/kg, p.o.) could decrease the paw swelling of collagen-induced arthritis (CIA) rats. Similar to tripterygium glycosides, the potential molecular mechanisms of FSBT are closely correlated to the regulation of Caspase-8, Fas and FasL in synovial tissues of CIA rats [42].

Monomers from Herbal Medicine
In 2008, Zhao et al. reported that Resveratrol (1), which is a natural active compound existed in Polygounm cuspidatum [43], has promising curative effects on RA symptoms of CIA rats and could induce the primary cultured FLS (rFLS) from CIA rats via up-regulation of Caspase-8, whereas down-regulation of FLICE inhibitory protein (FLIP) [44] which is an important anti-apoptotic protein that can suppresses the death receptors ligands (such as TNF-α, FasL, and TRAIL) induced apoptosis in FLS of RA patients (RA-FLS) (Figure 1), and is also an important reason for the resistance of RA-FLS to apoptosis [45][46][47]. Later, in 2013, it is reported that Propyl gallate (2) (64 µg/mL) which is an active component in Radix paeoniae rubra could induce the apoptosis of RA-FLS via up-regulating the mRNA expressions of Fas [48,49]. Furthermore, a study reported that Daphnetin (3) (40 µg/mL) significantly induced apoptosis in rFLS from CIA rats, and the related molecular mechanisms were closely related to up-regulation of Caspase -3, -8, and -9 and FasL [50,51]. The potential mechanisms of herbal medicine for inducing apoptosis in this part are summarized in Figure 1 and Table 1.

Mitochondrial Dependent Apoptotic Pathway
Mitochondrion, as an important organelle of cell, is the energy factory of cell activities. The integrity of its structure and function is an important premise to ensure the normal life activities of cells. Mitochondria mediated cell apoptosis is also known as the endogenous pathway of cell apoptosis, which is one of the important ways of cell apoptosis. In 1994, Newmeyer et al. reported that mitochondria in the extract of Xenopus afroensis egg could coagulate the nucleus chromatin, and the nucleus showed contractile and fragmented, suggesting that mitochondria were closely correlated to programmed cell death [52]. Since then, increasing studies have also proved that mitochondria play an important role in the process of cell apoptosis, which is the key element of apoptosis. Briefly, when cells receive apoptosis signals from p53-PUMA signal or death receptor signal pathways (Figure 2), the pro-apoptotic proteins of Bcl-2 family were up-regulated and activated, whereas expressions of anti-apoptotic proteins were down-regulated. The pro-apoptotic proteins, such as Bax and Bak, were transferred from the cytoplasm to the mitochondrial membrane, forming transmembrane pores and decreasing the mitochondrial membrane potential (MCMP). Meanwhile, due to breaking of the balance of pro-/anti-apoptotic proteins, the permeability transition pore (PT) was induced to open, which further reduced the mitochondrial membrane potential (MCMP), increased the permeability of the mitochondrial membrane, resulting in the releases of cytochrome C (Cyto C). With the participation of ATP or dATP, Cyto C enters the cytoplasm and subsequently binds to Apaf-1 and other apoptotic protease activators to form apoptotic complexes. Apoptosis complexes recruit and activate the initiator cysteinyl aspartate specific proteinase (Caspase), such as Caspase 9, etc. Then, the activated initiators activate executioner Caspases (such as Caspase-3, -6, -7), and start the Caspase cascade, cutting poly ADP-ribose polymerase (PARP), actin and other substrates, eventually leading to the cell death and cell lysis [20,26,27]. As natural products, herbal medicines and its active ingredients are considered as effective strategies for RA treatment due to their good efficacy and low toxicity. Meanwhile, increasing studies have proved that these drugs can play anti-RA effects by mediating mitochondrial apoptosis pathway in FLS.

