Magnolol as STAT3 inhibitor for treating multiple sclerosis by restricting Th17 cells.

OBJECTIVE: Multiple sclerosis (MS) is an immune disease in the central nervous system (CNS) associated with Th17 cells. Moreover, STAT3 initiates Th17 cell differentiation and IL-17A expression through facilitating RORγt in MS. Here, we reported that magnolol, isolated from Magnolia officinalis Rehd. Et Wils, was regarded as a candidate for MS treatment verified by both in vitro and in vivo studies. METHODS: In vivo, experimental autoimmune encephalomyelitis (EAE) model in mice was employed to evaluate the alleviation of magnolol on myeloencephalitis. In vitro, FACS assay was employed to evaluate the effect of magnolol on Th17 and Treg cell differentiation and IL-17A expression; network pharmacology-based study was applied to probe the involved mechanisms; western blotting, immunocytochemistry, and luciferase reporter assay was used to further confirm the regulation of magnolol on JAK/STATs signaling pathway; surface plasmon resonance (SPR) assay and molecular docking were applied to manifest affinity with STAT3 and binding sites; overexpression of STAT3 was employed to verify whether magnolol attenuates IL-17A through STAT3 signaling pathway. RESULTS: In vivo, magnolol alleviated loss of body weight and severity of EAE mice; magnolol improved lesions in spinal cords and attenuated CD45 infiltration, and serum cytokines levels; correspondingly, magnolol focused on inhibiting Th17 differentiation and IL-17A expression in splenocyte of EAE mice; moreover, magnolol selectively inhibited p-STAT3(Y705) and p-STAT4(Y693) of both CD4+ and CD8+ T cells in splenocyte of EAE mice. In vitro, magnolol selectively inhibited Th17 differentiation and IL-17A expression without impact on Treg cells; network pharmacology-based study revealed that magnolol perhaps diminished Th17 cell differentiation through regulating STAT family members; western blotting further confirmed that magnolol inhibited p-JAK2(Y1007) and selectively antagonized p-STAT3(Y705) and slightly decreased p-STAT4(Y693); magnolol antagonized both STAT3 nucleus location and transcription activity; magnolol had a high affinity with STAT3 and the specific binding site perhaps to be at SH2 domain; overexpression of STAT3 resulted in failed inhibition of magnolol on IL-17A. CONCLUSION: Magnolol selectively inhibited Th17 differentiation and cytokine expression through selectively blocking of STAT3 resulting in decreased the ratio of Th17/Treg cells for treating MS, suggesting that the potential of magnolol for treating MS as novel STAT3 inhibitor.

Characterization of a Novel Gja8 (Cx50) Mutation in a New Cataract Rat Model.

Purpose: To describe a novel spontaneous cataract inbred strain isolated from large-scale breeding SD rats, identify the responsible gene mutation, and understand how this mutation affects lens function. Methods: Exome sequencing of 12 cataract-associated genes was performed in the affected and healthy relatives. Sequences of rat wild-type or mutant gap junction protein alpha 8 gene (Gja8) were transfected into cells. The expression level of protein was assayed by Western blot analysis. Subcellular localization of connexin 50 (Cx50) was analyzed in confocal fluorescent images. Wound-healing, 5-ethynyl-2'-deoxyuridine incorporation, and attachment assay were performed to characterize the cell migration, proliferation and adhesion. Results: The abnormality was found to be inheritable in an autosomal semi-dominant pattern through different mating patterns. We found a G to T transversion at codon 655 in Gja8, leading to a substitution of valine by phenylalanine (p.V219F). Gja8V219F/+ heterozygotes expressed nuclear cataract while Gja8V219F/V219F homozygotes manifested microphthalmia in addition to cataract. Histology revealed fiber disorders and loss of organelle-free zone in the mutant lens. Cx50V219F altered its location in HeLa cells and inhibited the proliferation, migration and adhesion abilities of HLEB3 cells. The mutation also reduced the expression of focal adhesion kinase and its phosphorylation. Conclusions: The c.655G>T mutation (p.V219F) is a novel mutation in Gja8, inducing semi-dominant nuclear cataracts in a new spontaneous cataract rat model. The p.V219F mutation altered Cx50 distribution, inhibited lens epithelial cell proliferation, migration, and adhesion, and disrupted fiber cell differentiation. As a consequence, the nuclear cataract and small lens formed.

Importance of Gedunin in Antagonizing Rheumatoid Arthritis via Activating the Nrf2/ARE Signaling.

