Anti-Inflammatory Effects of Traditional Chinese Medicines against Ischemic Injury in In Vivo Models of Cerebral Ischemia

Inflammation plays a crucial role in the pathophysiology of acute ischemic stroke. In the ischemic cascade, resident microglia are rapidly activated in the brain parenchyma and subsequently trigger inflammatory mediator release, which facilitates leukocyte-endothelial cell interactions in inflammation. Activated leukocytes invade the endothelial cell junctions and destroy the blood-brain barrier integrity, leading to brain edema. Toll-like receptors (TLRs) stimulation in microglia/macrophages through the activation of intercellular signaling pathways secretes various proinflammatory cytokines and enzymes and then aggravates cerebral ischemic injury. The secreted cytokines activate the proinflammatory transcription factors, which subsequently regulate cytokine expression, leading to the amplification of the inflammatory response and exacerbation of the secondary brain injury. Traditional Chinese medicines (TCMs), including TCM-derived active compounds, Chinese herbs, and TCM formulations, exert neuroprotective effects against inflammatory responses by downregulating the following: ischemia-induced microglial activation, microglia/macrophage-mediated cytokine production, proinflammatory enzyme production, intercellular adhesion molecule-1, matrix metalloproteinases, TLR expression, and deleterious transcription factor activation. TCMs also aid in upregulating anti-inflammatory cytokine expression and neuroprotective transcription factor activation in the ischemic lesion in the inflammatory cascade during the acute phase of cerebral ischemia. Thus, TCMs exert potent anti-inflammatory properties in ischemic stroke and warrant further investigation.


Introduction
Stroke is the third leading cause of death in developed countries [1] and the major cause of severe long-term disability worldwide [1][2][3]. Approximately 15 million people experience stroke annually. Of these, one-third die and one-third experience permanent disabilities, thus imposing considerable social and economic burden [4]. Approximately 80%-85% of all stroke events are ischemic caused by cerebral arterial thrombosis or embolism [5,6]. To date, recombinant tissue plasminogen activator (rtPA) is the only Food and Drug Administration-approved medical therapy for acute ischemic stroke. However, rtPA has severe disadvantages, including the narrow therapeutic time window of 4.5 h and potential risk of hemorrhagic transformation; therefore, the eligibility of rtPA is reduced to only 4%-7% in all the patients with acute ischemic stroke [5]. Thus, potential therapeutic strategies for ischemic stroke are urgently needed.
Increasing evidence has demonstrated that inflammation plays a pivotal role in the pathophysiology of acute ischemic stroke [3,5,7]. During acute ischemic stroke, the brain is injured by ischemia-and inflammation-related primary and secondary insults [5]. The primary injury occurs at the beginning of ischemia; it rapidly interrupts the cerebral blood flow to the ischemic core and subsequently causes a significant decrease in oxygen and glucose supply to 2 Evidence-Based Complementary and Alternative Medicine cerebral neurons [8,9]. The secondary injury is attributed to the postischemic inflammatory cascade, which produces various proinflammatory mediators, including cytokines, chemokines, proteases, and cell adhesion molecules, leading to an exacerbated ischemic brain injury [10]. However, the postischemic inflammatory response has a disadvantage and an advantage, exacerbating ischemic brain damage in the early phase and triggering tissue regeneration in the delayed phase, respectively [1,2].
The lack of effective and widely applicable therapeutic strategies for the treatment of ischemic stroke has triggered increasing interest in traditional medicines, particularly traditional Chinese medicine (TCM) [11,12]. Several centuries ago, TCM was used in China to treat cerebrovascular disorders, including stroke. Evidence revealed that TCM preparation, Chinese herb medicine, and TCM-derived active compounds exert anti-inflammatory effects by inhibiting inflammatory mediators, leukocyte infiltration, and bloodbrain barrier (BBB) disruption in experimental cerebral ischemia [13]. These potent effects of TCMs against cerebral ischemic injury highlight their potential in clinical applications. Therefore, this review summarized the origin and development of the postischemic inflammatory cascade and delineated the anti-inflammatory effects of TCMs (namely, TCM-derived active compounds, Chinese herbs, and TCM formulations) on the basis of the in vivo literature.