Mitochondrial Dependent Apoptotic Pathway
Mitochondrion, as an important organelle of cell, is the energy factory of cell activities. The integrity of its structure and function is an important premise to ensure the normal life activities of cells. Mitochondria mediated cell apoptosis is also known as the endogenous pathway of cell apoptosis, which is one of the important ways of cell apoptosis. In 1994, Newmeyer et al. reported that mitochondria in the extract of Xenopus afroensis egg could coagulate the nucleus chromatin, and the nucleus showed contractile and fragmented, suggesting that mitochondria were closely correlated to programmed cell death [52]. Since then, increasing studies have also proved that mitochondria play an important role in the process of cell apoptosis, which is the key element of apoptosis. Briefly, when cells receive apoptosis signals from p53-PUMA signal or death receptor signal pathways (Figure 2), the pro-apoptotic proteins of Bcl-2 family were up-regulated and activated, whereas expressions of anti-apoptotic proteins were down-regulated. The pro-apoptotic proteins, such as Bax and Bak, were transferred from the cytoplasm to the mitochondrial membrane, forming transmembrane pores and decreasing the mitochondrial membrane potential (MCMP). Meanwhile, due to breaking of the balance of pro-/anti-apoptotic proteins, the permeability transition pore (PT) was induced to open, which further reduced the mitochondrial membrane potential (MCMP), increased the permeability of the mitochondrial membrane, resulting in the releases of cytochrome C (Cyto C). With the participation of ATP or dATP, Cyto C enters the cytoplasm and subsequently binds to Apaf-1 and other apoptotic protease activators to form apoptotic complexes. Apoptosis complexes recruit and activate the initiator cysteinyl aspartate specific proteinase (Caspase), such as Caspase 9, etc. Then, the activated initiators activate executioner Caspases (such as Caspase-3, -6, -7), and start the Caspase cascade, cutting poly ADP-ribose polymerase (PARP), actin and other substrates, eventually leading to the cell death and cell lysis [20,26,27]. As natural products, herbal medicines and its active ingredients are considered as effective strategies for RA treatment due to their good efficacy and low toxicity. Meanwhile, increasing studies have proved that these drugs can play anti-RA effects by mediating mitochondrial apoptosis pathway in FLS.
from Glycyrrhiza uralensis, and recently, Zhai et al. reported that Liquirtin (0.345-34.5 μM) induced apoptosis in RA-FLS via down-regulating the ratio of Bcl-2/Bax [74]. Besides, it is reported that Cryptotanshinone (17) (5 μM), a fat soluble anthraquinone derivative isolated from the Salvia miltiorrhiza, could induce the apoptosis in MH7A cells and RA-LFS cells via reactive oxygen species (ROS) induced apoptosis by regulation of Bcl-2, Bad, cleaved-Caspase-3 and PARP [75]. The potential mechanisms of herbal medicine for inducing apoptosis in this part are summarized in Figure 2 and Table 1.  [53]. Later in 2013, it is also reported that an Chinese patent drug called Feng-shi-ning capsule has significant anti-rheumatic activities and the related mechanisms were related to induction of mitochondrial dependent apoptosis in via increasing the releases of Cyt-C, up-regulating Caspase-3, as well as down-regulating Bcl-2 [54]. In 2018, Gao and Lu studied effect of medicated serum of Duhuo Jisheng decoction (DHJS medicated serum, 0.75, 1.5 and 3 g/kg) on rFLS from Freund's adjuvant-induced arthritis (AIA) rats, the results showed that 20% DHJS medicated serum can induce apoptosis in rFLS (AIA), and the mechanisms are related to regulating Bax, Bcl-2 and Caspase-3 [55]. Furthermore, a research by Ci et al. isolated an interesting polysaccharide from the Saposhnikovia divaricate (SDP), and the SDP (5-15 mg/mL) dose-dependently induced apoptosis in the rFLS from AIA rats. The possible mechanisms are closely related to up-regulating p53, Bax and Caspase-3, whereas down-regulating Bcl-2 [56]. Later in 2019, Wu et al. investigated the apoptosis inducing effects of Pterocarya Hupehensis Skan extracts (PHSE) on MH7A cells, and the results showed that PHSE (25, 50, 100 µg/mL) resulted in obvious anti-proliferative and apoptotic effects in MH7A cells. The further mechanisms research revealed that PHSE can up-regulate p53, Bak, Cyt-C, Caspase-3, -9 and Bax, whereas down-regulate Bcl-2 and Bcl-xL, and activate the Caspase-3 in MH7A cells [57]. Recently, Zhang et al. investigated the curative effects of Guizhishaoyaozhimu Decoction (GZSD), which is a known classical prescription of traditional Chinese medicine (TCM), on type II collagen-induced arthritis (CIA) in rats. It is found that GZSD has a significant anti-arthritic effect on CIA rats, and the further in vitro study in MH7A cells revealed GZSD (0.4, 0.8, 1.6 mg/mL) could induce apoptosis in MH7A cells and up-regulate pro-apoptotic proteins such as Caspase-3, -9, Bax and down-regulate Bcl-2 [16].