Objective: This study assessed the anti-arthritic effect and protection of Gedunin (GDN) on joint tissues and revealed the possible mechanism in suppressing rheumatoid arthritis (RA). Methods: LPS-induced macrophages and TNF-α-stimulated synovial fibroblasts (MH7A) or IL-1ß-stimulated primary rheumatoid arthritis synovial fibroblasts (RASFs) were used to evaluate the antiinflammatory effect of GDN. In addition, CIA-induced arthritis was employed here to evaluate the anti-arthritic effect. MTT and BRDU assays were utilized to evaluate the cell viability and proliferation, Q-PCR was conducted to detect the mRNA expression of cytokines, FACS was adopted to monitor ROS production, while western blotting (WB) and siRNA interference were applied in confirming the anti-arthritic effects of GDN via the Nrf2 signaling. Results. In vitro, cell viability was inhibited in macrophages and MH7A cells, but not in RASFs; but the proliferation of RASFs was significantly suppressed in time- and dose-dependent manners. GDN suppressed cytokine levels in LPS-stimulated macrophages and TNF-α-stimulated MH7A cells or RASFs. GDN suppressed ROS expression. Furthermore, GDN treatment notably dose-dependently decreased the mRNA and protein expression of iNOS in LPS-induced macrophages. sip62 interference results showed that GDN cause the less expression of HO-1 and Keap1 and also fail to inhibit cytokines after sip62 interference. In vivo, GDN effectively inhibited paw swelling, arthritis score, and arthritis incidence and cytokines. Conclusions: Our study suggested that GDN exhibited strong antagonistic effect on arthritis both in vitro and in vivo via activation of Nrf2 signaling. Our work will provide a promising therapeutic strategy for RA.

Neobaicalein Inhibits Th17 Cell Differentiation Resulting in Recovery of Th17/Treg Ratio through Blocking STAT3 Signaling Activation.

Huangqin is the dried root of Scutellaria baicalensis Georgi, which has been widely utilized for heat-clearing (Qingre) and dewetting (Zaoshi), heat-killed (Xiehuo) and detoxifying (Jiedu) in the concept of Traditional Chinese Medicine and is used for treating inflammation and cancer in clinical formulas. Neobaicalein (NEO) is of flavonoid isolated from Huangqin and has been reported to possess prominent anti-inflammatory effects in published work. Th17/Treg balance shift to Th17 cells is an essential reason for autoimmune inflammatory diseases. However, the role NEO plays in Th17 and Treg and the underlying mechanism has not been elucidated yet. Network pharmacology-based study revealed that NEO predominantly regulated IL-17 signaling pathway. Moreover, our result shown that NEO (3-30 µmol/L) down-regulated Th17 differentiation and cellular supernatant and intracellular IL-17A level and tumor necrosis factor α production in a concentration-dependent manner. The further mechanism research revealed that NEO also specifically inhibited phosphorylation of STAT3(Tyr725) and STAT4 (Y693) without influence on activation of STAT5 and STAT6 in splenocytes. Immunofluorescence results illuminated that NEO effectively blocked STAT3 translocated into nucleus. Interestingly, NEO at appreciated dose could only inhibit Th17 cell differentiation and have no effect on Treg differentiation. The present study revealed that NEO effectively inhibited Th17 cell differentiation through specifically blocking the activation of STAT3 signaling without inactivation of STAT5 and STAT6. Additional inhibitory effect on activation of STAT4 by NEO also suggested the potential for antagonism against Th1 differentiation. All work suggested that NEO may be a potential candidate for immunoregulation and treating autoimmune inflammatory diseases through inhibiting immune cell viability and T cell differentiation.

Pimpinellin Inhibits Collagen-induced Platelet Aggregation and Activation Through Inhibiting Granule Secretion and PI3K/Akt Pathway.

Pimpinellin is a coumarin-like compound extracted from the root of Toddalia asiatica. Its effects on platelet function has not been investigated. This study found that pimpinellin pretreatment effectively inhibited collagen-induced platelet aggregation, but did not alter ADP- and thrombin-induced aggregation. Platelets pretreated with pimpinellin showed reduced α granule (CD62) level and secretion of dense granule (ATP release). Pimpinellin-treated platelets also exhibited decreased clot reaction and TxB2 production. Pimpinellin pretreatment suppressed adhesion and spreading of human platelets on the fibrinogen coated surface. Analysis of tail bleeding time of mice administered with pimpinellin (40 mg/kg) revealed that pimpinellin did not change tail bleeding time significantly, number of blood cells, and APTT and PT levels. Pimpinellin inhibited collagen-induced ex vivo aggregation of mice platelets. Immunoblotting results showed that pimpinellin suppressed collagen-induced phosphorylation of PI3K-Akt-Gsk3ß and PKC/MAPK in platelets.

Two Isostructural Flexible Porous Coordination Polymers Showing Contrasting Single-Component and Mixture Adsorption Properties for Propylene/Propane.

Solvothermal reactions of 3-(3-methylpyridin-4-yl)benzoic acid (Hmpba) with Mn(NO3)2 or Co(NO3)2 yielded isostructural porous coordination polymers, [Mn(mpba)2]·guest (MCF-56, 1·g) and [Co(mpba)2]·guest (MCF-57, 2·g), respectively. X-ray diffraction revealed that 1·g and 2·g possess similar one-dimensional ultramicroporous channels, and guest-free [Mn(mpba)2] (1') and [Co(mpba)2] (2') possess significantly and slightly contracted channels, respectively. Single-component C3H6/C3H8 adsorption isotherms and computational simulations showed the typical nonporous-to-porous structural transformations for 1', in which C3H6 exhibits a significantly lower threshold pressure, and the typical small-pore-to-large-pore structural transformations for 2', in which C3H6 exhibits a slightly lower threshold pressure. Mixture column breakthrough experiments showed that the C3H6/C3H8 separation performances of 2' are obviously better than those of 1', because the latter cannot adsorb C3H6 below the threshold pressure for pore opening, and the pore opened by C3H6 can adsorb C3H8.