Activation of Microglia in the Initial Phase of Cerebral Ischemia.
In the acute phase (min to h) of cerebral ischemia, ischemic injury triggers a rapid activation of resident microglia in the brain parenchyma [3,14]. During cerebral ischemia, microglial morphology changes from a ramified to an amoeboid shape upon activation [15]. In the initial stage of ischemia, the injured neurons expose damage-associated molecular patterns (DAMPs), which are subsequently recognized by toll-like receptors (TLRs), such as TLR4, and other pattern recognition receptors on the surface of the reactive microglia; this recognition triggers microgliamediated inflammatory mediators release, contributing to secondary damage after stroke [6,10,16]. Reactive microglia/ macrophages can be detected as early as 2 h after cerebral ischemia and maintained up to 1 week after the ischemic insult [6]. Reactive microglia are divided into two phenotypes: the classically and alternatively activated phenotypes (M1 and M2, resp.) [17]. The M1 microglia produce proinflammatory mediators, such as cytokines [interleukin-(IL-) 1 , IL-6, IL-18, and tumor necrosis factor-(TNF-) ], chemokines [monocyte chemoattractant protein-(MCP-) 1, and macrophage inflammatory protein-(MIP-) 1 ], interferon-(IFN-) , matrix metalloproteinase-(MMP-) 9, and reactive oxygen species (ROS) [18], exerting detrimental functions in the early phase. By contrast, the M2 microglia secrete anti-inflammatory mediators, such as IL-4, IL-10, IL-13, transforming growth factor-(TGF-) , and insulin growth factor-1, exerting neuroprotective effects in the delayed phase [2,3,19]. In cerebral ischemia, microglial activation is accompanied by reactive astrogliosis, which also produces an excessive amount of cytokines and causes the exacerbation of ischemic brain injury [18].  [20]. Pretreatment with tetramethylpyrazine (TMP), an active compound isolated from Ligusticum wallichii Franch (Chuan Xiong), effectively reduces the cerebral volume by inhibiting myeloperoxidase (an inflammation marker) and ED1 (a microglia/macrophage marker) expression in the ischemic core 72 h after reperfusion. The effect of TMP against microglial activation-mediated neurotoxicity can be further attributed to the suppression of prostaglandin E2 (PGE2) in the ischemic core [21]. A later study reported that TMP provides neuroprotection against ischemic brain injury partially by inhibiting microglial activation and subsequently downregulating MCP-1 expression in the ischemic cortex 72 h after reperfusion [22]. Andrographolide, the major active compound derived from Andrographis paniculata (Chuan Xin Lian) protects against cerebral infarction and ameliorates neurological deficits 24 h after permanent middle cerebral artery occlusion (MCAo). Andrographolide exerts neuroprotective effects partially by inhibiting microglial activation and microglia-mediated IL-1 and TNF-expression in the ischemic area [23]. Pretreatment with Sophora japonica L. (Huai Hua; intraperitoneal injection) effectively reduced the cerebral infarct area and neurological deficits at 1.5 h of ischemia and 24 h of reperfusion. The effects of S. japonica involve the suppression of microglial activation and microglia-mediated IL-1 expression in the ischemic cortex [24]. Lim et al. demonstrated that an intragastric administration of total isoflavones isolated from Pueraria lobata (Ge Gen; TIPL) significantly reduces the cerebral infarct volume at 2 h of ischemia and 48 h of reperfusion. The anti-inflammatory effect of TIPL is partially attributed to the inhibition of astrocyte and microglial activation in the hippocampal CA1 region 7 d after MCAo [25]. On the basis of these studies, the anti-inflammatory effects of TCMs against cerebral ischemic injury could be attributed to the downregulation of microglial activation and microglia-mediated proinflammatory cytokines production in the ischemic area during the initial phase of MCAo ( Figure 1 and Table 1).