NF-κB Mediated Apoptotic Pathways
Under normal physiological conditions, cells receive extracellular signal stimulation, and receptor proteins on the membrane polyaggregate and transmit the signal to IKK (IκB kinase). IKK, a kinase, phosphorylates IκB, which is subsequently dissociated from the trimer complex formed with Nuclear factor-kappa B (NF-κB) [76]. The released NF-κB regenerates its activity, quickly enters the nucleus from the cytoplasm, and combines with specific sequences on DNA in the nucleus to control protein transcription, and participate in physiological processes such as cell proliferation and apoptosis, stress response, and release of cytokines. However, when NF-κB is abnormally activated, the body can develop a series of serious diseases, such as cancer, atherosclerosis, and rheumatoid arthritis. Beg et al. showed that fetal mice with defects in the Re1A subunit of NF-κB would show programmed cell death or apoptosis in the second trimester, leading to large-scale degradation of the liver and ultimately the death of the embryo. It was the first time that NF-κB was involved in the process of cell apoptosis. Subsequently, more and more studies have found that NF-κB is closely related to apoptosis [77].
Currently, NF-κB has attracted more and more attention as a potential therapeutic target in the management of RA [78][79][80]. Modern pharmacological studies have proved that natural products can induce FLS apoptosis to prevent synovial hyperplasia via regulation of NF-κB pathway.  [84]. Furthermore, Klosech et al. systemically investigated the apoptotic effects of Curcumin on MH7A cells and its related molecular mechanisms, and the results showed that Curcumin has notable pro-apoptotic effects on MH7A via modulation of the NF-κB and MAPK signal pathways [85]. In 2017, Fang et al. studied the effects of Celastrol (9) on activated FLS from RA patients. The results showed that Celastrol attenuated the proliferation of RA-FLS, and inhibited phosphorylation of IKK and IκBα as well as down-regulated NF-κB p65 [86]. The monomer of 1,7-Dihydroxy-3,4-dimethoxyxanthone (XAN, 20) is isolated from Securidaca inappendiculata Hassk, and can induce apoptosis in RA-FLS by inhibiting the activities of NF-κB and p38. It is worth noting that XAN can also regulate the transcription of X-linked inhibitor of apoptosis protein (XIAP), mediate the occurrence of Caspase cascade reaction, and induce cell apoptosis [87,88]. Furthermore, previous research in 2017 also found that Jinwu-Jiangu decoction (JJD) could reduce inflammatory symptoms of CIA rats and inhibit tumor like hyperplasia of FLS, and the in-depth study found that JJD medicated serum could promote FLS apoptosis by interfering with the expressions of NF-κB p65, IKK-α, and IKK-β [89,90]. Another paper by Wang et al., in 2017, reported that Baicalin (21) (10, 20, and 30 µM) could inhibit the proliferation of RA-FLS by decrease of NF-κB p65, phospho-NF-κB p65 and acetyl-NF-κB p65, as well as pro-inflammatory cytokines [91]. Piperlongumine 10, and 20 µM) can trigger apoptosis in MH7A cells by blocking activation of NF-κB and protein kinase B (Akt)/mammalian target of rapamycin (mTOR) pathways (Akt/mTOR) [93]. In 2018, Zhang et al. reported that by photodynamic therapy, Hypericin (24) (0.25-4 µM) could induce NF-κB mediated apoptosis in MH7A cells via increasing ROS, cleaved Caspase-9, Cleaved PARP, whereas decreasing NF-κB p65 [94]. The α-Mangostin (25) is an active monomer isolated from the Garcinia mangostana Linn, and an interesting study by Zuo et al. reported that α-Mangostin (15 µg/mL) decreased XIAP and increased cleaved Caspase-3, as well as inhibited phosphorylation of NF-κB p65, IκB, and IKK in FLS, suggesting the pro-apoptotic potential of α-mangostin in FLS which is closely related to inhibition of NF-κB [95]. The potential mechanisms of herbal medicine for inducing apoptosis in this part are summarized in Figure 3 and Table 1.  [94]. The α-Mangostin (25) is an active monomer isolated from the Garcinia mangostana Linn, and an interesting study by Zuo et al. reported that α-Mangostin (15 μg/mL) decreased XIAP and increased cleaved Caspase-3, as well as inhibited phosphorylation of NF-κB p65, IκB, and IKK in FLS, suggesting the pro-apoptotic potential of α-mangostin in FLS which is closely related to inhibition of NF-κB [95]. The potential mechanisms of herbal medicine for inducing apoptosis in this part are summarized in Figure 3 and Table 1.

MAPK Mediated Apoptotic Pathway
MAPK pathway is an important signal transduction pathway in eucaryotic cells, which exists in most cells and is regulated step by step by MAP kinases kinases kinases (MAPKKK) and mapkinases kinases (MAPKKK). In brief, extracellular signal stimulation phosphorylates receptors on cell membrane, such as Ras, binds and activates intracellular MAPKKK, such as Raf. Activated MAPKKK then activates MAPKK, and MAPKK reactivates MAPKs to transmit signals to cells and their nuclei, and this pathway playing a crucial role in the physiological process of cell proliferation, differentiation, as well as apoptosis [96][97][98]. MAPKs mainly include extracellular regulated protein kinases (ERK), P38, and c-Jun N-terminal kinase (JNK). Generally speaking, ERK regulates Bcl-2 expression and is mainly involved in cell proliferation and differentiation. JNK and p38 are mainly involved in the stress response and apoptosis of cells by regulating the expression of downstream proteins, such as Bax, GADD153, and c-Jun [99,100]. It has been reported that MAPKs also play crucial roles in activation of Caspase cascades [101,102]. Currently, effects of the MAPK pathway in immune diseases such as RA have been comprehensively investigated. An increasing number of studies have found that MAPKs expressions in synovial tissues of RA patients are abnormally increased, which promotes synovial proliferation. Consequently, MAPKs are considered as promising potential treatment targets for treating RA [103][104][105][106].

