Isovitexin, a main Flavonoid Compound from Celtis Sinensis Pers. Leaves, Exerts Activity Against Inammation caused by Contacting Ginkgolic Acids in Ginkgo Fruit through Downregulating SHP2 Activation

It has been reported that Celtis sinensis Pers. is employed as a folk medicine for the treatment of inammation diseases. But the mechanism supporting its use as anti-inammatory remain unclear. To investigate the anti-inammatory of Celtis sinensis Pers. ICR mice were provided Celtis sinensis Pers. leaf extract (CLE) at 100, 200mg/kg after ginkgolic acids (GA) sensitization. Our data showed that CLE and the main avonoid isovitexin in CLE could ameliorate GA-induced contact dermatitis in mice. Ear swelling, inammatory cell inltration and splenomegaly were inhibited signicantly by isovitexin, while the weight loss of mice in the isovitexin-treated group was much better than that in the dexamethasone-treated group (positive control drug). It has been reported in previous research that GA-induced inammation is closely related to the T cell response. Therefore, T cells were the focus of the anti-inammatory effect of isovitexin in this paper. The in vivo results showed that isovitexin (10, 20mg/kg) inhibited the expression of proinammatory cytokines (TNF-α, IFN-γ, IL-2 and IL-17A) in lymph nodes, inhibited the secretion of cytokines into the serum from mice with contact dermatitis and promoted the expression of apoptosis-related proteins. In vitro, isovitexin also induced apoptosis and proinammatory cytokine expression in Con A-activated T cells. Further study showed that the MAPK and STAT signaling pathways and the phosphorylation of SHP2 were inhibited by isovitexin. Both molecular docking and biological experiments indicated that SHP2 may be an anti-inammatory target of isovitexin in T cells. Taken together, isovitexin can serve as a potential natural agent for the treatment or prevention of GA-induced inammatory problems. 100μM 10μM SHP2 or not; (A) The levels of proinammatory cytokines in cell culture supernatants were determined using ELISA kits after 24 hours cultivation. (B) Cells were stained with Annexin V and PI, and the apoptosis of the cells was determined by ow cytometry assay of Annexin V/PI staining. Annexin V positive cells of three independent experiments were shown in column statistics. (C-D) Lymph nodes cells were harvested and lysed and the phosphorylation of STAT3 and STAT6 was evaluated by Western blot. The grouping of blots was cropped from different parts of the same gel. Data are mean ± SEM and are representative of two independent experiments. Signicant differences are expressed as ##P (cid:0) 0.01 vs Con A group, *P < 0.05, **P < 0.01 vs the isovitexin group.