Electrochemical Exfoliation of Pillared-Layer Metal-Organic Framework to Boost the Oxygen Evolution Reaction.

Two-dimensional (2D) materials and ultrathin nanosheets are advantageous for elevating the catalysis performance and elucidating the catalysis mechanism of heterogeneous catalysts, but they are mostly restricted to inorganic or organic materials based on covalent bonds. We report an electrochemical/chemical exfoliation strategy for synthesizing metal-organic 2D materials based on coordination bonds. A catechol functionalized ligand is used as the redox active pillar to construct a pillared-layer framework. When the 3D pillared-layer MOF serves as an electrocatalyst for water oxidation (pH 13), the pillar ligands can be oxidized in situ and removed. The remaining ultrathin (2 nm) nanosheets of the metal-organic layers are an efficient catalyst with overpotentials as low as 211 mV at 10 mA cm-2 and a turnover frequency as high as 30 s-1 at an overpotential of 300 mV.

[Clinical and experimental study on wuling pill in treating gestation period intrahepatic cholestasis].

OBJECTIVE: To evaluate the curative effect of Wuling pill (WLP), a traditional Chinese patent medicine, in treating gestation period intrahepatic cholestasis (GPI). METHODS: In the clinical study, 90 GPI patients were divided into the treated group treated by conventional therapy plus WLP and the control group treated by conventional therapy plus compound Yiganling (YGL) with a ratio of 2:1. Clinical symptoms and accouchement condition were observed. Levels of cholyglycine acid (CGA), total and direct bilirubin (TB and DB), alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP) were detected before and after treatment. For the experimental study, GPI rat model was induced by injecting estradiol benzoate to pregnant Wistar rats. The model rats in the treated group were administrated with WLP by gastro-perfusion and those in the control group, were administrated with YGL. Levels of CGA,TB,DB, ALT, AST and ALP in the mother and fetal rats, as well as in the amnionic fluid were determined. Besides, the volume of bile excreted by the mother rats was observed. RESULTS: In clinical trials, the markedly effective rate in the treated group (47 cases, 78.3% ) was higher than that in the control group (15 cases, 50%, chi2 = 7.17286, P < 0.01). Levels of blood CGA, TB, ALT and AST were all decreased in both groups after treatment, but WLP showed a better efficacy than YGL (P < 0.05) in lowering CGA, ALT and AST. Moreover, the occurrence of meconium contaminated amnionic fluid and premature delivery were lower, while weight and Apgar grade of newborn babies were higher in the treated group than those in the control group. In animal experiment, WLP showed significant effects in decreasing CGA level in amniotic fluid, and in blood of the mother and fetal rats. In addition, it could also decrease the levels of bilirubin, ALT and AST, and promote the bile excretion to reduce CGA concentration in bile. All the above effects showed a dose-dependent pattern. CONCLUSION: WLP could effectively lower the serum bile acid, improve the hepatic function and better the pregnant outcome in treating GPI.

Effect of intermittent injection of recombinant human parathyroid hormone on bone histomorphometry of ovariectomized rats.

AIM: To observe the effect of intermittent parathyroid hormone (PTH) administration on bone histomorphology of relatively old ovariectomized rats. METHODS: The 6-month-old female SD rats were randomly divided into 5 groups: (1) sham-operated for baseline (ShamB, n=5), (2)ovariectomized for baseline (OVXB, n=6), (3) Sham-operated for end point (ShamE, n=6), (4) ovariectomized for end point (OVXE, n=6), (5) ovariectomized for PTH treatment (OVXEP, n=6). ShamB and OVXB rats were sacrificed 3 months after operation, ShamE, OVXE and OVXEP rats were sacrificed 4.5 months after operation. During 3-4.5 months after operation, OVXEP rats received daily subcutaneous injection of rhPTH1-84, while ShamE and OVXE received vehicle injection. The proximal tibiae of all rats were processed without decalcification for quantitative bone histomorphometry. RESULTS: The percent trabecular area (TbAr) of OVXEP was significantly greater than that of OVXE (P <0.05), and was similar to that of OVXB (P >0.05), but was smaller than that of ShamE (P <0.05); the trabecular thickness (TbTh) of OVXEP was thicker than any other group (all, P <0.05); the trabecular number (TbN) of OVXEP was only slightly higher than that of OVXE; the percent labeled perimeter (LPm), mineral apposition rate (MAR) and bone formation rate with bone area as referent (BFR/BV) of OVXEP were all higher than those of ShamE and OVXE respectively (P <0.01), whereas the osteoclast number (N of Oc) of OVXEP was similar to those of ShamE and OVXE (P >0.05). CONCLUSION: Short-term intermittent injection of rhPTH1-84 can prevent further bone loss in 9-month-old rats 3 months after ovariectomy, the mechanisms of this therapy are that PTH could increase TbTh while not alter TbN, and promote bone-forming activity while not influence bone-resorptive activity.