Modulatory effects of the fruits of Tribulus terrestris L. on the function of atopic dermatitis-related calcium channels,Orai1 and TRPV3

Objective: To examine the effects of Tribulus terrestris L.(T. terrestris) extract on the modulation of calcium channels to evaluate its use in topical agents for treatment of atopic dermatitis.Methods: The 70% methanol extract of T. terrestris was prepared. Human HEK293 T cells with over-expressed calcium release-activated calcium channel protein 1(Orai1),transient receptor potential vanilloid 1, or transient receptor potential vanilloid 3(TRPV3)were treated with T. terrestris extract. Modulation of ion channels was measured using a conventional whole-cell patch-clamp technique.Results: T. terrestris extract(100 mg/m L) significantly inhibited Orai1 activity in Orai1-stromal interaction molecule 1 co-overexpressed HEK293 T cells. In addition, T. terrestris extract significantly increased the TRPV3 activity compared with 2-Aminoethyl diphenylborinate(100 mmol/L), which induces the full activation of TRPV3.Conclusions: Our results suggest that T. terrestris extract may have a therapeutic potential for recovery of abnormal skin barrier pathologies in atopic dermatitis through modulating the activities of calcium ion channels, Orai1 and TRPV3. This is the first study to report the modulatory effect of a medicinal plant on the function of ion channels in skin barrier.

Clean-DM1, a Korean Polyherbal Formula, Improves High Fat Diet-Induced Diabetic Symptoms in Mice by Regulating IRS/PI3K/AKT and AMPK Expressionsin Pancreas and Liver Tissues

Objective:To investigate the effects of Clean-DM1(C-DM1),a polyherbal formulation of Radix Scrophulariae,Radix Astragali,Rhizoma Atractylodis,and Radix Salviae Miltiorrhizae,on high-fat diet(HFD)-induced diabetes mice.Methods:The information about active components of C-DM1 extract and molecular mechanism was obtained from network pharmacology analysis.Main compounds of C-DM1 extract by high performance liquid chromatography-mass spectrometry(HPLC-MS)analysis were conducted for quality control.For in vivo study,mice were induced diabetes by HFD for 12 weeks.The mice in the normal group(Nor)were maintained with a regular diet and treated with saline by gavage.The HFD model mice were randomly divided into 3 groups,including a HFD diabetic model group,a C-DM1 extract-administered group(C-DM1,500 mg/kg),and metformin-administered groups(Met,500 mg/kg),8 mice in each group.Food intake,body weight(BW),and fasting blood glucose(FBG)levels were recorded weekly for 4 weeks.After 4 weeks of treatment,alanine aminotransferase(ALT),aspartate aminotransferase(AST),blood glucose,low-density lipoprotein cholesterol(LDL-C)were determined using an automated clinical chemistry analyzer,and homeostatic model for assessing insulin resistance(HOMA-IR)levels and oral glucose tolerance test(OGTT)were detected.The histopathological changes of liver and pancreatic tissues were observed by hematoxylin-eosin staining.Insulin receptor substrate(IRS)/phosphatidylinositol 3 kinase(PI3K)/protein kinase B(AKT)and adenosine 5'-monophosphate-activated protein kinase(AMPK)expressions in liver and pancreas tissues were detected by Western blot analysis.Results:HPLC-MS identified dihydroisotanshinone,dihydroisotanshinone I,cryptotanshinone,harpagoside,and atractyloside A in C-DM1 extract.The administration of C-DM1 extract significantly decreased body weight,calorie intake,and the levels of blood glucose and insulin in the diabetic mice(P<0.05 or P<0.01).The C-DM1 extract administration improved the impaired glucose tolerance and insulin resistance in the diabetic mice and significantly decreased the levels of LDL-C,ALT and AST(P<0.01).The C-DM1 extract inhibited the histopathological changes of fatty liver and hyperplasia of pancreatic islets in the diabetic mice.The C-DM1 extract significantly increased the phosphorylation of IRS,AKT,and AMPK and the expression of PI3K in pancreas and liver tissues(P<0.05 or P<0.01),which was consistent with the analysis results of network pharmacology.Conclusion:C-DM1 extract improved diabetes symptoms in longterm HFD-induced mice by regulation of IRS/PI3K/AKT and AMPK expressions in pancreas and liver tissues,suggesting that C-DM1 formulation may help prevent the progression of T2DM.

Effect of the semen extract of Cuscuta chinensis on inflammatory responses in LPS-stimulated BV-2 microglia

AIM: To investigate the anti-inflammatory activities of the semen extract of Cuscuta chinensis Lam.(Cuscutae Semen; CS) on the production of inflammatory mediators, nitric oxide(NO), prostaglandin 2(PGE2), and proinflammatory cytokines in lipopolysaccharide(LPS)-stimulated BV-2 microglia. METHOD: BV-2 cells were treated with CS extract for 30 min, and then stimulated with LPS or without for 24 h. The levels of NO, PGE2 and proinflammatory cytokines were measured by Griess assay and ELISA. The expression of inducible nitric oxide synthase(iNOS), and cyclooxygenase(COX)-2 mRNA and protein was determined by RT-PCR and Western blot, respectively. The phosphorylation of extracellular signal-regulated kinase 1/2(ERK1/2), Jun N-terminal kinase(JNK), and p38 mitogen-activated protein kinase(MAPK), and the nuclear expression of nuclear factor(NF)-κB p65 were investigated by Western blot analysis. RESULTS: CS extract significantly decreased the production of NO and PGE2 by suppressing the expression of iNOS and COX-2 in activated microglia. CS extract decreased the production of TNF-α, IL-1β, and IL-6 by down-regulating their transcription levels. In addition, CS extract suppressed the phosphorylation of ERK1/2, JNK, and p38 MAPK, and the nuclear translocation of NF-κB p65 in activated microglia. CONCLUSION: These results indicate that CS extract is capable of suppressing the inflammatory response by microglia activation, suggesting that CS extract has potential in the treatment of brain inflammation.

