Gansui-Banxia Decoction extraction inhibits MDSCs accumulation via AKT /STAT3/ERK signaling pathways to regulate antitumor immunity in C57bl/6 mice.

BACKGROUND: Gansui-Banxia Decoction (GSBXD) is a classic formula of traditional Chinese medical (TCM) sage Zhang Zhongjing to treat stagnation of evil heat and obstruction of qi. At present GSBXD is wildly used to treat cancerous ascites, pleural effusion, peritoneal effusion, pericardial effusion, cranial cavity effusion and several types of cancers, such as hepatocellular carcinoma (HCC) and esophageal cancer. Myeloid-derived suppressor cells (MDSCs) are a kind of immature and heterogeneous cells which can suppress lymphocytes activation by forming a suppressive environment. MDSCs accumulation in peripheral blood and tumors are closely related to the cancer stage and low survival rate of clinical patients. The antitumor immune effect of GSBXD has not received widespread attention. PURPOSE: To investigate the effects of GSBXD on MDSCs accumulation and the mediators including AKT/STAT3/ERK signaling pathways. METHODS: The chemical components of GSBXD were analyzed by UHPLC-MS, and the putative pathways of GSBXD based on Network pharmacology were predicted. Mice were vaccinated with Hepatoma 22 (H22) to establish tumor growth model, which were then administrated with GSBXD ethanol extraction (0.49 mg/kg/day, 1.75 mg/kg/day), sorafenib (60 mg/kg) or saline for 14 days. The cell morphology was evaluated by hematoxylin and eosin (H&E) staining, and immunity cells were determined through flowcytometry analysis. The levels of cytokines production in blood were evaluated by using ELISA kits. STAT3, ERK and AKT/mTOR signaling transduction associated proteins were determined by Western blot. RESULTS: GSBXD could inhibit tumor growth and splenomegaly in H22 tumor model mice. Importantly, GSBXD reduced MDSCs accumulation and differentiation, and inhibited proliferation of F4/80+ CD11b+ macrophages and apoptosis of T cells and B cells, and increased the percentage of CD 3- NK1.1+ NK cells. To better understand the active component of GSBXD, the ethanol-extraction powdered GSBXD was prepared and analyzed by UHPLC-MS. Combined with these main chemical compounds, we predicted that the anti-tumor effect of GSBXD mainly mediated PI3K-AKT and RAS-MAPK signal pathways based on Network Pharmacology. Western blot analysis of tumor tissues and MDSCs cells demonstrated that phosphorylation of AKT, ERK and STAT3 were significantly reduced, specially the activation of ERK. The levels of IL-1ß and IFN-γ were significantly decreased by ELISA analysis. CONCLUSION: GSBXD exhibited antitumor immune activity by reducing the accumulation of MDSCs in vivo, which is possible via down-regulation of AKT/STAT3/ERK signaling pathway and suppression of IL-1ß and IFN-γ.

Matrine-Type Alkaloids from the Seeds of Sophora alopecuroides and Their Potential Anti-inflammatory Activities.

Three new matrine-type alkaloids, 8ß-hydroxyoxysophoridine (1), 9ß-hydroxysophoridine (2), 9ß-hydroxyisosophocarpine (3), together with one known analog, 11,12-dehydromatrine (4), were isolated from the seeds of Sophora alopecuroides L. The structures of new compounds were elucidated using extensive spectroscopic techniques including the experimental and calculated ECD data. The anti-inflammatory activities of all the isolates on NO production in RAW 264.7 cells stimulated by lipopolysaccharide were evaluated. Among them, 8ß-hydroxyoxysophoridine (1) showed a significant inhibitory effect with an IC50 value of 18.26 µM.

Astragalus mongholicus Bunge and Panax notoginseng (Burkill) F.H. Chen Formula for Renal Injury in Diabetic Nephropathy-In Vivo and In Vitro Evidence for Autophagy Regulation.