Introduction
Ginkgo fruit, the seeds of Ginkgo biloba L., has been used as a nutritious food for thousands of years, and a variety of medicinal effects have been attributed to the ginkgo fruit. The main active pharmaceutical ingredients in Ginkgo biloba include oxyglycoside avonoids, terpene trilactones, proanthocyanidins and so on 1,2 . However, food poisoning by ginkgo fruit has been reported in Japan and China, which presents as frequent vomiting and generalized convulsions 3 . Ginkgolic acids (GA), the alkylphenol constituents in ginkgo fruit (ginkgo seeds), have been considered one of the potential toxic components in Ginkgo biloba. The functional disorders are probably due to GA 4 . It has also been reported that contact allergic dermatitis (ACD) can be induced by ginkgolic acids when people contact ginkgo leaves during the picking of ginkgo fruit 5,6 . Ginkgo biloba leaves exhibit useful applications in health, food and dietary supplements but are also controversial in the application of some aspects because of the existence of GA. Under some conditions, GA can be separated by organic solvent extraction or column chromatography 7 . However, for raw material products such as ginkgo fruits and ginkgo tea, the GA removal process cannot be performed 8,9 . Other methods, such as detoxi cation by compatibility, need to be taken to solve the GA-caused toxicity problems in the application of ginkgo fruits and ginkgo tea.
Although a high dose of GA causes some diseases, it also shows pharmacological effects, such as antibacterial and antitumor effects 10,11 . Therefore, a strategy to reduce the side effects of GA should be different from other inactive compounds. The strategy could be a therapeutic that ameliorates GAinduced diseases in the form of a compound added to ginkgo products where GA cannot be separated effectively, such as ginkgo fruits and ginkgo tea.
GA, a derivative of alkyl-substituted salicylic acid, is similar to the chemical structure of urushiol and differs by carbon number and unsaturation, classi ed as C13:0, C15:1, C15:0, and C17:1 12 . Allergies caused by paint occur frequently in our daily lives. As an allergen from paint, urushiol can cause intense, persistent itch, skin rashes and a burning sensation in severe cases 13 . The clinical manifestations of GAinduced ACD are similar to urushiol-induced ACD, followed by the appearance of erythematous edematous plaques and papulovesicles accompanied by intense pruritus on the forearms in severe cases 14 . These in ammatory reactions can lead to a series of problems, such as skin injury, immune liver injury, gastrointestinal in ammatory injury and so on. With an important role in the immune response, T cells appear to proliferate and differentiate. However, overdeveloped T cells are associated with cutaneous allergic and in ammatory responses, which exacerbate skin in ammation, tissue injury and other immunopathies. In a previous study, reports suggested that the mechanism of GA-induced ACD may be consistent with urushiol-induced ACD. Numerous experimental animal models have validated GA as a hapten with the ability to activate T cells against innocuous or autoantigens and induce type IV allergic reactions 15 . A study has shown that T cells in rats sensitized with GA are differentiated into CD4 + T cells, suggesting that GA may act as an allergen to enhance body sensitivity, induce T cell division and proliferation and enhance the cellular immune response.
Celtis sinensis Pers. has been used as a traditional herbal remedy for thousands of years in China. It was used to treat urushiol-induced dermatitis. There is a folk tale, from the Maolan karst forest, that if someone contacted poison ivy inadvertently, he could be treated by putting the leaves of Celtis sinensis Pers. into his mouth. Isovitexin, the most abundant avonoid in the leaves of Celtis sinensis 16 , is a widely found natural carbon glycoside avonoid. Isovitexin has various pharmacological activities, such as antineoplastic 17 , antioxidant 18 , and neurological protective effects 19 . This compound has also shown anti-in ammatory e cacy against human diseases 20 . It has been reported that isovitexin inhibits the MAPK and NF-κB pathways in macrophages in acute lung injury 20 . In lymphocytes, a number of ACDassociated cytokines are dependent on JAK-STAT signaling, and antigen presentation is dependent on MAPK signaling for the effects on cellular transcription and activation. In this study, a contacthypersensitivity mouse model induced by GA was established to reveal the mechanisms underlying the anti-in ammatory effect of isovitexin. In summary, this study provides supporting data for isovitexin ameliorating GA-induced allergic contact dermatitis.

Reagents
Isovitexin (chemical structure is shown in Fig. 2A,CAS No. 38953-85-4, purity:98.01%) was obtained from Mansite Biotechnology, ChengDu, China) was dissolved in 100% DMSO at a concentration of 100 mM as a stock solution and diluted test concentration with culture medium before each experiment. Celtis sinensis Pers. leaves were collected from GuiZhou, China. The concentration of nal DMSO did not extend 0.1% throughout the trail. Ginkgolic acids was purchased from Jingzhu Medical Technology (Nanjing, China). The Annexin V-FITC/PI apoptosis kit and ELISA kits for murine (TNF-α, IFN-γ, IL-2, IL