The Process of Leukocyte Infiltration during Cerebral
Ischemia. The microglial and astrocytic production of proinflammatory mediators rapidly increase the expression of  Figure 1: Schematic representation of the effects of traditional Chinese medicines on inflammation responses in the inflammatory cascade after cerebral ischemia. TCMs, traditional Chinese medicines; DAMPs, damage-associated molecular patterns; TLRs, toll-like receptors; LFA-1, leukocyte function-associated antigen-1 (CD11a/CD18); ICAM-1, intercellular adhesion molecule-1; MMPs, matrix metalloproteinases; iNOS, inducible nitric oxide synthase; COX-2, cyclooxygenase-2; 5-LO, 5-lipoxygenase; BBB, blood-brain barrier. Thick solid lines with arrowheads indicate activation, and thin dotted lines indicate inhibition. adhesion molecules on the endothelium [26,27]. During acute cerebral I/R injury, the peripheral leukocytes first roll, become activated, and consequentially attach to the endothelial cells in the ischemic lesion. Leukocyteendothelial cell interactions in inflammation are mediated by various adhesion molecules, including selectins, integrins, intercellular adhesion molecule-(ICAM-) 1, and vascular cell adhesion molecule-1 [28]. Selectins, comprising L-selectin on leukocytes and E-and P-selectins on endothelial cells, are a family of lectin-like adhesion glycoproteins that regulate leukocyte rolling and recruitment [28,29]. Integrins, including leukocyte function-associated antigen-1 (CD11a/CD18) expressed on all leukocytes and macrophage-1 (MAC-1; CD11b/CD18) expressed on neutrophils and monocytes, are transmembrane glycoproteins and mediate leukocyteendothelium interactions [30]. In acute cerebral ischemia, the upregulation of integrins facilitates a firm adherence of leukocytes to endothelial ICAM-1; the leukocytes subsequently penetrate the endothelial basement membrane into the brain parenchyma. Thus, the tight junctions (TJs) between endothelial cells of the BBB are disrupted and become more permeable, leading to leukocyte infiltration [30]. This evidence revealed that circulating leukocytes adhere to the damaged endothelium as early as 4 h and achieve the peak at approximately 12-48 h after ischemic brain injury. Meanwhile, reactive microglia, platelets, and infiltrating leukocytes further release IL-1 , IL-6, TNF-, ROS, MCP-1, MIP-1 , IL-8, and MMPs (mainly MMP-9) that exacerbate ischemic injury in MCAo [3,5,6].  The beneficial effects of Borneol involve the reduction of leukocyte infiltration and ICAM-1 expression in the ischemic area [37].

The Effects and Mechanisms of TCMs on Inhibiting
These results indicate that TCMs provide beneficial effects against leukocyte infiltration mainly by downregulating ICAM-1 expression and activated leukocyte-induced cytokines, such as IL-1 , TNF-, and IL-8, in the ischemic lesion in the acute phase of cerebral ischemia ( Figure 1 and Table 2).

Blood-Brain Barrier Disruption during Cerebral Ischemia.
Under normal conditions, leukocyte recruitment across the BBB into the brain parenchyma contributes to the maintenance of the central nervous system immune privilege [9]. The BBB comprising endothelial cells, the basement membrane, the astrocyte end-feet, and pericytes provides a highly selective permeability barrier that separates the blood cells from the brain interstitial fluid and maintains brain homeostasis [38]. However, during the acute phase of cerebral ischemia, the infiltrated leukocytes and reactive microglia synthesize and secrete MMPs (mainly MMP-2 and MMP-9) and ROS, thus increasing BBB permeability [72,73]. Previous studies have reported that MMP-9 activation is initiated as early as 4 h, which reaches the maximum level at 24 h and persists for at least 5 d after cerebral ischemia [74,75], whereas activated MMP-2 reaches the highest level 5 d after MCAo [75]. Active MMPs disrupt BBB integrity by degrading the Evidence-Based Complementary and Alternative Medicine 5 ICAM-1, intercellular adhesion molecule-1; BCCAo, bilateral common carotid artery occlusion. extracellular matrix and TJs in endothelial cells and result in vascular and BBB leakage. The BBB disruption facilitates the entry of circulating leukocytes and intravascular fluid into the brain, which cause vasogenic edema and hemorrhagic transformation, leading to the exacerbation of cerebral infarction [6,9,76]. TJs, including claudin-5, occludin, and zonula occludens-(ZO-) 1, play a pivotal role in maintaining the structural and functional integrity of the BBB [77]. Previous studies have reported that decreased claudin-5, occludin, and ZO-1 expression is closely related to BBB disruption and ischemic brain edema formation [77,78]. Thus, MMPs, claudin-5, occludin, and ZO-1 could present the potential targets for pharmacological intervention to stabilize BBB integrity in cerebral ischemic injury and the regulation of their activity may yield therapeutic effects.