Effects of Flower Buds Extract of Tussilago farfara on Focal Cerebral Ischemia in Rats and Inflammatory Response in BV2 Microglia

Objective： To investigate the effects of the flower buds extract of Tussilago farfara Linné（Farfarae Flos; FF） on focal cerebral ischemia through regulation of inflammatory responses in activated microglia. Methods： Brain ischemia was induced in Sprague-Dawley rats by a transient middle cerebral artery occlusion（t MCAO） for 90 min and reperfusion for 24 h. Twenty rats were randomly divided into 4 groups（n=5 per group）： normal, t MCAO-induced ischemic control, t MCAO plus FF extract 300 mg/kg-treated, and t MCAO plus MK-801 1 mg/kg-treated as reference drug. FF extract（300 mg/kg, p.o.） or MK-801（1 mg/kg, i.p.） was administered after reperfusion. Brain infarction was measured by 2,3,5,-triphenyltetrazolium chloride staining. Neuronal damage was observed by haematoxylin eosin, Nissl staining and immunohistochemistry using anti-neuronal nuclei（Neu N）, anti-glial fibril ary acidic protein（GFAP）, and antiCD11 b/c（OX42） antibodies in ischemic brain. The expressions of inducible nitric oxide synthase（i NOS）, tumor necrosis factor（TNF-α）, and hypoxia-inducible factor-1 a（HIF-1α） were determined by Western blot. BV2 microglial cel s were treated with FF extract or its main bioactive compound, tussilagone with or without lipopolysaccharide（LPS）. Nitric oxide（NO） production was measured in culture medium by Griess assay. The expressions of i NOS, COX-2 and pro-inflammatory cytokines mRNA were analyzed by reverse transcription-polymerase chain reaction. The expression of i NOS, and COX-2 proteins, the phosphorylation of ERK1/2, JNK, and p38 MAPK and the nuclear expression of NF-κB p65 in BV2 cells were determined by Western blot. Results： FF extract significantly decreased brain infarctions in ischemic rats（P〈0.01）. The neuronal death and the microglia/astrocytes activation in ischemic brains were inhibited by FF extract. FF extract also suppressed i NOS, TNF-α, and HIF-1α expression in ischemic brains. FF extract（0.2 and 0.5 mg/m L, P〈0.01） and tussilagone 20 and 50 μmol/L, P〈0.01） significantly decreased LPS-induced NO production in BV2 microglia through downregulation of i NOS m RNA and protein expression. FF extract and tussilagone significantly inhibited LPS-induced expression of TNF-α, IL-1β, and IL-6 m RNA, and also suppressed the phosphorylation of ERK1/2, JNK and p38 MAPK and the nuclear expression of NF-κB in a dosedependent manner. Conclusion： FF extract has a neuroprotective effect in ischemic stroke by the decrease of brain infarction, and the inhibition of neuronal death and microglial activation-mediated inflammatory responses.

Inhibitory Effect of the Extract of Phellodendron amurense Ruprecht Root on Collagen-Induced Arthritis in Mice

Objective： To investigate whether the dried root of Phellodendron amurense Ruprecht（Phellodendri cortex; PC） extract improves arthritic symptoms through anti-inflammatory and immune-modulatory effects in collagen-induced arthritis in mice. Methods： Rheumatoid arthritis（RA） was induced in male DBA/1 mice by immunization with type Ⅱ collagen（ColⅡ）. CIA mice were divided into 5 groups（n=10 per a group） with normal, CIA control, PC extract（50 mg/kg and 100 mg/kg）-treated, and meloxicam（50 mg/kg）-treated as the reference drug. The PC extract or meloxicam were administered orally in CIA mice once a day for 14 days after arthritis induction. Arthritic score, levels of anti-ColⅡ IgG2a antibody, prostaglandin E2（PGE2）, tumor necrosis factor（TNF）-α, and interleukin（IL）-17 in the sera of CIA mice were measured. Histopathological changes in the ankle joints of CIA mice were also analyzed by staining with hematoxylin and eosin（H and E）, safranin-O and immunohistochemistry using anti-TNF-α and anti-IL-17 antibodies. Results： The arthritic score was increased in CIA mice in a time-dependent manner, as were the serum levels of anti-ColⅡ IgG2a antibody, PGE2, TNF-α, and IL-17. However, the oral administration of PC extract at 50 and 100 mg/kg in CIA mice significantly decreased the arthritic scores, and the serum levels of anti-ColⅡ IgG2a, PGE2, TNF-α, and IL-17 compared with those in the CIA group（P〈0.05 or P〈0.01）. Furthermore, histopathological improvement of the joint architecture in CIA mice was observed after administration of PC extract. PC extract also significantly inhibited the expression of TNF-α and IL-17 in the joints of CIA mice by suppressing the expression of their m RNA and proteins. Conclusion： PC extract may improve the pathological progression of RA through the inhibition of joint destruction by synovial inflammation and immune-stimulation, therefore, it would be a potential anti-arthritic agent in RA.