Animal
Female ICR mice and BALB/c mice, 18-23 g, 6-8 weeks old, were obtained from the Experimental Animal Center of Yangzhou University (Yangzhou, China). The mice were maintained in plastic cages with free access to eat food and drink water, temperature kept at 21 ± 2°C and kept on a 12 h light/dark cycle. Animal welfare and experimental procedures were subjected to the Guide for the Care and Use of Laboratory Animals (National Institutes of Health, USA), and the study protocol was approved by the Animal Care and Protection Committee of Nanjing University-Gulou Hospital (SYXK 2004-0013). The authors con rmed that all animals received human care and all animal experiments were performed in accordance with the relevant guidelines and regulations. All the authors complied with the ARRIVE guidelines experiments.
Experimental design for GA-induced contact hypersensitivity is shown in Fig. 1A. On the rst day (day 0), female ICR mice were sensitized with FAC emulsi ed GA (GA/FAC, 1 mg/ml, 100 µl) in the right ank by subcutaneous injection. On Day 6 and day 13, mice were sensitized with FAC emulsi ed GA (GA/FAC, 1 mg/ml, 200 µl) in the back by subcutaneous injection of multipoints. On the last day (day 20), mice were challenged on their left ears with GA/FAC (10 mg/ml, 30 µl). 24 hours after the challenge, the ear thickness was measured with a digimatic micrometer. Ears' swelling was evaluated by the thickness difference between the left and right. Mice in normal group were normally sensitized with FAC and challenged with olive oil without GA. CLE (100, 200 mg/kg, intragastrically) or isovitexin (10, 20 mg/kg, intraperitoneally) or dexamethasone (0.5 mg/kg, intraperitoneally) was administered once a day from day 6 to day 20. Mice in the normal group and model group were given saline as control.

Cell culture and proliferation assay
T cells, isolated from female Balb/c mice Lymph nodes, were cultured in 96-well plates in 1640 medium at the density of 5×10 6 cells/well and stimulated with 5 µg/ml of Con A for 48 h under a humidi ed 5% (v/v) CO 2 atmosphere at 37℃. MTT with concentration of 4 mg/ml dissolves in PBS, 20 µl of MTT was added in each well 4 h before the end of incubation. Then culture media was removed and 200 µl DMSO was added to dissolve the crystals. Measuring the absorbance value at 540 nm.

Extraction of Celtis leaves
The method of extraction followed with few modi cations as described by Lee et al 21 . The air-dried leaves of Celtis sinensis were ground in a cutting mill and soaked with ethyl alcohol (25L'3, for 1.5h each) at 70℃. After the crude extract was dried under reduced pressure, a crude dark green residue was suspended in water with freeze-dried preservation.

RT-PCR
Total RNA was obtained from lymph node cells of mice, and reverse transcribed to cDNA. After cDNA was ampli ed by PCR, the expression of speci c RNA was detected by 5% agarose gel electrophoresis. The primer sequences were used: il-2 forward GTGCTCCTTGTCAACAGCG, il-2 reverse GGGGAGTTTCAGGTTCCTGTA; il-17a forward TTTAACTCCCTTGGCGCAAAA, il-17a reverse CTTTCCCTCCGCATTGACAC.

Cytokine assay
Cytokine levels were measured using ELISA kits from MultiSciences (Lianke) Biotech Co., Ltd. (Hangzhou, China) according to the manufacturer's instructions.

Western blot analysis
Cells isolated from lymph node were cultured in 6-well plates at a density of 1×10 7 cells/well in RPMI1640 medium and stimulated with Con A (5 µg/ml). Proteins lysed from cultured cells were separated by SDS-PAGE and electrophoreticically transferred onto PVDF membranes (Millipore, Bedford, MA). After treatment with blocking buffer in 5% BSA at RT for 1 h, membranes were incubated with primary antibodies at 4°C overnight, and the secondary antibody incubation at RT for 2 h. Protein content was detected by ECL luminescence method.

Histological analysis
The 5 mm thickness of ear sections were obtained by formalin-xed, para n-embedded and stained with hematoxylin and eosin. Histological parameter was following as described before: (1) the level of leukocyte in ltration and vascular congestion; (2) the erosion and anabrosis of epidermal cells; (3) affection of the other side of the ears 22 . We scored each of the histological assessment on a scale of 1 to 4 and the higher score means more serious in ammation.

Molecular docking analysis
The molecular docking analysis was conducted in Maestro v11.1 (Schrödinger, LLC) by the default protocols 23 . We prepared the ligand isovitexin and SHP2 protein (Protein Data Bank ID: 3o5x). The docking grid was generated based on the position of the tyrosine phosphatase SHP2 with the default protocol. Subsequently, glide docking was performed and induce-t docking was conducted based on the results of glide docking 2.10 Statistical analysis All Data were represented means ± SEM from triplicate experiments performed in a parallel manner. Data were statistically compared and one-way analysis of variance ANOVA followed by Dunnett's test between the vehicle group and multiple dose groups. The level of signi cance was set at P<0.05.