BACKGROUND: Diabetic nephropathy (DN) is a serious complication of diabetes mellitus (DM) with limited treatment options. DN leads to progressive renal failure and accelerates rapidly into end-stage renal disease. Astragalus mongholicus Bunge and Panax notoginseng (Burkill) F.H. Chen formula (APF) is a traditional Chinese medicine (TCM) formula widely used to treat chronic kidney diseases (CKD) in the clinic in the southwest of China. The aim of this study is to explore how APF and its related TCM theory work on DN and whether mTOR/PINK1/Parkin signaling plays a part in this process. METHODS: HPLC was used for preliminary chemical analysis and quantitative analysis of the five components of APF. An in vivo autophagy deficiency model was established in C57BL/6 mice by streptozocin (STZ) combined with a high-fat and high-sugar diet, while the in vitro autophagy deficiency model was induced with high glucose (HG) in renal mesangial cells (RMCs). Renal histopathology staining was performed to investigate the extents of inflammation and injury. Real time-PCR and Western blotting techniques were utilized to assess autophagy-related proteins. RESULTS: APF significantly ameliorated renal injury in DN mice, specifically restoring blood urea nitrogen, serum creatinine, and 24-hour albuminuria. APF also reduced the mRNA and protein expressions of TNFα, IL-1ß, and IL-6 in STZ-induced DN mice. Furthermore, APF improved the autophagy deficiency induced by STZ in vivo or HG in vitro, as revealed by changes in the expressions of mTOR, PINK1, Parkin, Beclin 1, p62, and LC3B. Notably, inhibition of autophagy with 3-methyladenine in APF-treated RMCs aggravated cellular damage and altered mTOR/PINK1/Parkin signaling, indicating that APF rescued HG damage through promoting autophagy. CONCLUSION: APF may protect the kidneys from inflammation injuries in DN by upregulating autophagy via suppressing mTOR and activating PINK1/Parkin signaling. This experimental evidence strongly supports APF as a potential option for the prevention and treatment of DN.

Curcumin relieved cisplatin-induced kidney inflammation through inhibiting Mincle-maintained M1 macrophage phenotype.

BACKGROUND: Acute kidney injury (AKI) is a common kidney disease with a high risk of death and can develop into chronic kidney disease (CKD) and renal failure eventually. Curcumin, an herbal supplement, has been reported exhibiting a renoprotective role in AKI. However, the underlying mechanism is largely unclear. PURPOSE: Recent research showed that Mincle (Macrophage-inducible C-type lectin) maintained M1 macrophage polarization, which plays a key role in kidney injury of AKI through up-regulating the expression and secretion of inflammatory cytokines. Here, we investigated the effects of Curcumin on Mincle expression and macrophage polarization in vitro using lipopolysaccharide (LPS) induced macrophage inflammatory cell model and in vivo using a cisplatin induced murine AKI (cis-AKI) model. METHODS: Cell activation, inflammatory cytokines expression and secretion, protein levels, macrophage polarization and renal pathology were analyzed. RESULTS: Our results showed that Curcumin markedly reduced the mRNA expression and secretion of IL-1ß, IL-6, TNFα and MCP-1 in LPS stimulated RAW264.7 cell and the supernatant. The same results were found in Curcumin treated cis-AKI kidney and blood. The data also demonstrated that Curcumin remarkably down-regulated mRNA expression and protein level of Mincle in cis-AKI kidney and also reduced expression of iNOS (M1 macrophage marker) as well as inhibited the activation of Syk and NF-kB. Interestingly, although Mincle deletion in RAW264.7 cell largely decreased the LPS-induced protein level of iNOS, Curcumin cannot further reduce expression of iNOS without Mincle, indicating that Curcumin inhibits M1 macrophage with a Mincle-dependent pattern. Furthermore, flow cytometry results showed that Curcumin significantly decreased the iNOS positive macrophages and increased the CD206 (M2 macrophage marker) positive macrophages in vivo and in vitro. CONCLUSION: Our findings prove that Curcumin protects kidney from cisplatin induced AKI through inhibiting Mincle maintained M1 macrophage phenotype, that may provide a specific renoprotection mechanism for Curcumin to develop it as a new therapeutic candidate for AKI.