MAPK Mediated Apoptotic Pathway
MAPK pathway is an important signal transduction pathway in eucaryotic cells, which exists in most cells and is regulated step by step by MAP kinases kinases kinases (MAPKKK) and mapkinases kinases (MAPKKK). In brief, extracellular signal stimulation phosphorylates receptors on cell membrane, such as Ras, binds and activates intracellular MAPKKK, such as Raf. Activated MAPKKK then activates MAPKK, and MAPKK reactivates MAPKs to transmit signals to cells and their nuclei, and this pathway playing a crucial role in the physiological process of cell proliferation, differentiation, as well as apoptosis [96][97][98]. MAPKs mainly include extracellular regulated protein kinases (ERK), P38, and c-Jun N-terminal kinase (JNK). Generally speaking, ERK regulates Bcl-2 expression and is mainly involved in cell proliferation and differentiation. JNK and p38 are mainly involved in the stress response and apoptosis of cells by regulating the expression of downstream proteins, such as Bax, GADD153, and c-Jun [99,100]. It has been reported that MAPKs also play crucial roles in activation of Caspase cascades [101,102]. Currently, effects of the MAPK pathway in immune diseases such as RA have been comprehensively investigated. An increasing number of studies have found that MAPKs expressions in synovial tissues of RA patients are abnormally increased, which promotes synovial proliferation. Consequently, MAPKs are considered as promising potential treatment targets for treating RA [103][104][105][106].
In recent years, the effects of herbal medicine and its active monomers on MAPK pathway in articular synovium have been comprehensively reported in vivo and in vitro. In 2007, Liagreh et al. found that Diosgenin (26) can induce apoptosis in RA-FLS cells, and Diosgenin (40 µM) can activate p38 and JNK, but inhibit phosphorylation of ERK. In addition, DNA fragmentation induced by Diosgenin could be reduced by SB203580 and SP600125 (p38 and JNK inhibitors) [107]. Another similar study also reported that Hecogenin (27) and Tigogenin (28), which are similar to Diosgenin (26) in structure, have anti-proliferation and pro-apoptotic potential in FLS of arthritis patients cultured in vitro, but these two saponins only seem to activate p38, and have no significant effect on the phosphorylation of JNK and ERK [108].  [110]. β-Elemene (30) is a known natural sesquiterpene compound, treatment with β-Elemene (10-200 µg/mL) can improve the phosphorylation level of p38 and promote cell apoptosis in RA-FLS, and p38 inhibitors can significantly reverse the pro-apoptotic effect of β-Elemene, consequently these results suggested that the MAPK pathway is closely involved in the pro-apoptotic effects of β-Elemene on RA-FLS [111].
However, there are also some investigations that showed that suppression of the MAPK signal pathway might be beneficial for induction of apoptosis in FLS. Triptolide (31) is a known natural monomer isolated from the Tripterygium wilfordii with immunosuppressive and anti-inflammatory activities. It is reported that Triptolide (0.28-140 nM) possessed notable anti-proliferative effects on RA-FLS, as well as induced cell cycle arrest and apoptosis in RA-FLS. Furthermore, the possible mechanism is related to suppression of Ras-MAPK signaling [100,112]. Brucine (32) is an important alkaloid of Strychni Semen, and is reported by Tang that Brucine (0.125-2 mg/mL) has potential inhibitory effect on proliferation of TNF-αstimulated RA-FLS cells via down-regulating JNK MAPK and p-JNK MAPK [113]. Previous literatures have showed that the MAPK signal could activate the NF-κB signal which is also a known anti-apoptosis signal pathway in FLS cells [114], consequently we speculated that in a certain condition, blocking MAPK signal might also induce cell proliferation suppression or apoptosis in FLS. The potential mechanisms of herbal medicine for inducing apoptosis in this part are summarized in Figure 4 and Table 1. alkaloid of Strychni Semen, and is reported by Tang that Brucine (0.125-2 mg/mL) has potential inhibitory effect on proliferation of TNF-αstimulated RA-FLS cells via down-regulating JNK MAPK and p-JNK MAPK [113]. Previous literatures have showed that the MAPK signal could activate the NF-κB signal which is also a known anti-apoptosis signal pathway in FLS cells [114], consequently we speculated that in a certain condition, blocking MAPK signal might also induce cell proliferation suppression or apoptosis in FLS. The potential mechanisms of herbal medicine for inducing apoptosis in this part are summarized in Figure 4 and Table 1.