The Effects and Mechanisms of TCMs on Ameliorating Blood-Brain Barrier Disruption in In Vivo Models of Cerebral
Ischemia. Posttreatment methylophiopogonanone-(MO-) A, an active compound isolated from Ophiopogon japonicus (Mai Men Dong), effectively reduces the infarct volume and brain edema and improves neurological deficits 7 d after transient MCAo. The results indicate that MO-A protects against cerebral I/R injury mainly through its property to ameliorate BBB disruption through MMP-9 downregulation, and claudin-3 and claudin-5 upregulation in the ischemic cortex [38]. Tan et al. reported that pretreatment with ligustrazine, an active ingredient of L. wallichii Franchat (Chuan Xiong), effectively preserves BBB integrity by downregulating MMP-9 expression and upregulating claudin-5 and occludin expression in the ischemic area in a rat model of focal cerebral I/R injury [39]. Levo-tetrahydropalmatine (l-THP), a major active ingredient of Rhizoma corydalis (Yan Hu Suo), protects against cerebral I/R-induced BBB injury at 1.5 h of ischemia and 24 h of reperfusion. The protective effect of l-THP can be partially attributed to MMP-2 and MMP-9 downregulation and claudin-5, occludin, and ZO-1 upregulation in the ischemic area [40]. From these results, we conclude that MMP-9 downregulation and claudin-5, occludin, and ZO-1 upregulation are the potential effects of TCMs on the stabilization of BBB integrity to ameliorate inflammatory responses in the ischemic area during the acute and subacute phases of cerebral I/R injury ( Figure 1 and Table 3).  MMP-9, matrix metalloproteinase-9; ZO-1, zonula occludens-1.

TCM-Mediated Regulation of Proinflammatory Mediator Release
cytokines in response to ischemic injury [73,80,81]. TLRs are pivotal components in the innate immune system, and TLRs (mainly TLR2 and TLR4) stimulation in microglia/ macrophages and T-lymphocytes also exerts strong regulatory effects on postischemic inflammatory responses [2,73]. During cerebral ischemic injury, TLRs facilitate cytokine and chemokine release and trigger transcription factor activation by activating intercellular signaling pathways. According to recruitment of specific adaptors, TLR signaling can be classified into either myeloid differentiation primary response gene 88-(MyD88-) dependent or independent pathways [81]. The binding of HSPs, such as HSP60 and HSP70, or HMGB1 with TLR2 and TLR4 initiate the expression of nuclear factor-(NF-) B, TNF-, IL-1 , IL-6, inducible nitric oxide synthase (iNOS), and ICAM-1 by activating the MyD88-dependent signaling pathway and aggravating cerebral ischemic injury [82]. TLR2 and TLR4 levels markedly increase in the ischemic brain at 6 h, peak at 24 h, and decline at 72 h after MCAo [83].

The Effects and Mechanisms of TCMs on Downregulating Proinflammatory Mediators in In Vivo Models of Cerebral
Ischemia. Notoginseng saponins isolated from the root of Panax notoginseng (San Qi) provide beneficial effects Evidence-Based Complementary and Alternative Medicine 7 against cerebral I/R injury partially through IL-1 mRNA downregulation in the ischemic area after 22 h of reperfusion [43]. Chang et al. reported that pretreatment with puerarin effectively reduces the cerebral infarct size and neurobehavioral deficits 24 h after MCAo. The anti-infarct effect of puerarin is, at least partially, because of the inhibition of TNF-and iNOS expression in the ischemic area [44]. Li et al. explored the effect of osthole, a major active ingredient in Cnidium monnieri (L.) Gusson (She Chuang Zi), on acute cerebral I/R injury and reported that pretreatment with osthole markedly reduces the brain infarct volume and ameliorates neurological scores 24 h after MCAo. The neuroprotective effects of osthole are accompanied by the downregulation of proinflammatory mediators, including TNF-, IL-1 , COX-2, and iNOS, expressed in the ischemic cortex [45]. The caffeic acid ester (Caf) fraction from Erigeron breviscapus (Deng Zhan Hua) significantly reduces the cerebral infarct volume and improves neurobehavioral performance at 1 h of ischemia and 24 h of reperfusion. The inhibition of iNOS, TNF-, and IL-1 mRNA expression is one of the mechanisms underlying the neuroprotective effects of Caf against cerebral infarction [46]. Pretreatment with arctigenin, an active agent from Arctium lappa (Nu Bang Zi), effectively inhibits microglial activation and subsequently downregulates TNF-and IL-1 expression in the penumbra region 24 h after transient MCAo [47]. Lee et al. reported that schisandrin B isolated from Fructus schisandrae (Wu Wei Zi) markedly reduces the cerebral infarct size and neurological deficits 24 h after transient focal cerebral ischemia. The anti-inflammatory effect of schisandrin B involves the inhibition of TNF-, IL-1 , MMP-2, and MMP-9 expression and suppression of microglial activation in the ischemic area [48]. Posttreatment with asiaticoside, an active compound isolated from Centella asiatica (L.) (Ji Xue Cao), attenuates memory deficits by suppressing iNOS, TNF-, IL-1 , and IL-6 expression in the hippocampus 7 d after transient bilateral common carotid artery occlusion [49]. Chen et al. reported that posttreatment with magnolol, an active ingredient of Magnolia officinalis (Hou Pu), ameliorates cerebral infarction partially by dose-dependently inhibiting iNOS, TNF-, IL-1 , and IL-6 expression in the ischemic area 24 h after transient global ischemia [50]. Posttreatment with danhong, extracted from Radix salviae miltiorrhizae (Dan Shen) and Flos carthami (Hong Hua), exerts beneficial effects in cerebral I/R injury, at least partially, through dose-dependent IL-1 and TNF-downregulation in the ischemia area at 1.5 h of ischemia and 14 d of reperfusion [51]. Gastrodin, an active constituent of Gastrodia elata Blume (Tian Ma), exerts an initial anti-inflammatory effect by suppressing TNF-and IL-1 expression in the ischemic hemispheres 6 h after cerebral I/R injury [52].

