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.

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.

[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.