ERS Mediated Apoptotic Pathway
As an important organelle of the cell, the endoplasmic reticulum (ER) can guide the synthesis, folding, and secretion of proteins in eukaryotic cells. The endoplasmic reticulum stress (ERS) of cells caused by endoplasmic reticulum dysfunction can enhance protein folding ability, retards translation of most proteins, as well as accelerate protein degradation, etc., which is the self-protective mechanism of cells [115]. When ERS function is abnormal, Caspase-12 can be activated to directly induce apoptosis. It can also activate the unfolded protein response (UPR), induce the expression of molecular chaperones such as glucose-2 regulated protein 78kD (GRP78), GRP94, Bip, etc., regulate the Irel/xBPT pathway, p-ERK/eIF2 α pathway, and up-regulate the expression of CCAAT/enhancer binding protein epsilon (CHOP), indirectly promoting apoptosis. In addition, ESR can induce the opening of calcium channels, promote Ca 2+ outflow, and break the Bax/bcl-2 balance. These responses are collectively called endoplasmic reticulum associated with death (ERAD) [116][117][118].
Currently, it is abundantly reported that herbal medicines and its monomers can induce apoptosis of FLS through ERS pathway to exert anti-RA activity. In 2014, Jeong et al. found that Hempseed oil (0-2.5%) can reduce the survival rate of MH7A cells via promoting apoptosis in MH7A with a time-, dose-dependent manner. Further mechanism studies of the pro-apoptotic activities of Hempseed oil suggested that Hempseed oil can increase CHOP expression in MH7A cell, which is an important transcription factor closely related to apoptotic effect of Hempseed oil in MH7A cells [119]. Later, in 2015, Kim et al. investigated the effects of a novel chalcone derivative named (E)-3-(3,5-dimethoxyphenyl)-1-(1-hydroxynaphthalen-2-yl)-prop-2-en-1-one (DK-59, 33) on MH7A cells. The results showed that DK-59 reduced cell viability and induced ROS production and apoptosis in MH7A cells via up-regulating ATF4 and CHOP, activating Caspase-7 and PARP, as well as increasing phosphorylation of eIF2α [120]. Similarly, the extracts of Eupatorium japonicum Thunb (EJTE, 37.5 µg/mL) also showed apoptosis inducing activities in RA-FLS via ERS-mediated apoptotic pathway, such as up-regulation of ATF4 and CHOP [121]. Another in vitro experiment by Lu et al. showed that in the presence of H 2 O 2 , resveratrol (1) (50-400 µM) could induce the apoptosis in FLS with a dose-dependent manner. Meanwhile, resveratrol treatment increased the expression of Caspase-12 and CHOP, suggesting that ERS was involved in the occurrence of FLS apoptosis. Additionally and interestingly, resveratrol did not seem to cause intracellular calcium overload [122]. The potential mechanisms of herbal medicine for inducing apoptosis in this part are summarized in Figure 5 and Table 1.

PI3K-Akt Mediated Apoptotic Pathway
Phosphoinositide 3 kinase (PI3K) is an important kinase, when phosphorylated, activates inositol and phosphatidylinositol, catalyzing Akt migration to the membrane and complete activation. Activated Akt is involved in physiological activities such as cell survival, growth, proliferation, cell migration, and angiogenesis by activating a series of downstream intracellular proteins. However, the abnormal regulation of the PI3K-Akt pathway may lead to the increase of these activities and induce serious diseases in the body. Studies have found that the PI3K-Akt pathway is abnormally activated in synovial cells of RA patients, which can also lead to increased expression of anti-apoptotic genes in cells [123,124]. More and more studies have proved that inhibiting the abnormally activated PI3K-Akt pathway can induce RA-HFLS apoptosis, and improve synovial hyperplasia, cartilage destruction, and other pathological phenomena of RA patients [125,126]. Currently, increasing evidence has revealed that regulation of PI3K-Akt pathway is also an important way for herbal medicines to treating RA.