The Effects and Mechanisms of TCMs on Regulating Anti-Inflammatory Cytokines in In Vivo Models of Cerebral
Ischemia. IL-4, IL-10, IL-13, and TGF-reduce microglia/ macrophages-induced proinflammatory cytokines, such as IL-8 [99]. Moreover, IL-4 promotes long-term recovery after ischemic stroke [100]. IL-10 can inhibit IL-1 and TNF-expression [55] and prevent the downregulation of the antiapoptotic protein Bcl-2 expressed in ischemic brain lesion [101]. IL-4 mRNA generates as early as 1 h, reaches a peak at 3-24 h, and gradually declines 2 d following ischemic stroke [102]. Pretreatment with danshen, an aqueous extract of the root and rhizome of Salvia miltiorrhiza Bunge (Dan Shen), protects against cerebral I/R injury in association with decreased IL-10 and TNF-mRNA and protein expression in the ischemic area 24 h after transient MCAO [53]. Guizhi fuling capsules, composed of Cinnamomum cassia Blume

The Effects and Mechanisms of TCMs on Downregulating Proinflammatory Enzymes in In Vivo Models of Cerebral Ischemia. COX-2 and 5-lipoxygenase (5-LO)
are rate-limiting enzymes that convert arachidonic acid to prostaglandins and leukotrienes [57]. In the delayed phase of cerebral ischemia, microglia/macrophages produce 5-LO, which converts arachidonic acid to leukotrienes. Leukotrienes are potent inflammatory mediators that trigger chemotaxis of leukocytes and BBB damage and subsequently cause vasogenic edema, thus exacerbating cerebral ischemia [103]. COX-2 and 5-LO expression is markedly enhanced in the ischemic cortex 24 h after cerebral I/R injury [57]. Guo et al. explored the anti-infarct effect of paeoniflorin (PF), the principle component of P. radix (Shao Yao), in the subacute phase of cerebral I/R injury and reported that PF protects against cerebral infarction mainly through TNF-, IL-1 , iNOS, COX-2, and 5-LO downregulation in the ischemic area 14 d after reperfusion [56]. Chen et al. reported that pretreatment with PF effectively ameliorates the cerebral infarct volume and neurological deficits 24 h after reperfusion in a model of pharmacological preconditioning. The neuroprotective effects of PF against cerebral I/R injury are partially related to the inhibition of COX-2, 5-LO, and iNOS expression in the ischemic lesion [57].

NF-B Activation during Cerebral Ischemia.
NF-B is a classic transcription factor and plays a crucial role in the regulation of hundreds of genes involved in cell survival and death [104]. Thus, NF-B can be activated via several intracellular signaling pathways associated with host defense, inflammation, and apoptosis [58]. In the brain, NF-B regulates the expression of different sets of genes, such as antiapoptotic, proapoptotic, and proinflammatory genes, thereby playing a dual role in neuronal survival and death [63]. The NF-B family includes five members, namely, p65 (RelA), RelB, c-Rel, p50/p105 (NF-B1), and p52/p100 (NF-B2), which form various homo-and heterodimeric complexes [2]. The most common form of NF-B is the p65/p50 heterodimer [6]. Under an unstimulated condition, the inhibitor of NF-B proteins (I Bs), including mainly I B , I B , and I B , retain inactive NF-B dimmers in the cytosol, whereas in response to various extracellular stimuli, including infection, proinflammatory cytokines, and antigen receptor engagement, the activated I B kinase complexes phosphorylate I B proteins, resulting in their ubiquitination and proteasomal degradation and consequently inducing the release of NF-B for nuclear translocation and the activation of target gene transcription [6,105]. During cerebral ischemia, the activated NF-B dimers are subsequently translocated into the nucleus where they selectively bind to specific DNA sequences called B sites; promoter domains present a large number of proinflammatory genes and subsequently cause TNF-, IL-1 , IL-6, ICAM-1, PGE2, COX-2, and iNOS translation [6,105,106]. NF-B activators include some proinflammatory cytokines, such as TNF-and IL-1 , whose genes are regulated by NF-B itself, inducing a positive feedback loop and resulting in the amplification of the inflammatory response and exacerbation of cerebral ischemic insults [107]. Previous studies have indicated that NF-B activation occurs as early as 1 h, reaches a peak at 6 h, and sustains for at least 72 h in the cerebral ischemic area in rats [62,107].

