Fufang Zhenzhu Tiaozhi (FTZ) suppression of macrophage pyroptosis: Key to stabilizing rupture-prone plaques.

BACKGROUND: Research on the Chinese herbal formula Fufang Zhenzhu Tiaozhi (FTZ) has demonstrated its effectiveness in treating hyperlipidemia and glycolipid metabolic disorders. Additionally, FTZ has shown inhibitory effects on oxidative stress, regulation of lipid metabolism, and reduction of inflammation in these conditions. However, the precise mechanisms through which FTZ modulates macrophage function in atherosclerosis remain incompletely understood. Therefore, this study aims to investigate whether FTZ can effectively stabilize rupture-prone plaques by suppressing macrophage pyroptosis and impeding the development of M1 macrophage polarization in ApoE-/- mice. METHODS: To assess the impact of FTZ on macrophage function and atherosclerosis in ApoE-/- mice, we orally administered FTZ at a dosage of 1.2 g/kg body weight daily for 14 weeks. Levels of interleukin-18 and interleukin-1ß were quantified using ELISA kits to gauge FTZ's influence on inflammation. Total cholesterol content was measured with a Cholesterol Assay Kit to evaluate FTZ's effect on lipid metabolism. Aortic tissues were stained with Oil Red O, and immunohistochemistry techniques were applied to assess atherosclerotic lesions and plaque stability. To evaluate the effects of FTZ on macrophage pyroptosis and oxidative damage, immunofluorescence staining was utilized. Additionally, we conducted an analysis of protein and mRNA expression levels of NLRP3 inflammasome-related genes and macrophage polarization-related genes using RT-PCR and western blotting techniques. RESULTS: This study illustrates the potential therapeutic effectiveness of FTZ in mitigating the severity of atherosclerosis and improving serum lipid profiles by inhibiting inflammation. The observed enhancements in atherosclerosis severity and inflammation can be attributed to the suppression of NLRP3 inflammasome activity and M1 polarization by FTZ. CONCLUSION: The current findings indicate that FTZ provides protection against atherosclerosis, positioning it as a promising candidate for novel therapies targeting atherosclerosis and related cardiovascular diseases.

The 15-hydroxyprostaglandin dehydrogenase inhibitor SW033291 ameliorates abnormal hepatic glucose metabolism through PGE2-EP4 receptor-AKT signaling in a type 2 diabetes mellitus mouse model.

Type 2 diabetes mellitus (T2DM) is associated with high rates of morbidity and mortality worldwide. Prostaglandin E2 (PGE2) is a lipid signaling molecule that can ameliorate the symptoms of some metabolic diseases, including T2DM, and improve tissue repair and regeneration. Although SW033291 can increase PGE2 levels through its action as a small molecule inhibitor of the PGE2-degrading enzyme 15-hydroxyprostaglandin dehydrogenase, its effects on T2DM remain unclear. In the present study, we evaluated whether SW033291 treatment exerts a protective effect against T2DM and explored the underlying mechanisms. A T2DM mouse model was established using a high-fat diet combined with streptozotocin treatment. Palmitic acid-treated LO2 cells were used as an insulin-resistant cell model. SW033291 treatment reduced body weight and fasting blood glucose levels as well as serum triglyceride, total cholesterol, and low-density lipoprotein cholesterol levels in vivo. In addition to ameliorating glucose and insulin tolerance, SW033291 treatment reversed the T2DM-induced decrease in glycogen synthesis and increase in gluconeogenesis in the liver. Furthermore, SW033291 administration increased hepatic glycogen synthase kinase 3 beta (GSK3ß) phosphorylation levels to promote glycogen synthesis. SW033291 treatment also inhibited gluconeogenesis by upregulating AKT serine/threonine kinase (AKT) and forkhead box O1 (FOXO1) phosphorylation and reducing glucose-6-phosphatase and phosphoenolpyruvate carboxykinase 1 expression in the livers of T2DM model mice. Additionally, SW033291 treatment improved abnormal hepatic glucose metabolism through the PGE2-EP4 receptor-AKT-GSK3ß/FOXO1 signaling pathway in vitro. These results suggest a novel role of SW033291 in improving T2DM and support its potential as a novel therapeutic agent.