Herbal Medicine Extracts
In 2012, an in vivo experiment showed that the apoptosis rate of rat synovial fibroblasts induced by IL-1β increased by intervention of total saponin of Dioscoreae Nipponicae Rhizoma (TSDN, 100 μg/L), and at the same time, the phosphorylation levels of PI3K and Akt protein in FLS cells were down-regulated, suggesting that PI3K-AKT pathway was involved in the apoptotic activity of TSDN [127]. Recently, Pan et al. reported that the intervention of Duan tengyimu decoction (DTYD, 100 and 200 μg/mL) significantly inhibited the activation of PI3K and Akt, further enhanced the expression of Bax, and reduced the expression of Bcl-2 in RA-FLS [128]. In addition, it is reported that medicated serum of Shuangwuxuan bi granule also showed similar pro-apoptotic effect on RA-FLS, and the mechanism was related to the exception of PI3K/Akt signal pathway [129]. Heiguteng-Zuifenghuoluo Capsule (HGTZFC) is a Chinese patent medicine used for treating RA, Liu et al. reported that HGTZFC (0.315 g/kg) could down-regulate the HIF-α, p-PI3K, p-Akt, and Bcl-2, whereas up-regulate Bax in synovial tissue of CIA rats [130].

PI3K-Akt Mediated Apoptotic Pathway
Phosphoinositide 3 kinase (PI3K) is an important kinase, when phosphorylated, activates inositol and phosphatidylinositol, catalyzing Akt migration to the membrane and complete activation. Activated Akt is involved in physiological activities such as cell survival, growth, proliferation, cell migration, and angiogenesis by activating a series of downstream intracellular proteins. However, the abnormal regulation of the PI3K-Akt pathway may lead to the increase of these activities and induce serious diseases in the body. Studies have found that the PI3K-Akt pathway is abnormally activated in synovial cells of RA patients, which can also lead to increased expression of anti-apoptotic genes in cells [123,124]. More and more studies have proved that inhibiting the abnormally activated PI3K-Akt pathway can induce RA-HFLS apoptosis, and improve synovial hyperplasia, cartilage destruction, and other pathological phenomena of RA patients [125,126]. Currently, increasing evidence has revealed that regulation of PI3K-Akt pathway is also an important way for herbal medicines to treating RA.

Herbal Medicine Extracts
In 2012, an in vivo experiment showed that the apoptosis rate of rat synovial fibroblasts induced by IL-1β increased by intervention of total saponin of Dioscoreae Nipponicae Rhizoma (TSDN, 100 µg/L), and at the same time, the phosphorylation levels of PI3K and Akt protein in FLS cells were down-regulated, suggesting that PI3K-AKT pathway was involved in the apoptotic activity of TSDN [127]. Recently, Pan et al. reported that the intervention of Duan tengyimu decoction (DTYD, 100 and 200 µg/mL) significantly inhibited the activation of PI3K and Akt, further enhanced the expression of Bax, and reduced the expression of Bcl-2 in RA-FLS [128]. In addition, it is reported that medicated serum of Shuangwuxuan bi granule also showed similar pro-apoptotic effect on RA-FLS, and the mechanism was related to the exception of PI3K/Akt signal pathway [129]. Heiguteng-Zuifenghuoluo Capsule (HGTZFC) is a Chinese patent medicine used for treating RA, Liu et al. reported that HGTZFC (0.315 g/kg) could down-regulate the HIF-α, p-PI3K, p-Akt, and Bcl-2, whereas up-regulate Bax in synovial tissue of CIA rats [130].

Monomers from Herbal Medicine
In 2011, Zhang et al. found that Genistein (34) (50, 100, and 200 µM) can induce apoptosis in vitro cultured FLS of CIA rats, and the mechanism regulates the expression of apoptosis-related proteins Bax and Bcl-2 by inhibiting Akt expression [131]. Another study by Li et al. reported that Tanshinone IIA (12, 40 µM) can promote apoptosis in RA-FLS via up-regulating lncRNA GAS5, cleaved Caspase-3 and -9 and inhibiting PI3K/Akt signaling [132]. In addition, Yang studied the effect of Anacardic acid (35) on RA, and found that anacardic acid has good therapeutic effect on symptoms of CIA rats, and the Anacardic acid (5, 30, and 60 µM) can induce apoptosis of the RA-FLS, and the possible mechanism is closely related to the PI3K/Akt signal pathway [133]. Another study in 2019 by Zuo and Wang reported that Juglone (30 µM) can promote the apoptosis in RA-FLS via inhibiting the phosphorylation of Akt and increasing expression of p21 [134]. Besides, from the results of Feng et al., Diosgenin (26) (10, 20, and 40 µg/mL) could also result in apoptosis in RA-FLS via regulation of the proteins related to PI3K-Akt signal [135]. In a recent study, Wang et al. investigated the effect of Pectolinarin (36) (20 µM) on RA-FLS, and found that besides mitochondrial dependent apoptotic pathway, regulation of PI3K/Akt signaling might be also an important molecular mechanism responding to the apoptosis inducing effects of Pectolinarin [136]. The potential mechanisms of herbal medicine for inducing apoptosis in this part are summarized in Figure 6 and Table 1.

