Triptolide inhibits the proinflammatory potential of myeloid-derived suppressor cells via reducing Arginase-1 in rheumatoid arthritis.

Triptolide (TPT) is widely used in the treatment of rheumatoid arthritis (RA). However, its regulatory mechanisms are not fully understood. This study demonstrated that Myeloid-derived suppressor cells (MDSCs) were expanded in both RA patients and arthritic mice. The frequency of MDSCs was correlated with RA disease severity and T helper 17 (Th17) responses. MDSCs from RA patients promoted the polarization of Th17 cells in vitro, which could be substantially attenuated by blocking arginase-1 (Arg-1). TPT inhibited the differentiation of MDSCs, particularly the monocytic MDSCs (M-MDSCs) subsets, as well as the expression of Arg-1 in a dose dependent manner. Alongside, TPT treatment reduced the potential of MDSCs to promote the polarization of IL-17+ T cell in vitro. Consistently, TPT immunotherapy alleviated adjuvant-induced arthritis (AIA) in a mice model, and reduced the frequency of MDSCs, M-MDSCs and IL-17+ T cells simultaneously. The presented data suggest a pathogenic role of MDSCs in RA and may function as a novel and effective therapeutic target for TPT in RA.

Formononetin, a bioactive isoflavonoid constituent from Astragalus membranaceus (Fisch.) Bunge, ameliorates type 1 diabetes mellitus via activation of Keap1/Nrf2 signaling pathway: An integrated study supported by network pharmacology and experimental validation.

ETHNOPHARMACOLOGICAL RELEVANCE: Type 1 diabetes mellitus (T1DM) results from insulin deficiency due to the destruction of pancreatic ß-cells. Previously, our studies showed that inhibition of Keap1/Nrf2 signaling pathway promoted the onset of T1DM, which suggests that finding drugs that can activate the Keap1/Nrf2 signaling may be a promising therapeutic strategy for the T1DM treatment. Astragalus membranaceus (Fisch.) Bunge is a common traditional Chinese medicine that has been frequently applied in Chinese clinics for the treatment of diabetes and other diseases. Formononetin (FMNT), one of the major isoflavonoid constituents isolated from this herbal medicine, possesses diverse pharmacological benefits and T1DM therapeutic potential. However, the exact molecular mechanisms underlying the action of FMNT in ameliorating T1DM have yet to be fully elucidated. AIMS OF THE STUDY: This study is to investigate the regulation of FMNT on the Keap1/Nrf2 signaling pathway to ameliorate T1DM based on network pharmacology approach combined with experimental validation. MATERIALS AND METHODS: A mouse-derived pancreatic islet ß-cell line (MIN6) was used for the in vitro studies. An alloxan (ALX)-induced T1DM model in wild-type and Nrf2 knockout (Nrf2-/-) C57BL/6J mice were established for the in vivo experiments. The protective effects of FMNT against ALX-stimulated MIN6 cell injury were evaluated using MTT, EdU, apoptosis and comet assays. The levels of blood glucose in mice were measured by using a blood monitor and test strips. The protein expression was detected by Western blot analysis. Furthermore, the binding affinity of FMNT to Keap1 was evaluated using cellular thermal shift assay (CETSA), drug affinity responsive target stability (DARTS) assay, and solvent-induced protein precipitation (SIP) assay. The interaction pattern between FMNT and Keap1 was assessed by molecular docking and molecular dynamics simulation techniques. RESULTS: Network pharmacology analysis revealed that FMNT exerted its therapeutic effect against T1DM by mainly regulating oxidative stress response-associated signaling molecules and pathways, such as Nrf2 regulating anti-oxidant/detoxification enzymes and Keap1-Nrf2 signaling pathway. The in vivo results showed that FMNT significantly deceased the ALX-induced high blood glucose levels and conversely increased the ALX-induced low insulin contents. In vitro, FMNT markedly protected MIN6 cells from ALX-induced cytotoxicity, proliferation inhibition and DNA damage and reduced the ALX-stimulated cell apoptosis. FMNT also inhibited ALX-induced overproduction of intracellular ROS to alleviate oxidative stress. In addition, FMNT could bind to Keap1 to notably activate the Keap1/Nrf2 signaling to upregulate Nrf2 expression and promote the Nrf2 translocation from the cytoplasm to the nucleus, resulting in enhancing the expression of antioxidant proteins HO-1 and NQO1. Inhibition of Keap1/Nrf2 signaling by ALX was also markedly abolished in the cells and mice exposed to FMNT. Moreover, these effects of FMNT in ameliorating T1DM were not observed in Nrf2-/- mice. CONCLUSIONS: This study demonstrates that FMNT could bind to Keap1 to activate the Keap1/Nrf2 signaling to prevent intracellular ROS overproduction, thereby attenuating ALX-induced MIN6 cell injury and ameliorating ALX-stimulated T1DM. Results from this study might provide evidence and new insight into the therapeutic effect of FMNT and indicate that FMNT is a promising candidate agent for the treatment of T1DM in clinics.