The Effects and Mechanisms of TCMs on Downregulating NF-B Activation in In Vivo Models of Cerebral Ischemia.
Wogonin, a flavonoid derived from Scutellaria baicalensis Georgi (Huang Qin), exerts neuroprotective effects by inhibiting the inflammatory activation of microglia in an in vitro cell culture model. The anti-inflammatory effects of wogonin are partially attributed to the downregulation of NF-B-mediated iNOS and TNF-expression in the ischemic hippocampal CA1 area in transient global cerebral ischemia in rats [58]. Tanshinone IIA (Ts IIA) and IIB, the key compounds of S. miltiorrhiza Bunge, effectively reduce the cerebral infarct volume and improve the neurological function 24 h after transient MCAo [59]. Dong et al. further reported that pretreatment with Ts IIA protects against cerebral infarction partially associated with the reduction of ROS-mediated NF-B activation, leading to the inhibition of iNOS expression in the ischemic area 24 h after permanent MCAo [60]. Silymarin, a bioactive component isolated from Silybum marianum (Shui Fei Ji), provides neuroprotection against cerebral I/R injury by inhibiting oxidative and nitrosative stress in the ischemic area 24 h after cerebral ischemia. The antioxidative and antinitrosative effects of silymarin are partially attributed to the reduction of NF-B-mediated iNOS, COX-2, ICAM-1, TNF-, and IL-1 expression in the injured tissues [61]. In addition, Guan et al. reported that ruscogenin, a major effective compound isolated from O. japonicus Ker-Gawl, ameliorates cerebral I/R injury through the downregulation of NF-B target genes, including ICAM-1, iNOS, COX-2, TNF-, and IL-1 , in the ischemic area 24 h after reperfusion [62]. Hydroxysafflor yellow A, a major active component of C. tinctorius L. (Hong Hua), reduces cerebral infarction by suppressing cytosolic NF-Bp65 translocation to the nucleus and subsequently downregulates NF-B-mediated TNF-, IL-1 , and IL-6 expression in the ischemic area 24 h after permanent MCAo [63]. Chern et al. reported that 2-methoxystypandrone (2-MS), a major active component of Polygonum cuspidatum (Hu Zhang), attenuates the brain infarct size and improves the neurological function, at least partially, by preventing I B degradation and a reducing NF-B-mediated iNOS and COX-2 expression in the peri-infarct cortex 24 h after transient MCAo. The anti-inflammatory effects of 2-MS can further contribute toward the preserving BBB integrity [64]. Previous studies have indicated that p38 mitogen-activated protein kinase (MAPK), one of the MAPK family members, upregulates NF-B expression (p38 MAPK/NF-B signaling) and subsequently causes the transcription of genes encoding proinflammatory cytokines, resulting in the exacerbation of cerebral infarction in the acute phase of transient MCAo [108][109][110]. In addition, activated p38 MAPK occurs in the ischemic area as early as 2 h and reaches a peak 24-48 h after reperfusion [110]. Piperlonguminine from Piper longum (Bi Bo) alkaloids protects against cerebral ischemic injury by inhibiting the activation of p38 MAPK/NF-B signaling cascade in the ischemic region 24 h after permanent MCAo [65].

