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.

Perspectives of herbs and their natural compounds, and herb formulas on treating diverse diseases through regulating complicated JAK/STAT signaling.

JAK/STAT signaling pathways are closely associated with multiple biological processes involved in cell proliferation, apoptosis, inflammation, differentiation, immune response, and epigenetics. Abnormal activation of the STAT pathway can contribute to disease progressions under various conditions. Moreover, tofacitinib and baricitinib as the JAK/STAT inhibitors have been recently approved by the FDA for rheumatology disease treatment. Therefore, influences on the STAT signaling pathway have potential and perspective approaches for diverse diseases. Chinese herbs in traditional Chinese medicine (TCM), which are widespread throughout China, are the gold resources of China and have been extensively used for treating multiple diseases for thousands of years. However, Chinese herbs and herb formulas are characterized by complicated components, resulting in various targets and pathways in treating diseases, which limits their approval and applications. With the development of chemistry and pharmacology, active ingredients of TCM and herbs and underlying mechanisms have been further identified and confirmed by pharmacists and chemists, which improved, to some extent, awkward limitations, approval, and applications regarding TCM and herbs. In this review, we summarized various herbs, herb formulas, natural compounds, and phytochemicals isolated from herbs that have the potential for regulating multiple biological processes via modulation of the JAK/STAT signaling pathway based on the published work. Our study will provide support for revealing TCM, their active compounds that treat diseases, and the underlying mechanism, further improving the rapid spread of TCM to the world.

Identification of Novel Cannabinoid CB2 Receptor Agonists from Botanical Compounds and Preliminary Evaluation of Their Anti-Osteoporotic Effects.

As cannabinoid CB2 receptors (CB2R) possess various pharmacological effects-including anti-epilepsy, analgesia, anti-inflammation, anti-fibrosis, and regulation of bone metabolism-without the psychoactive side effects induced by cannabinoid CB1R activation, they have become the focus of research and development of new target drugs in recent years. The present study was intended to (1) establish a double luciferase screening system for a CB2R modulator; (2) validate the agonistic activities of the screened compounds on CB2R by determining cAMP accumulation using HEK293 cells that are stably expressing CB2R; (3) predict the binding affinity between ligands and CB2 receptors and characterize the binding modes using molecular docking; (4) analyze the CB2 receptors-ligand complex stability, conformational behavior, and interaction using molecular dynamics; and (5) evaluate the regulatory effects of the screened compounds on bone metabolism in osteoblasts and osteoclasts. The results demonstrated that the screening system had good stability and was able to screen cannabinoid CB2R modulators from botanical compounds. Altogether, nine CB2R agonists were identified by screening from 69 botanical compounds, and these CB2R agonists exhibited remarkable inhibitory effects on cAMP accumulation and good affinity to CB2R, as evidenced by the molecular docking and molecular dynamics. Five of the nine CB2R agonists could stimulate osteoblastic bone formation and inhibit osteoclastic bone resorption. All these findings may provide useful clues for the development of novel anti-osteoporotic drugs and help elucidate the mechanism underlying the biological activities of CB2R agonists identified from the botanical materials.

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.

Anti-Osteoporotic Activity of an Edible Traditional Chinese Medicine Cistanche deserticola on Bone Metabolism of Ovariectomized Rats Through RANKL/RANK/TRAF6-Mediated Signaling Pathways.

