Study on the Antitumor Mechanism of Tanshinone IIA In Vivo and In Vitro through the Regulation of PERK-ATF4-HSPA5 Pathway-Mediated Ferroptosis.

As a traditional Chinese medicine, Salvia miltiorrhiza Bunge was first recorded in the Shennong Materia Medica Classic and is widely used to treat "the accumulation of symptoms and masses". The main active ingredient of Salvia miltiorrhiza Bunge, Tanshinone IIA (TIIA), has shown anti-inflammatory, antitumor, antifibrosis, antibacterial, and antioxidative activities, etc. In this study, the results showed that TIIA could inhibit the proliferation and migration of HepG2 cells and downregulate glutathione (GSH) and Glutathione Peroxidase 4 (GPX4) levels; besides, TIIA induced the production of Reactive Oxygen Species (ROS), and upregulated the total iron content. Based on network pharmacology analysis, the antitumor effect of TIIA was found to be focused on the endoplasmic reticulum (ER)-mediated ferroptosis signaling pathway, with protein kinase R (PKR)-like ER kinase (PERK)-activating transcription factor 4 (ATF4)-heat shock 70 kDa protein 5 (HSPA5) as the main pathway. Herein, TIIA showed typical ferroptosis characteristics, and a ferroptosis inhibitor (ferrostatin-1) was used to verify the effect. The antitumor effects of TIIA, occurring through the inhibition of the PERK-ATF4-HSPA5 pathway, were further observed in vivo as significantly inhibited tumor growth and the improved pathological morphology of tumor tissue in H22-bearing mice. In summary, the antitumor mechanism of TIIA might be related to the downregulation of the activation of PERK-ATF4-HSPA5 pathway-mediated ferroptosis.

Effects of Shenling Baizhu powder on intestinal microflora metabolites and liver mitochondrial energy metabolism in nonalcoholic fatty liver mice.

Background & purpose: Non-alcoholic fatty liver disease (NAFLD) is characterised by the excessive accumulation of triglycerides in the liver. Shenling Baizhu powder (SLBZP) is formulated from various natural medicinal plants that protect the liver and are used to treat intestinal diseases. SLBZP improves the symptoms of NAFLD. However, its mechanism of action remains unclear. Herein, we investigated the ameliorative effect of SLBZP on model mice with high-fat-diet (HFD)-induced NAFLD. Additionally, we evaluated the impact of SLBZP on the intestinal flora and its metabolites and mitochondrial energy metabolism in NAFLD. Methods: We used HFD to establish a mouse model of NAFLD. Different drug interventions were administered. We measured serum biochemical indices. Liver sections were visualised with hematoxylin-eosin and oil red O staining. 16S rDNA amplicon sequencing technology was used to analyse the diversity and abundance of the intestinal flora. Short-chain fatty acids (SCFAs) in the intestinal contents were detected using GC-MS. Liver tissue was sampled to detect mitochondrial membrane functional indices. Western blotting was used to determine the levels of mitochondrial pathway-related proteins, namely, uncoupling protein 2 (UCP2), adenosine monophosphate-activated protein kinase (AMPK) and inhibitory factor 1 (IF1) of F1Fo ATP synthesis/hydrolase, in the liver. Results: The spleen-invigorating classic recipe of SLBZP reduced liver lipid deposition in mice with HFD-induced NAFLD. Additionally, SCFAs produced by intestinal flora metabolism regulated the UCP2/AMPK/IF1 signalling pathway involved in liver mitochondrial energy metabolism to improve the liver mitochondrial membrane permeability, respiratory state and oxidative phosphorylation efficiency of mice with NAFLD. Finally, SLBZP increased the liver ATP level. Conclusion: Our results suggest that the therapeutic effect of SLBZP on NAFLD is related to the regulation of hepatic mitochondrial energy metabolism by intestinal flora and its metabolites and is possibly associated with the UCP2/AMPK/IF1 signalling pathway.

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.

Role of the p-Coumaroyl Moiety in the Antioxidant and Cytoprotective Effects of Flavonoid Glycosides: Comparison of Astragalin and Tiliroside.

The aim of this study was to explore the role of p-coumaroyl in the antioxidant and cytoprotective effects of flavonoid glycosides. The antioxidant effects of astragalin and tiliroside were compared using ferric ion reducing antioxidant power, DPPH• scavenging, ABTS•⁺ scavenging, •O2- scavenging, and Fe2+-chelating assays. The results of these assays revealed that astragalin and tiliroside both exhibited dose-dependent activities; however, tiliroside exhibited lower IC50 values than astragalin. In the Fe2+-chelating assay, tiliroside gave a larger shoulder-peak at 510 nm than astragalin, and was also found to be darker in color. Both of these compounds were subsequently evaluated in a Fenton-induced mesenchymal stem cell (MSC) damaged assay, where tiliroside performed more effectively as a cytoprotective agent than astragalin. Tiliroside bearing a 6''-O-p-coumaroyl moiety exhibits higher antioxidant and cytoprotective effects than astragalin. The 6''-O-p-coumaroyl moiety of tiliroside not only enhances the possibility of electron-transfer and hydrogen-atom-transfer-based multi-pathways, but also enhances the likelihood of Fe-chelating. The p-coumaroylation of the 6"-OH position could therefore be regarded as a potential approach for improving the antioxidant and cytoprotective effects of flavonoid glycosides in MSC implantation therapy.

[Preclinical evaluation of pseudoallergic reactions on Chinese herbal injections: study on animal strain and gender difference].

Pseudoallergic reactions occured after the first administration of patients, and the pathogenic mechanisms of them were different from the allergic reactions which needed excitation after antigen sensitization. To provide a basis for evaluation, clinical use and drug development of pseudoallergic reactions, the models were established by two kinds of Chinese herbal injections (CHI) both on different strain or gender mice. With the use of ICR, Kunming, BALB/C, C57 mice, pseudoallergic tests of two CHI were conducted to compare the sensitivity of four strains mice, and compared the differences in male and female animals. Test substances contain 0.8% Evans blue (EB) were intravenously injected into different strain and gender mice. Scores of ear blue staining and quantitation of ear EB exudation were the parameters for pseudoallergic reaction. Results of strain difference indicated that both CHI A and B could cause severe pseudoallergic reactions indicated by obvious vascular hyperpermeability on ICR mice. The pseudoallergic reactions in ICR mice are more obvious under the the same dose of injection, which stated the sensibility of ICR mice. And the reactions of KM mice and BALB/C mice were slightly reduced which compared to ICR mice, even alomost nothing on C57 mice. Comparison results of gender difference showed that one CHI was not have significant difference in male and female animals, but male animals were more susceptible than females on another CHI. Therefore, ICR mice were preferable experimental strain on the model of pseudoallergic reactions induced by CHI A and B. Because of female animals were easily influenced by estrous cycle, the pseudoallergic reactions induced by CHI A and B select and use male mice befittingly.