The Effects and Mechanisms of TCMs on Upregulating Peroxisome Proliferator-Activated Receptor Activation in In
Vivo Models of Cerebral Ischemia. Peroxisome proliferatoractivated receptors (PPARs) include PPAR , PPAR , and PPAR / isoforms, which are members of the nuclear receptor superfamily and represent ligand-activated transcription factors. PPAR is predominantly expressed in the central nervous system and binds to peroxisome proliferator response elements to regulate its target gene expression [66]. During cerebral ischemia, PPAR is detected in the peri-infarct area as early as 4 h and is sustained for at least 14 d after ischemia [111]. PPAR exerts neuroprotective effects against inflammatory mediators to initiate responses by inhibiting the activation of NF-B signaling in the ischemic area after focal cerebral ischemia [66,111]. Liu et al. reported that pretreatment with curcumin, a natural polyphenolic component of curcuma longa (Jiang Huang), markedly reduces the cerebral infarct volume by activating PPAR signaling in the ischemic cortex 24 h after reperfusion. The effects of curcumin on the regulation of PPAR signaling further contribute to the downregulation of NF-Bp65-mediated TNF-, IL-1 , iNOS, PGE2, and COX-2 expression [66]. Another study revealed that pretreatment with icariin, a natural flavonoid compound extracted from Epimedium brevicornum maxim (Yin Yang Huo), protects against cerebral I/R injury by activating PPAR and PPAR and subsequently suppressing NF-Bp65-mediated IL-1 expression in the ischemic cortex at 2 h of ischemia and 24 h of reperfusion [67].

The Effects and Mechanisms of TCMs on Regulating
Signal Transducer and Activator of Transcription Activation in In Vivo Models of Cerebral Ischemia. Signal transducer and activator of transcription (STAT) proteins are a family of transcription factors comprising seven members, namely, STAT1, STAT2, STAT3, STAT4, STAT5a, STAT5b, and STAT6 [112]. Some controversy exists on whether STAT signaling is neuroprotective or neurotoxic in cerebral ischemic injury [113]. Growing evidence has revealed that cytokines induce the activation of the receptor-associated Janus kinases (JAKs), including JAK1, JAK2, JAK3, and tyrosine kinase 2, which consequently activate STAT proteins; the activated STAT proteins undergo dimerization and translocation to the nucleus, thereby regulating the expression of proapoptotic and proinflammatory genes [114,115]. Among STAT protein isoforms, STAT3 is the most-conserved isoform [68]. In the transient cerebral ischemic rat model, STAT3 is activated as early as 30 min and sustained 24 h after reperfusion, and activated STAT3 triggers cerebral I/R injury by amplifying inflammatory responses [116]. However, other studies have reported that activated STAT3 signaling provides neuroprotection by upregulating Bcl-2 and vascular endothelial growth factor expression in the peri-infarct area after transient cerebral ischemia [113,117]. Kaempferol-3-O-rutinoside (KRS) and kaempferol-3-O-glucoside (KGS) are also the active components of C. tinctorius L. Both KRS and KGS markedly reduce the cerebral infarct volume at least partially associated with the inhibition of STAT3 and NF-Bp65 activation, and subsequently, proinflammatory mediators (TNF-, IL-1 , iNOS, MMP-9, and ICAM-1) production in the cortical penumbra 24 h after transient MCAo [68]. Astragaloside IV [the active component of Astragalus (Huang Qi)] combined with Ginsenoside Rg1, Ginsenoside Rb1, and Notoginsenoside R1 (the active components of . notoginseng) effectively restores cell survival partially related to the inhibition of JAK1/STAT1 and NF-B signaling and consequently suppresses TNF-, IL-1 , and ICAM-1 mRNA expression in the ischemic area 24 h after transient global cerebral ischemia [69]. By contrast, Li et al. reported that curcumin reduces cerebral infarction and attenuates neurological deficits by activating JAK2/STAT3 signaling and downregulating IL-1 and IL-8 in the injured region after 24 h of reperfusion [70].