Celtis sinensis leaf extract (CLE) protects mice from allergic contact dermatitis
Celtis sinensis is employed as a folk medicine for treating in ammation, skin infections and other diseases 24 . To study the possibility of using Celtis sinensis to treat in ammation in GA-induced allergic contact dermatitis, a mouse model of GA-induced hypersensitive contact dermatitis was established. We stimulated the popliteal lymph nodes and the left ear of ICR mice by GA (Fig. 1A). Mice challenged by GA displayed de nite in ammation, as indicated by splenomegaly and ear swelling, compared with the vehicle group.
Celtis sinensis leaf extract (investigational ingredient, CLE) and dexamethasone (positive control drug, Dex) were used to treat the degree of ear swelling. Notably, mice that received CLE exhibited signi cantly reduced susceptibility to GA-induced allergic contact dermatitis, as shown by the milder splenomegaly and ear swelling compared with the model group ( Fig. 1B and C). Indeed, CLE did not decrease the body weight of mice, but it appeared in mice that received dexamethasone (Fig. 1D). Histopathologically, mice that received CLE displayed signi cantly alleviated leukocyte in ltration, epidermal anabrosis and affection damage compared with the vehicle group ( Fig. 1E and F). Collectively, intragastric instillation of CLE confers protection from GA-induced allergic contact dermatitis in ICR mice.
3.2 Isovitexin, the main avonoid in the leaves of Celtis sinensis, ameliorates GA-induced allergic contact dermatitis in mice Since isovitexin ( Fig. 2A) is the predominant avone of Celtis sinensis 25 , we further examined the possible role of isovitexin in dermal in ammation. Mice that received isovitexin exhibited ameliorative ear swelling and splenomegaly compared with the model group ( Fig. 2B and C). Meanwhile, consistent with the CLE experiments, isovitexin did not show body weight loss, suggesting that compared with dexamethasone, isovitexin displayed signi cant anti-in ammatory function without serious side effects (Fig. 2D). Histopathologically, mice that received isovitexin displayed directly reduced leukocyte chemotaxis and ameliorated proin ammatory cytokine release (Fig. 2E and F). Therefore, isovitexin, a natural avone, exerts immunosuppressive effects in GA-induced allergic contact dermatitis

Isovitexin inhibits T cell-mediated in ammation in mice with GA-induced allergic contact dermatitis
Stimulation with GA also exacerbated the release of in ammatory cytokines (TNF-α, IFN-γ, IL-2, and IL-17A) in serum. The upregulation of these T cell-speci c cytokines indicates that GA-induced allergic contact dermatitis mainly triggers in ammatory activation of T cells rather than macrophage-mediated innate in ammation. The anti-in ammatory effects of isovitexin were presumably attributed to a decrease in T cell-speci c cytokines in serum (Fig. 3A). We then evaluated the mRNA level of T cellassociated cytokines in popliteal lymph nodes. Compared with in amed dermatitis mice, the contents of tnf-α, ifn-γ, il-2, and il-17a were signi cantly lower in mice that received isovitexin ( Fig. 3B and C). Moreover, we observed that proapoptotic proteins (cleaved caspase-3 and cleaved PARP) were activated and that proliferation proteins (phosphor-AKT and phospho-ERK1/2) were inhibited in isovitexin-treated T cells from allergic mice ( Fig. 3D and E). Taken together, these data clearly demonstrate that isovitexin may inhibit T cell-mediated in ammatory responses in dermatitis.