Other Reported Pathways
Additionally to the apoptotic pathways mentioned above, other mechanisms also reported to be closely related to the apoptosis inducing activities of herbal medicines on FLS. Previous research has also revealed that janus kinase/signal transducers and activators of transcription (JAK-STAT) signal is closely correlated to the apoptosis induced by herbal medicines. Ren et al. reported that TAHP (6) (50 and 250 µM) treatment induced apoptosis in FLS, and can down-regulate Jak2 and STAT3, resulting in inhibition of the Jak2/STAT3 signal pathway [60]. Another report by Yang et al. also revealed that the pro-apoptotic effect of Matrine (37) (0.75 mg/mL) which is a known natural monomer from Sophora flavescens on rFLS in CIA rats was achieved by inhibiting the JAK/STAT pathway [137]. Recently, Zhang et al. suggested that suppression of JAK-STAT signaling is also an important molecular mechanism for the pro-apoptotic effects of GZSD in MH7A cells [16]. In 2013, Celastrol (9) (1-5 µM) was reported to be an active neutral compound that could dose-dependently induce apoptosis of RA-FLS, the possible mechanism is the induction of DNA damage, the G2/M phase of the cell cycle arrest and apoptosis related proteins (Bax/Bcl-2, Caspase-3 and -9) [138]. In 2016, Ren studied the inhibitory effect of 10-Hydroxycamptothecine (10-HCPT) (38) on proliferation of RA-FLS, and the results showed that 10-HCPT (1 and 10 µg/mL) induced obvious apoptosis in RA-FLS. In addition, the further investigation revealed that the pro-apoptotic effects of this compound might be related to down-regulation of VEGF and MMP-3 [139]. In 2017, Yao et al. reported that three natural compounds including Tamaractam (39), Cis-N-feruloyl-3-O-methylaids (40), and Trans-N-feruloyl-3-O-methylaids (41) isolated from Tamarix ramosissima showed in vitro pro-apoptotic effects on RA-FLS and up-regulated caspase-3 and-7. Flow cytometry results showed these compounds can increase the sub-G1 fraction in the cell cycle, suggesting that activation of the Caspase family and induction of cycle arrest are involved in pro-apoptotic effects [140]. Later, in 2018, it was also reported that Huangqichongteng Drink (HQCD) can induce the synovial cells in G0/G1 stage of cell growth, promoting apoptosis in RA-FLS [141]. Besides, it is also found resveratrol (9) (40-320 µM) down-regulated the autophagy related proteins (LC3A/B and ATG-5) in H 2 O 2 induced FLS cells, and increase the ROS production and Ca 2+ release, resulting in apoptosis of FLS [142]. Besides, it is reported that Wogonin (42), total glucosides of paeonia (TGP), Cinnamic aldehyde (CINA, 43), and Paclitaxel (44) also have pro-apoptosis effects on the RA-FLS [143][144][145][146].
The related monomers in the section (Effect of FLS apoptosis in RA) for inducing apoptosis in FLS and its possible molecular mechanism are summarized in Tables 1 and 2 and Figures 7 and 8. mechanism is closely related to the PI3K/Akt signal pathway [133]. Another study in 2019 by Zuo and Wang reported that Juglone (30 μM) can promote the apoptosis in RA-FLS via inhibiting the phosphorylation of Akt and increasing expression of p21 [134]. Besides, from the results of Feng et al., Diosgenin (26) (10, 20, and 40 μg/mL) could also result in apoptosis in RA-FLS via regulation of the proteins related to PI3K-Akt signal [135]. In a recent study, Wang et al. investigated the effect of Pectolinarin (36) (20 μM) on RA-FLS, and found that besides mitochondrial dependent apoptotic pathway, regulation of PI3K/Akt signaling might be also an important molecular mechanism responding to the apoptosis inducing effects of Pectolinarin [136]. The potential mechanisms of herbal medicine for inducing apoptosis in this part are summarized in Figure 6 and Table 1.