The Effects and Mechanisms of TCMs on Regulating
Nuclear Factor-Erythroid 2-Related Factor 2/Heme Oxygenase-1 and c-Jun N-Terminal Kinase/c-Jun/Activating Protein-1 Signaling in an In Vivo Model of Cerebral Ischemia. Nuclear factor-erythroid 2-related factor 2 (Nrf2), a potent cytoprotective transcription factor, induces the expression of genes encoding antioxidant and anti-inflammatory proteins [118]. Under stress, Nrf2 dissociates from its cytoplasmic inhibitory protein Kelch-like ECH-associated protein 1 and translocates into the nucleus, where it binds to an antioxidant response element and regulates target genes, including heme oxygenase-1 (HO-1). The Nrf2/HO-1 signaling pathway attenuates inflammatory responses in cerebral ischemia [71]. During transient focal cerebral ischemia, Nrf2 and HO-1 occur in the ischemic cortex as early as 6 h, up to a maximum 48 h and decline 72 h after cerebral I/R [119]. The activation of c-Jun N-terminal kinase (JNK), one of the MAPK family members, signaling plays a central role in ischemia-induced neuroinflammation. When stimulated, activated JNK translocates into the nucleus and phosphorylates c-Jun, the major component of activating protein-(AP-) 1, which comprises c-Jun and c-Fos proteins, leading to the expression of target genes encoding proinflammatory mediators. JNK/AP-1 signaling amplifies the inflammatory response during cerebral ischemia [71]. JNK/c-Jun/AP-1 signaling factors are predominantly expressed in the ischemic area 2 h after cerebral ischemia [120]. Kao et al. reported that TMP effectively reduces cerebral infarction by inhibiting microglia/macrophages activation in the ischemic cortex 72 h after permanent MCAo. The anti-inflammatory effects of TMP can be further attributed to the upregulation of Nrf2/HO-1 signaling and downregulation of JNK/c-Jun/AP-1 signaling in the ischemic cortex [71].
According to the aforementioned studies, NF-B and JNK/AP-1 signaling induced in the ischemic brain may amplify inflammatory responses, whereas PPARs and Nrf2/HO-1 signaling are considered to prevent postischemic inflammation and yield potent effects against cerebral ischemic injury. JAK/STAT signaling plays a dual role in the regulation of proinflammatory mediators depending on the experimental models of brain ischemia. TCMs protect against cerebral ischemic injury by inhibiting deleterious transcription factors (NF-B, JAK/STAT, and JNK/AP-1), activating neuroprotective transcription factors (PPARs and Nrf2/HO-1) and consequently regulating the expression of transcription factor-mediated proinflammatory genes (TNF-, IL-1 , IL-6, IL-8, iNOS, COX-2, PGE2, MMP-9, and ICAM-1) in the ischemic area in the early stage (24-72 h) of cerebral ischemia (Figure 2 and Table 5).

Conclusions
After the onset of cerebral ischemia, resident microglia are rapidly activated (within a few minutes) and subsequently produce large amounts of cytokines, chemokines, and ROS, thus causing the initial ischemic injury. TCMs can exert neuroprotective effects against the initial ischemic injury by rapidly downregulating ischemia-induced microglial activation and microglia-mediated proinflammatory cytokine production in the ischemic region. The microglial production of proinflammatory mediators subsequently increase adhesion molecule expression, facilitate leukocyte-endothelial cell interactions, and activated leukocytes that penetrate the endothelial cell barrier into the brain parenchyma (as early as 4 h after the ischemic onset). The infiltrating leukocytes further release inflammatory mediators in the  ischemic lesion, thus exacerbating ischemic injury. TCMs can effectively attenuate leukocyte infiltration by inhibiting ICAM-1 and activated leukocyte-induced cytokine expression in the ischemic region in the early phase of cerebral ischemia. Meanwhile, infiltrated leukocytes and activated microglia secrete MMPs, which cause the disruption of BBB integrity, worsening cerebral infarction. TCMs effectively inhibit MMPs expression and stabilize BBB integrity to ameliorate cerebral infarction. Increased TLRs stimulation in microglia/macrophages (activated microglia and recruited leukocytes) by the activation of intercellular signaling pathways robustly secretes various proinflammatory mediators (cytokines and enzymes) in the ischemic region 6-24 h after ischemia. TCMs can timely rescue the injured neurons by downregulating proinflammatory receptors (TLRs), cytokines, and enzymes and upregulating anti-inflammatory cytokine expression in the ischemic lesion. The proinflammatory transcription factors are subsequently activated by the secreted cytokines, whose genes are regulated by these transcription factors themselves, thus inducing a positive feedback loop, in which the inflammatory response is amplified and secondary brain injury is exacerbated 24-72 h after cerebral ischemia. TCMs protect against inflammatory response-induced secondary brain injury by inhibiting deleterious transcription factors, activating neuroprotective transcription factors, and consequently regulating the expression of transcription factor-mediated proinflammatory genes in the ischemic area. Therefore, TCMs provide promising antiinflammatory therapeutic strategies in the acute phase of cerebral ischemia. However, further studies are needed to elucidate the precise mechanisms of TCMs against inflammatory responses in the ischemic cascade after stroke. 4-VO, 4-vessel occlusion; I B , inhibitor of NF-B protein ; PPAR , peroxisome proliferator-activated receptor ; STAT3, signal transducer and activator of transcription 3; JAK1, Janus kinase 1; AP-1, activating protein-1.