FTZ promotes islet ß-cell regeneration in T1DM mice via the regulation of nuclear proliferation factors.

ETHNOPHARMACOLOGICAL RELEVANCE: Fufang-Zhenzhu-Tiaozhi capsule (FTZ), a Traditional Chinese Medicine (TCM) patent prescription commonly used in clinical practice, has a significant curative effect on hyperglycemia and hyperlipidemia. Previous studies have shown that FTZ can treat diabetes, but the effect of FTZ on ß-cell regeneration needs to be further explored in T1DM mice. AIM OF THE STUDY: The aim is to investigate the role of FTZ in promoting ß-cell regeneration in T1DM mice, and to further explore its mechanism. MATERIALS AND METHODS: C57BL/6 mice were used as control. NOD/LtJ mice were divided into the Model group and FTZ group. Oral glucose tolerance, fasting blood glucose, and fasting insulin level were measured. Immunofluorescence staining was used to detect the level of ß-cell regeneration and the composition of α-cells and ß-cells in islets. Hematoxylin and eosin staining was used to detect the infiltration degree of inflammatory cells. The apoptosis of islet cells was detected by terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end labeling. Western blotting was used to detect the expression levels of Pancreas/duodenum homeobox protein 1 (PDX-1), V-maf musculoaponeurotic fibrosarcoma oncogene homolog A (MAFA), and Neurogenin-3 (NGN3). RESULTS: FTZ could increase insulin levels and reduce the glucose level of T1DM mice and promote ß-cell regeneration. FTZ also inhibited the invasion of inflammatory cells and the islet cell apoptosis, and maintained the normal composition of islet cells, thus preserving the quantity and quality of ß-cells. Furthermore, FTZ promoting ß-cell regeneration was accompanied by increasing the expression of PDX-1, MAFA, and NGN3. CONCLUSION: FTZ can restore the insulin-secreting function of the impaired pancreatic islet, improve blood glucose level, possibly via the enhancing ß cell regeneration via upregulation of PDX-1, MAFA, and NGN3 in T1DM mice, and may be a potential therapeutic drug for T1DM.

The markers to delineate different phenotypes of macrophages related to metabolic disorders.

Macrophages have a wide variety of roles in physiological and pathological conditions, making them promising diagnostic and therapeutic targets in diseases, especially metabolic disorders, which have attracted considerable attention in recent years. Owing to their heterogeneity and polarization, the phenotypes and functions of macrophages related to metabolic disorders are diverse and complicated. In the past three decades, the rapid progress of macrophage research has benefited from the emergence of specific molecular markers to delineate different phenotypes of macrophages and elucidate their role in metabolic disorders. In this review, we analyze the functions and applications of commonly used and novel markers of macrophages related to metabolic disorders, facilitating the better use of these macrophage markers in metabolic disorder research.

Role of prostaglandin E2 in macrophage polarization: Insights into atherosclerosis.

Atherosclerosis, a trigger of cardiovascular disease, poses grave threats to human health. Although atherosclerosis depends on lipid accumulation and vascular wall inflammation, abnormal phenotypic regulation of macrophages is considered the pathological basis of atherosclerosis. Macrophage polarization mainly refers to the transformation of macrophages into pro-inflammatory (M1) or anti-inflammatory (M2) phenotypes, which has recently become a much-discussed topic. Increasing evidence has shown that M2 macrophage polarization can alleviate atherosclerosis progression. PGE2 is a bioactive lipid that has been observed to be elevated in atherosclerosis and to play a pro-inflammatory role, yet recent studies have reported that PGE2 promotes anti-inflammatory M2 macrophage polarization and mitigates atherosclerosis progression. However, the mechanisms by which PGE2 acts remain unclear. This review summarizes current knowledge of PGE2 and macrophages in atherosclerosis. Additionally, we discuss potential PGE2 mechanisms of macrophage polarization, including CREB, NF-κB, and STAT signaling pathways, which may provide important therapeutic strategies based on targeting PGE2 pathways to modulate macrophage polarization for atherosclerosis treatment.