Pharmacokinetic and Pharmacodynamics of Self-Assembled Cubic Liquid Crystalline Nanoparticle Gel After Transdermal Administration.

BACKGROUND The aim of this study was to assess the pharmacokinetics after transdermal administration by a novel skin microdialysis technology in rats. The guinea pig model was established by investigating the pharmacodynamics. MATERIAL AND METHODS Three different agents were given after hair removal, and the samples were extracted by microdialysis and detected by HPLC. Subcutaneous/plasma concentration-time curves of the 3 different agents were analyzed and the pharmacokinetic parameters were calculated. The SS-04B UV light therapy instrument was used in the modeling. Changes in melanin index and histopathology were observed with HE staining. RESULTS The increment and decrement results showed that the concentration had no significant effect on drug recovery both in vivo and in vitro. After the paeonol cubic liquid crystalline nanoparticles gel (PAE-LCNPs) was administered, the maximum peak time (tmax) of paeonol skin concentration appeared at 2.42±0.20 h, the maximum skin concentration Cmax was (926±105) ng/ml, and the area under the curve AUC0-8 was (8056±954) ng/h/ml. The tmax was shortened much more than in the other groups, and the performance of PAE-LCNPs targeting was good. Pharmacodynamic results showed that PAE-LCNPs can reduce melanocytes and reduce the melanin index, proving its utility in the treatment of melanin deposition. CONCLUSIONS The skin microdialysis study indicated PAE-LCNPs have good transdermal permeability and efficacy. Pharmacological experiments based on the study found that the topical pigmentation model of guinea pigs showed a better therapeutic effect.

Cuscuta chinensis seeds water extraction protecting murine osteoblastic MC3T3-E1 cells against tertiary butyl hydroperoxide induced injury.

ETHNOPHARMACOLOGICAL RELEVANCE: Cuscuta chinensis (C. chinensis) is a well-known traditional Chinese herb that has been used to treat heart disease, diabetes, liver injury, cancer, and aging. MATERIALS AND METHODS: Murine osteoblastic MC3T3-E1 cells were treated with various concentrations of C. chinensis water extraction at different time intervals. The antioxidant effect of C. chinensis on MC3T3-E1 cells was evaluated using MTT and TUNEL assays. The effect of C. chinensis on cell cycle was analyzed by flow cytometry with propidium iodide. Lipid peroxidation was measured by the HPLC method. The cellular redox status was determined from the reduced glutathione to oxidized glutathione ratio (GSH/GSSG) and the enzymes involved in glutathione metabolism, including glutathione reductase (GR), Glutathione S-transferase (GST), and Glucose-6-phosphate dehydrogenase (G6PD). The changes in relative mitochondrial transmembrane potential (ΔΨm) in the MC3T3-E1 cells were analyzed with rhodamine 123 staining. Western blot analysis was used to evaluate the levels of cytochrome c (cyto c), Bax, Bcl-2, caspase 3, Sirt3, and IDH2 expressions. RESULTS: The C. chinensis water extraction protects tertiary butyl hydroperoxide (TBHP)-treated MC3T3-E1 cells from death in a dose-dependent manner. C. chinensis treatment significantly inhibited the reactive oxygen species (ROS) generation, malondialdehyde (MDA) production, and increased the activity of superoxide dismutase (SOD), GR, GST, and G6PD. The release of cyto c from mitochondria was reduced by C. chinensis, which increased the expression of antiapoptotic IDH2, Sirt3, and Bcl-2 and decreased the expression of Bax, cyto c, and caspase 3. CONCLUSIONS: C. chinensis modulated the oxidative stress-induced apoptosis in MC3T3-E1 cells, probably due to its antioxidant activity and functioning via mitochondria-dependent pathways.