Isovitexin inhibits the proliferation and promotes apoptosis of Con A-activated T cells in vitro
The above ndings prompted us to suppose that isovitexin diminished T cell proin ammatory activities in response to GA stimulation. Furthermore, we used Con A-activated T cells to explore the mechanisms underlying the antagonizing effects of isovitexin on metabolic disorders. Con A is a plant lectin that induces the mitogenic activity of T lymphocytes and increases the production of in ammatory cytokines such as IL-2, TNF-α and IFN-γ 26 . The MTT assay was used to assess cell viability. As shown in Fig. 4B, Con A (5 μg/ml) strongly promoted T cell proliferation. In addition, cell culture with the addition of 0-100 μM isovitexin was not toxic to naïve T cells, but it inhibited the proliferation of Con A-activated T cells ( Fig. 4A and B). On the other hand, isovitexin triggered apoptosis of Con A-activated T cells, as analyzed by the Annexin V/PI staining assay (Fig. 4C). The percentages of early apoptotic T cells signi cantly increased with different doses of isovitexin after 24 h of incubation. To de ne the pathway of apoptosis, western blotting was used to analyze the cleavage of poly (ADP-ribose) polymerase (PARP) and caspase. Strong cleavage of PARP together with activation of caspase-3 and -8 were observed in the Con Aactivated T cells treated with isovitexin, which is consistent with the results of animal experiments ( Fig.  4D and E). These observations indicate that isovitexin inhibits the proliferation of Con A-activated T cells by promoting apoptosis.

Isovitexin inhibits the production of proinflammatory cytokines in Con A-activated T cells
Numerous apoptotic cells have been shown to inhibit proin ammatory cytokine production, preventing chronic in ammation 27 . For this claim, we examined whether isovitexin is linked with the production of proinflammatory cytokines, including TNF-α, IFN-γ, IL-2 and IL-17A. ELISA was performed to measure the release of cytokines in the culture supernatant, and RT-PCR was carried out to measure the expression of these cytokines from Con A-activated T cells (Fig. 5A, B and C). Interestingly, at a concentration of 100 μM, isovitexin signi cantly reduced the levels of proin ammatory cytokines (TNF-α, IFN-γ, IL-2 and IL-17A) at both the mRNA and protein levels.

The MAPK and STAT signaling pathways are regulated by isovitexin in Con A-activated T cells
The MAPK and STAT signaling pathways govern the expression of most proin ammatory genes 28,29 . To understand how isovitexin modulates in ammatory responses in T cells, we examined the relationship between isovitexin and these pathways. Mechanistically, T cells treated with 100 μM isovitexin exhibited markedly reduced phosphorylation of proteins such as P38, JNK, and ERK1/2 in Con A-induced T cells, which are the central kinases in the MAPK signaling pathway that cause in ammation. On the other hand, isovitexin dose-dependently decreased the level of IκB phosphorylation. The kinase is activated by a highly diverse group of extracellular signals, including in ammatory cytokines, growth factors, and chemokines. Unexpectedly, isovitexin did not affect the expression of p65 in whole cell lysate, and whether p65 is in the core needs to be further proven. Western blotting was also used to explore whether isovitexin decreased the phosphorylation of STAT3, STAT6, and SHP2, which play important roles in the proliferation and differentiation of T cells. Thus, isovitexin serves as a negative regulator of the MAPK and STAT signaling pathways ( Fig. 6A and B).

Molecular docking analysis of the interaction between isovitexin and SHP2 protein
Next, we sought to determine the molecular mechanisms by which isovitexin modulates the MAPK and STAT signaling pathways in Con A-activated T cells. We rst tested whether isovitexin might interact with SHP2. The interaction between the SHP2 protein and isovitexin was demonstrated by molecular docking analysis. As shown in the gure, there is a certain intensity of interaction between SHP2 and isovitexin, and the best induced-t docking score is -5.414 kcal/mol, where isovitexin can insert into the active domain of SH2 kinase and interact by hydrogen bonding and π-π interactions (Fig. 6C). The docking results suggest that there is obvious π-π conjugation between TYR279, the B ring and the C ring of isovitexin. There may be two hydrogen bond interactions between SHP2 and isovitexin: one formed between the ASN281 residue and 4'-OH on the C ring of isovitexin, and the other formed between GLN and the carbonyl group on the B ring of isovitexin. Indeed, the A ring of isovitexin forms seven kinds of hydrogen bonds with SHP2 with ASP425, LYS366, AGR465, TRP423 and GLY427, which confer stability to the docking conformation (Fig. 6D).