Other Reported Pathways
Additionally to the apoptotic pathways mentioned above, other mechanisms also reported to be closely related to the apoptosis inducing activities of herbal medicines on FLS. Previous research has also revealed that janus kinase/signal transducers and activators of transcription (JAK-STAT) signal is closely correlated to the apoptosis induced by herbal medicines. Ren et al. reported that TAHP (6) (50 and 250 μM) treatment induced apoptosis in FLS, and can down-regulate Jak2 and STAT3, resulting in inhibition of the Jak2/STAT3 signal pathway [60]. Another report by Yang et al. also revealed that the pro-apoptotic effect of Matrine (37) (0.75 mg/mL) which is a known natural monomer from Sophora flavescens on rFLS in CIA rats was achieved by inhibiting the JAK/STAT pathway [137]. Recently, Zhang et al. suggested that suppression of JAK-STAT signaling is also an important molecular mechanism for the pro-apoptotic effects of GZSD in MH7A cells [16]. In 2013, Celastrol (9) (1-5 μM) was reported to be an active neutral compound that could dose-dependently  Up-regulating p53, Bak, Cyt-C, Bax, Caspase-3, -9; Down-regulating Bcl-2 and Bcl-xL; Activating Caspase-3, -9 PHSE (25-100 µg/mL) MH7A p53, Bax, Bak, Bcl-2, Bcl-xL, Cyt-C, Caspase-3, -9 [57] Inhibiting proinflammatory cytokines; Up-regulating Caspase-3, -9 and Bax; Down-regulating Bcl

Conclusion and Perspectives
It is reported that after simulation by the pro-inflammatory cytokines such as TNF-α, the fibroblast-like synoviocytes (FLS) would grow rapidly like tumor cells, and development of RA is closely related to an imbalance between cell proliferation, survival, and death of FLS [16,147]. Thus, promoting death of FLS in RA patients might be a feasible way for treating RA, and compared with other therapeutic approaches, targeting FLS might ameliorate clinical symptoms of RA without suppressing systemic immunity. Apoptosis is the main typical programmed cell death ways (PCD) and physiological cell suicide processes. Recently, apoptosis is considered as an ideal way for treatment of cancers, and increasing studies have also indicated that inducing apoptosis in FLS of RA patients would be beneficial for controlling the symptoms and development of RA [16,18].
It is generally recognized that herbal medicines have played important roles in human health maintenance and disease therapy for thousands of years before the appearance of synthetic drugs [148]. Currently, natural products including extracts and monomers derived from herbal medicines are receiving increasing attention in the world, and are commonly considered as precious resource for screening and finding novel candidate drugs with less toxicity [14,149]. In addition, a large number of herbal medicines and its components have the pro-apoptotic activities on FLS, and we have summarized these herbal medicines in this present review. Taken together, apoptosis induction of FLS is an important molecular mechanism for herbal medicine and its active components in the treatment of rheumatoid arthritis, and the main related signal pathways are concluded as death receptors mediated apoptotic pathway, mitochondrial dependent apoptotic pathway, NF-κB mediated apoptotic pathways, MAPK mediated apoptotic pathway, ERS mediated apoptotic pathway, PI3K-Akt mediated apoptotic pathway, and other reported pathways such as

Conclusions and Perspectives
It is reported that after simulation by the pro-inflammatory cytokines such as TNF-α, the fibroblast-like synoviocytes (FLS) would grow rapidly like tumor cells, and development of RA is closely related to an imbalance between cell proliferation, survival, and death of FLS [16,147]. Thus, promoting death of FLS in RA patients might be a feasible way for treating RA, and compared with other therapeutic approaches, targeting FLS might ameliorate clinical symptoms of RA without suppressing systemic immunity. Apoptosis is the main typical programmed cell death ways (PCD) and physiological cell suicide processes. Recently, apoptosis is considered as an ideal way for treatment of cancers, and increasing studies have also indicated that inducing apoptosis in FLS of RA patients would be beneficial for controlling the symptoms and development of RA [16,18].
It is generally recognized that herbal medicines have played important roles in human health maintenance and disease therapy for thousands of years before the appearance of synthetic drugs [148]. Currently, natural products including extracts and monomers derived from herbal medicines are receiving increasing attention in the world, and are commonly considered as precious resource for screening and finding novel candidate drugs with less toxicity [14,149]. In addition, a large number of herbal medicines and its components have the pro-apoptotic activities on FLS, and we have summarized these herbal medicines in this present review. Taken together, apoptosis induction of FLS is an important molecular mechanism for herbal medicine and its active components in the treatment of rheumatoid arthritis, and the main related signal pathways are concluded as death receptors mediated apoptotic pathway, mitochondrial dependent apoptotic pathway, NF-κB mediated apoptotic pathways, MAPK mediated apoptotic pathway, ERS mediated apoptotic pathway, PI3K-Akt mediated apoptotic pathway, and other reported pathways such as JAK-STAT signal pathway. Understanding the apoptosis induction pathways in FLS of these natural medicines will not only help clear molecular mechanisms of herbal medicines for treating RA but also be beneficial for finding novel candidate therapeutic drugs from natural herbal medicines. Consequently, we expect the present review will highlight the importance of herbal medicines and its components for treating RA via induction of apoptosis in FLS, and provide some directions for the future development of these mentioned herbal medicines as anti-RA drugs in clinical.