Electro-Acupuncture Alleviates Chronic Unpredictable Stress-Induced Depressive- and Anxiety-Like Behavior and Hippocampal Neuroinflammation in Rat Model of Depression.

Depression is the second leading cause of disability worldwide. The effects of clinical depression may be mediated by neuroinflammation such as activation of microglia and high levels of proinflammatory cytokines in certain brain areas. Traditional Chinese medicine techniques such as electro-acupuncture (EA) are used extensively in Asia to treat mental health disorders. However, EA has not been rigorously studied in treatment of depression. This study was designed to assess the effectiveness of EA on depressive-like behavior and explore the role of hippocampal neuroinflammation in the potential antidepressant effect of EA. In this study, we used six chronic unpredictable stressors daily in a random sequence for 10 weeks. EA were performed on "Bai-Hui" (Du-20) (+) and "Yang-Ling-Quan" (GB-34, the right side; -) acupoints by an EA apparatus (HANS Electronic Apparatus, LH202H, 2/100 Hz, 0.3 mA) for 30 min once every other day for last 4 weeks. The behavior tests including open field test and forced swimming test, which are widely used to assess depressive and anxiety-like behavior were performed on the Monday and Tuesday of the eleventh week. The results showed that 10 week of chronic unpredictable stress (CUS) caused behavioral deficits in rats and neuroinflammation in hippocampus, such as increased expression of NLRP3 inflammasome components, upregulated mRNA level of IL-1ß and the protein level of IL-1ß mature form (p17) and activation of microglia. Moreover, 4 weeks of EA treatment significantly attenuated behavioral deficits caused by CUS. EA's antidepressant effect was accompanied by markedly decreased expression of certain NLRP3 inflammasome components and matured IL-1ß. Meanwhile, EA treatment can significantly reverse CUS-induced increases in P2X7 receptor, Iba-1, IL-18, TNFα and IL-6 expression and decreases in GFAP expression. In conclusion, EA exhibited the antidepressant effect and alleviated the hippocampal neuroinflammation. These findings may provide insight into the role of hippocampal neuroinflammation in the antidepressant effect of EA.

[Effect of Electroacupuncture Stimulation of "Baihui" (GV 20) , etc. on Changes of Behavior and Plasma Ghrelin Content in Depression Rats].

OBJECTIVE: To observe the effect of electroacupuncture (EA) stimulation of "Baihui" (GV 20) + "Anmian" (EX-HN 16) and "Baihui" (GV 20) + "Zusanli" (ST 36) on behavior reactions and plasma ghrelin level in depression rats, so as to explore the correlation between its antidepressant effect and plasma ghrelin level. METHODS: A total of 45 SD rats were randomly divided into 5 groups: normal control, model, Baihui (GV 20) + Anmian (EX-HN 16), Baihui (GV 20) + Zusanli (ST 36) and medication (clomipramine) groups, with 9 rats in each group. The depression model (unpredictable chronic mild stresses, UC-MS) was established by giving the animals with higher temperature environment (45 °C, 5 min), forced ice-water swimming (0- 4 °C, 5 min) , day and night reversal environment (12 h), stroboflash stimulation (12 h), noisy stimulation (12 h), rocking-bed movement (30 min) and damp pad dwelling (6-24 h), etc. for 4 weeks. EA was applied to GV 20-EX-HN 16, and GV 20-ST 36 for 30 min once every other day for 4 weeks after modeling. For rats of the medication group, clomipramine (5 mg/kg) was given (i. p. ) once a day for 4 weeks after modeling. The forced swimming test, sucrose preference test and open field test were used to evaluate the rats depressive-like behavior. Plasma ghrelin content was assayed by ELISA. RESULTS: After exposure to UCMS for 4 weeks, the immobility time was significantly increased, and the struggling time was significantly decreased in the model group (P < 0.05, P < 0.01). In comparison with the model group, the immobility time levels were obviously decreased, while the struggling time and sucrose preference were markedly increased in the Baihui (GV 20) + Anmian (EX-HN 16) , Baihui (GV 20) + Zusanli (ST 36) and medication groups (P < 0.05, P < 0.01). No significant changes were found in the rearing times and total distance of open-field test (locomotor activity) and plasma ghrelin content among the 5 groups among all the groups (P > 0.05). No significant differences were found among the two EA and medication groups in the decreased immobility time and the increased struggling times and sucrose preference levels (P > 0.05). CONCLUSION: EA intervention can improve the depression rats' hopeless behavior of forced swimming test and anhedonia behavior (sucrose preference test) , which may be not correlated to plasma ghrelin level at the late-stages and the antidepressant effect of EA intervention.