The effects of isovitexin on Con A-activated T cells can be reversed by the SHP2-specific inhibitor SHP099
SHP099, a selective SHP2 inhibitor, binds to protein tyrosine phosphatase domains, inhibiting SHP2 activity through an allosteric mechanism 30 . We investigated whether isovitexin suppressed the release of cytokines by regulating SHP2 in Con A-activated T cells, and the effects were examined in SHP099treated cells. The results showed that the inhibitory effects of isovitexin on T cell proliferation and proinflammatory cytokine production were partially reversed by SHP099 (Fig. 7A and B). Moreover, the regulation of the expression of p-STAT3 and p-STAT6 was partially reversed by SHP099 as well in T cells incubated with isovitexin ( Fig. 7C and D). In summary, our study proved that isovitexin restrains p-SHP2 and controls dermatitis in ammation (Fig. 8).

Discussion
Allergic contact dermatitis is a skin disease caused by environmental or occupational allergens. As a general concept of the antigen presentation process in the sensitization phase of ACD, allergens activate innate immunity through keratinocyte release of proin ammatory cytokines and chemokines to recruit T cells. Hapten-speci c T cells are guided to in ammatory sites and produce cytokines, such as TNF-α, IFNγ and IL-17A. In turn, these cytokines then stimulate skin-resident cells, which lead to further recruitment of T cells and induce an in ammatory cascade 31,32 . Indeed, dysregulation of the skin immune system occurs in nearly all ACD cases, which highlights the importance of appropriate immune regulation in preventing ACD. However, studies of contact-hypersensitivity mouse models have mostly used synthetic experimental allergens, such as DNFB. Different allergens cause widely divergent immune responses, resulting in the eventual failure of the therapeutic strategies for ACD. We therefore optimized a mouse model of GA-induced ACD to identify in ammation and ear swelling. We identi ed a critical role of proin ammatory cytokines such as TNF-α, IFN-γ, IL-2 and IL-17A in skin in ammation in this model and revealed that these cytokines may be regulated by SHP2 in vitro.
As an environmental allergen, GA-induced ACD is usually treated with NSAIDs or immunosuppressive drugs, and long-term use may result in serious side effects, including effects on the gastrointestinal tract and infection susceptibility 33 . In this context, new pharmacological strategies are being sought, such as resolution of in ammation with fewer side effects. In the present study, we have provided proof that CLE, especially isovitexin, may dampen proin ammatory signaling and the clearance of proin ammatory mediators to attenuate in ammation. In conclusion, isovitexin acts as an in ammatory inhibitor to ameliorate GA-induced ACD.
Isovitexin is the most abundant avone in the leaves of Celtis sinensis. It has anti-in ammatory pharmacological properties. In a previous study, isovitexin attenuated the LPS-induced phosphorylation of all three MAPKs, reduced NF-κB activation and promoted M2 polarization in macrophages 20,34 . However, few studies targeting the anti-in ammatory effects of T lymphocytes by isovitexin treatment have been reported. To reveal the regulatory effects of isovitexin on ACD, which is characterized by the Th1 response 35 , we used T cells from mouse lymph node cells under Con A stimulation. In this study, we found that isovitexin dose-dependently upregulated apoptosis and suppressed the cytokines TNF-α, IFN-γ, IL-2 and IL-17A. This result is consistent with animal experiments, indicating that isovitexin exerts an immunomodulatory effect when facing an in ammatory challenge.
To further reveal the underlying mechanisms of isovitexin on T cell apoptosis and signaling pathways.
We further examined the effects of isovitexin on cleaved caspase-3, cleaved caspase-8, MAPK signaling and STAT signaling in activated T cells. In this study, we have provided several lines of evidence that suggest that cleaved caspase-3 and cleaved caspase-8 are signi cantly enhanced by isovitexin treatment in a dose-dependent manner. These cleaved proteins lead to increased apoptosis. MAPK signaling and STAT signaling, along with countless studies linking to in ammatory pathologies, provide the rationale for applying potent inhibitors in the treatment of immune-system-mediated diseases 29,36 . Our research result is somewhat consistent with a previous report in which isovitexin normalized the phosphorylation of all three MAPKs and reduced NF-κB activation and the STAT signaling pathway. In addition, isovitexin dampens proin ammatory signaling and promotes the resolution of in ammation. SHP2, a nonreceptor protein tyrosine phosphatase, was the rst reported oncogenic tyrosine phosphatase and has attracted much attention. The function of SHP2 was reported to regulate cell survival and proliferation primarily through activation of the RAS-ERK signaling pathway and to mediate the immune checkpoint pathways 37 . Moreover, SHP2 is an important mediator in lupus erythematosus, ERK/MAPK signaling normalization and reduced production of IFN-γ and IL-17a cytokines involved in in ammation can occur by directly inhibiting SHP2 38 . Meanwhile, SHP2 is required for full activation of the JAK/STAT pathway, a major signaling cascade in in ammation 39 . SHP2 has been characterized as a positive regulator of JAK2/STAT3 signaling in rheumatoid arthritis diseases 40 . Here, molecular docking showed that isovitexin may bind to the PTP domain with hydrogen bonding and π-π interactions. Western blotting experiments suggested that isovitexin normalized Src-homology 2 domain-containing phosphatase tyrosine (Y542) phosphatase, which upregulates phosphatase activity 41 . Indeed, downregulated SHP2 reduces the downstream MAPK and STAT signaling pathways, which decreases the level of proin ammatory cytokine release and ameliorates ACD. It also prevents the T cell-decreased MAPK signaling, which would cause autoreactivity and autoimmunity 42 . In summary, isovitexin blockade of SHP2 may be a novel and effective therapy for the treatment of patients with GA-induced ACD.
SHP099, an inhibitor that binds to the allosteric site of SHP2 and stabilizes the closed form of SHP2 by interacting with the N-SH2 and PTP domains, has been used to provide several lines of evidence, which suggested functional expression and a physiological signaling role of SHP2 in Con A-active T cells. First, the Annexin V/PI staining results showed that T cells treated with isovitexin and SHP099 had an increased proportion of living cells versus the isovitexin group alone. These results indicated that isovitexin promotes T lymphocyte apoptosis by downregulating SHP2 activation. Moreover, SHP099 also reversed the inhibitory effects of isovitexin on MAPK and STAT signaling. Our results indicate that isovitexin exerts SHP2-dependent inhibitory effects in Con A-activated T cells.
In summary, our study demonstrates that isovitexin from Celtis sinensis is a potential therapeutic agent against GA-induced allergic contact dermatitis. The MAPK and STAT signaling pathways can be regulated by isovitexin, and SHP2 may be a potential anti-in ammatory target of isovitexin in T cells. Isovitexin can be used to solve anti-in ammatory problems induced by GA.    hours. (C) Cells were stained with Annexin V and PI, and the apoptosis of the cells was determined by ow cytometry assay of Annexin V/PI staining. Annexin V positive cells of three independent experiments were shown in column statistics. (D-E) Cells treated with different concentrations of isovitexin were harvested and lysed. The protein levels of p-AKT, Cleaved caspase-3, Cleaved caspase-8, Cleaved PARP in T cells were measured by western blotting, and the relative band density was analyzed using Image J.