Given the limitations of existing therapeutic agents for treatment of postmenopausal osteoporosis, there still remains a need for more options with both efficacy and less adverse effects. Cistanche deserticola Y. C. Ma is known as a popular tonic herb traditionally used to treatment deficiency of kidney energy including muscle weakness in minority area of Asian counties. Based on the theory of "kidney dominate bone," an ovariectomized (OVX) rat model of postmenopausal osteoporosis was used to evaluate the therapeutic effect of C. deserticola extract (CDE) on bone loss. Forty eight female Sprague-Dawley rats, aged about 12 weeks, were randomly assigned into six groups including sham group orally administrated with 0.5% carboxymethyl cellulose sodium (CMC-Na) (sham), positive group treated with 1 mg/kg of estradiol valerate (EV), low, moderate, and high dosage groups orally administrated with 200, 400, and 800 mg/kg/day of CDE, respectively. After 3 months of continuous intervention, CDE exhibited significant anti-osteoporotic activity evidenced by the enhanced total bone mineral density, ameliorated bone microarchitecture; increased alkaline phosphatase activity; decreased deoxypyridinoline, cathepsin K, tartrate-resistant acid phosphatase, and malondialdehyde levels; whereas the body, uterus, and vagina weights in OVX rats were not influenced by CDE intervention. In addition, a seemed contradictory phenomenon on levels of calcium and phosphorus between OVX and sham rats were observed and elucidated. Mechanistically, CDE significantly down-regulated the levels of TRAF6, RANKL, RANK, NF-κB, IKKß, NFAT2, and up-regulated the phosphatidylinositol 3-kinase (PI3K), AKT, osteoprotegerin, and c-Fos expressions, which implied CDE could suppress RANKL/RANK-induced activation of downstream NF-κB and PI3K/AKT pathways, and ultimately, preventing activity of the key osteoclastogenic proteins NFAT2 and c-Fos. All of the data suggested CDE possessed potential anti-osteoporotic activity and this effect was, at least in part, involved in modulation of RANKL/RANK/TRAF6-mediated NF-κB and PI3K/AKT signaling as well as c-Fos and NFAT2 levels. Therefore, CDE may represent a useful promising remedy candidate for treatment of postmenopausal osteoporosis.

Kaempferitrin prevents bone lost in ovariectomized rats.

BACKGROUND: Podocarpium podocarpum (DC.), an edible and medicinal plant popularly used for the treatment of bruises and fracture in Chinese folk medicine, has been proved to possess significant antiosteoporotic effect in our latest research. PURPOSE: Our study aimed to investigate the in vitro and in vivo antiosteoporotic effect of kaempfertrin (KN), a principal flavonoid in P. podocarpum obtained through bio-guided isolation. METHODS: An ovariectomized (OVX) rat model of osteoporosis as well as in vitro osteoblast and osteoclast cell lines were employed to evaluate the antiosteoporotic potency of KN. RESULTS: KN significantly improved the bone mass and microarchitecture in OVX rats, with little estrogen-like side effect compared with estradiol valerate. KN also exhibited stimulatory effect on osteoblastic cells and inhibitory action on osteoclastic cells, which down-regulated the phosphorylation level of I-κB. CONCLUSION: KN possessed significant antiosteoporotic activity. Combined with its limited estrogen-like side effect, KN can be regarded as an idealistic antiosteoporotic candidate for human osteoporosis diseases.

Antiosteoporotic activity and constituents of Podocarpium podocarpum.

Our study aimed to investigate the antiosteoporotic properties of the ethanol extract of Podocarpium podocarpum (DC.) Yang et Huang (PE) in ovariectomized (OVX) rats and to characterize the active constituents. As a result, PE significantly inhibited the increased urinary Ca excretion and activity of bone resorption markers including tartrate-resistant acid phosphatase (TRAP), deoxypyridinoline crosslinks and cathepsin K in OVX rats, whereas exhibited little effects on the body, uterus and vagina weight. Detailed micro-CT analysis showed that PE notably enhanced bone quality, with increased bone mineral content (BMC), bone volume fraction (BVF), connectivity density (CD), tissue mineral content (TMC), tissue mineral density (TMD) and trabecular number (Tb. N), and decreased trabecular separation (Tb. Sp), in OVX animal. Those findings implied that PE had notable antiosteoporotic effect, especially effective in preventing bone resorption, with little side-effects on reproductive tissue. Further chemical investigation led to the isolation of 17 flavonoids, most of which showed significantly stimulatory effect on osteoblastic proliferation, ALP activity and mineralized nodes formation as well as inhibitory effect on osteoclastic TRAP activity in osteoblastic and osteoclastic cells. Our results indicated that PE, with abundant flavonoids, had remarkable antiosteoporotic activity and therefore can be a promising candidate for the treatment of postmenopausal osteoporosis induced by estrogen deficiency through herbal remedy.