Abbreviations
The grouping of gels were cropped from different parts of the different gels. Data represented mean ± SEM of three independent experiments. ##P 0.01 vs negative control, *P < 0.05, **P < 0.01 vs control (Con A-activated T cells).

Figure 5
Isovitexin inhibits T cells proin ammatory cytokines production in activated mouse T cells. Con Aactivated T cells were treated with different concentrations of isovitexin for 24 hours; (A) Proin ammatory cytokines, in culture supernatants of the indicated groups, were determined by ELISA; (B-C) Relative tnf-α, ifn-γ, il-2, il-17a mRNA level was determined by RT-PCR, and relative band density was Page 22/26 analyzed using Image J. Data are mean ± SEM and are representative of at least three independent experiments. Signi cant differences are expressed as *P < 0.05, **P < 0.01 vs the Con A group. P65, p-STAT3, p-STAT6, p-SHP2 in T cells were measured by western blotting, and the relative band density was analyzed using Image J Data are mean ± SEM and are representative of at least three independent experiments. The grouping of gels were cropped from different parts of the different gels.
Signi cant differences are expressed as *P < 0.05, **P < 0.01 vs the Con A group. (C-D) The induced t docking analysis of isovitexin and SHP2 protein (PDB:3o5x). Hydrogen bonds and π-π stacking were indicated with green and pink dot line, respectively.  On the other hand, isovitexin bind to SHP2 inhibits MAPK signaling, STAT signaling and its dependent