[Identification of Volatile Chemical Constituents from Hui Formula "Ha Hei Lili" by GC-MS].

OBJECTIVE: The volatile components of the Hui formula "Ha Hei Lili" were extracted by steam distillation extraction (SD) and supercritical CO2 fluid extraction, and the structures were analyzed and identified by GC-MS. METHODS: The GC-MS conditions were set as follows: Rxi-5Sil MS quartz capillary column (30 m x 0.25 mm, 0.25 µm), the initial temperature of 50 degrees C to keep 1 min, to 10 degrees C/min heating to 120 degrees C, maintained 3 min, then to 3 degrees C/min heating to 200 degrees C, maintained 3 min, and then to 5 degreesC/min heating to 290 degrees C, maintained until completion of analysis; helium as the carrier gas, column flow rate 1.0 ml/min, split ratio 25: 1, inlet temperature 250 degrees C, EI ionization source 70 eV, ion source temperature 230 degrees C, scan range of m/z 35 - 500. RESULTS: Yield of volatile oil were 0.21% and 5.44% extracted by SD and SFE methods, respectively; and for SD method, 36 kinds of compounds were identified, accounted for 87.02% of total mass of volatile oil; for SFE method, 38 kinds of constituents were identified, accounted for 97.47% of total mass of volatile oil. CONCLUSION: The type of constituents contained in the volatile oil extracted by SD and SFE methods are totally different; and GC-MS can be used to identify the structures and relative content of volatile components, the results of this study can provide an experimental basis for development and utilization of Hui formula "Ha Hei Lili".

Anti-arthritic activity of Xanthium strumarium L. extract on complete Freund׳s adjuvant induced arthritis in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Xanthium strumarium L. fruit (Xanthiu fruit) has been traditionally used as a medicinal herb in China for the treatment of many ailments including rheumatoid arthritis. However, the anti-arthritic activity of Xanthium strumarium fruit has still not been demonstrated. In the present study, we confirmed that the extract of Xanthium strumarium (EXS) prevents rheumatoid arthritis induced by Complete Freund׳s Adjuvant (CFA) in rats. MATERIALS AND METHODS: Male Wistar rats (160±10 g) were immunized by intradermal injection of 0.1 mL of CFA into the left hind metatarsal footpad. EXS was administered orally at a dose of 300 and 75 mg/kg once a day after the induction of adjuvant arthritis. Methotrexate (3 mg/kg, twice a week) was used as a positive control. Paw swelling, arthritic score, body weight loss, spleen index, thymus index, serum cytokines, inflammatory mediators and histological change were measured. The chemical profile of EXS was analyzed by HPLC-DAD. RESULTS: We found that the EXS significantly suppressed paw swelling and arthritic score, increased body weight loss and decreased the thymus index. The overproduction of TNF-α and IL-1ß were remarkably suppressed in the serum of all EXS-treated rats, and in contrast IL-10 was markedly increased. The level of COX-2 and 5-LOX was also decreased with EXS treatment. Ten phenolic acid derivatives were identified from 14 detected peaks by HPLC-DAD with the reference substances and verified by LC-MS. CONCLUSIONS: These results suggest the potential effect of EXS as an anti-arthritis agent towards CFA-induced arthritis in rats. Xanthium strumarium has the potential to be regarded as a candidate for use in general therapeutics and as an immune-modulatory medicine in rheumatoid arthritis.

Inhibitory effects of the root extract of Litsea cubeba (lour.) pers. on adjuvant arthritis in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: The dried root of Litsea cubeba (Lour.) Pers. (Family Lauraceae) has long been used as a folk remedy in Traditional Chinese Medicine (TCM) and Dai Ethnopharmacy for the treatment of rheumatic diseases in southwestern China. AIM OF THE STUDY: This study investigated the preventive efficacy of Litsea cubeba root in treating rheumatoid arthritis using Freund's complete adjuvant (CFA) induced arthritis (AA) in rat model. MATERIALS AND METHODS: Arthritis was induced in male Wistar rats by immunization with CFA. Ethanol extract (EELC) and water extract (WELC) of Litsea cubeba root both at 50mg/kg and 200mg/kg were orally administered from a day after the induction of arthritis. Paw swelling, arthritic score, body weight growth rate, index of thymus and spleen were observed, and the production of TNF-α, IL-1ß, IL-6 and IL-10 in serum were measured by enzyme-linked immunosorbent assay. The expression levels of inflammatory enzymes like cyclooxygenase and lipoxygenase were also measured by enzyme-linked immunosorbent assay. Moreover, histological changes in the ankle joint were analyzed in AA rats. RESULTS: Both EELC and WELC significantly suppressed paw swelling and arthritic score, increased the loss in body weight and decreased the index of thymus. Histopathological improvement in joint architecture was also observed in EELC, WELC-treated AA rats. The expression levels of cyclooxygenase-2 (COX-2) and 5-lipoxygenase (5-LOX) were decreased on treatment with EELC and WELC. Furthermore, the overproduction of TNF-α, IL-1ß and IL-6 were remarkably attenuated in serum of all Litsea cubeba-treated rats, however, IL-10 was markedly increased at doses of 50mg/kg of EELC and WELC. CONCLUSIONS: These results indicate that extract of Litsea cubeba root significantly attenuates adjuvant arthritis in rats by decreasing the levels of TNF-α, IL-1ß and IL-6 and increasing of IL-10 in serum as well as down-regulate the levels of inflammatory enzymes such as COX-2 and 5-LOX. This suggests that Litsea cubeba root might be used as a therapeutic agent for the treatment of human arthritis.

[Study on molecular recognition technology in active constituents extracted and isolated from Aconitum pendulum].

OBJECTIVE: To investigate CD molecular recognition technology applied in active constituents extracted and isolated from traditional Chinese medicine--Aconitum pendulum. METHODS: The inclusion constant and form probability of the inclusion complex of Aconitum pendulum with p-CD was calculated by UV spectra method. The active constituents of Aconitum pendulum were extracted and isolated by molecular recognition technology. The inclusion complex was identified by UV. The chemical constituents of Aconitum pendulum and inclusion complex was determined by HPLC. The analgesic effects of inclusion complex was investigated by experiment of intraperitoneal injection of acetic acid in rats. RESULTS: The inclusion complex was identified and confirmed by UV spectra method, the chemical components of inclusion complex were simple, and the content of active constituents increased significantly, the analgesic effects of inclusion complex was well. CONCLUSION: The molecular recognition technology can be used for extracting and isolating active constituents of Aconitum pendulum, and the effects are obvious.

[Optimization of extraction process of total alkaloids from radix of Aconitum flavum using response surface methodology].

OBJECTIVE: To study the technological parameters of the extraction process of total alkaloids from Radix of Aconitum flavum. METHODS: Using response surface methodology (RSM) of three factors and three levels to optimize ethanol heat reflux applied for alkaloids extraction from Radix of Aconitum flavum. RESULTS: A quadratic polynomial mathematical model with good correlation was constructed and employed to the optimization. The optimal extraction conditions were as follows: the extraction time was 1.72 h, the concentration of ethanol was 64.00%, the ratio of ethanol to raw material was 8.18 mL/g, under which the predictive value of the rate of extraction yield of alkaloids was 0.526% and the measured value was 0. 521%, respectively, and there was a good agreement with their predicted values. CONCLUSION: This reveals the good predictability of the mathematical model, the selected process also has a good reproducibility.