Traditional Chinese Medicine Fufang-Zhenzhu-Tiaozhi capsule prevents renal injury in diabetic minipigs with coronary heart disease.

BACKGROUND: Renal injury is one of the common microvascular complications of diabetes, known as diabetic kidney disease (DKD) seriously threatening human health. Previous research has reported that the Chinese Medicine Fufang-Zhenzhu-Tiaozhi (FTZ) capsule protected myocardia from injury in diabetic minipigs with coronary heart disease (DM-CHD). And we found significant renal injury in the minipigs. Therefore, we further investigated whether FTZ prevents renal injury of DM-CHD minipig and H2O2-induced oxidative injury of HK-2 cells. METHODS: DM-CHD model was established by streptozotocin injection, high fat/high-sucrose/high-cholesterol diet combined with balloon injury in the coronary artery. Blood lipid profile, fasting blood glucose (FBG), and SOD were measured with kits. The levels of blood urea nitrogen (BUN), serum creatinine (Scr), urine trace albumin (UALB), urine creatinine (UCR) (calculate UACR), cystatin (Cys-C), and ß-microglobulin (ß-MG) were measured by ELISA kits to evaluate renal function. TUNEL assay was performed to observe the apoptosis. qPCR was used to detect the mRNA expression levels of HO-1, NQO1, and SOD in kidney tissue. The protein expressions of Nrf2, HO-1, NQO1, Bax, Bcl-2, and Caspase 3 in the kidney tissue and HK-2 cells were detected by western blot. Meanwhile, HK-2 cells were induced by H2O2 to establish an oxidative stress injury model to verify the protective effect and mechanisms of FTZ. RESULTS: In DM-CHD minipigs, blood lipid profile and FBG were elevated significantly, and the renal function was decreased with the increase of BUN, Scr, UACR, Cys-c, and ß-MG. A large number of inflammatory and apoptotic cells in the kidney were observed accompanied with lower levels of SOD, Bcl-2, Nrf2, HO-1, and NQO1, but high levels of Bax and Cleaved-caspase 3. FTZ alleviated glucose-lipid metabolic disorders and the pathological morphology of the kidney. The renal function was improved and the apoptotic cells were reduced by FTZ administration. FTZ could also enhance the levels of SOD, Nrf2, HO-1, and NQO1 proteins to promote antioxidant effect, down-regulate the expression of Bax and Caspase3, as well as up-regulate the expression of Bcl-2 to inhibit cell apoptosis in the kidney tissue and HK-2 cells. CONCLUSIONS: We concluded that FTZ prevents renal injury of DM-CHD through activating anti-oxidative capacity to reduce apoptosis and inhibiting inflammation, which may be a new candidate for DKD treatment.

Hepatocyte glutathione S-transferase mu 2 prevents non-alcoholic steatohepatitis by suppressing ASK1 signaling.

BACKGROUND & AIMS: Non-alcoholic fatty liver disease (NAFLD) has become the most common chronic liver disease worldwide. The advanced stage of NAFLD, non-alcoholic steatohepatitis (NASH), has been recognized as a leading cause of end-stage liver injury for which there are no FDA-approved therapeutic options. Glutathione S-transferase Mu 2 (GSTM2) is a phase II detoxification enzyme. However, the roles of GSTM2 in NASH have not been elucidated. METHODS: Multiple RNA-seq analyses were used to identify hepatic GSTM2 expression in NASH. In vitro and in vivo gain- or loss-of-function approaches were used to investigate the role and molecular mechanism of GSTM2 in NASH. RESULTS: We identified GSTM2 as a sensitive responder and effective suppressor of NASH progression. GSTM2 was significantly downregulated during NASH progression. Hepatocyte GSTM2 deficiency markedly aggravated insulin resistance, hepatic steatosis, inflammation and fibrosis induced by a high-fat diet and a high-fat/high-cholesterol diet. Mechanistically, GSTM2 sustained MAPK pathway signaling by directly interacting with apoptosis signal-regulating kinase 1 (ASK1). GSTM2 directly bound to the N-terminal region of ASK1 and inhibited ASK1 N-terminal dimerization to subsequently repress ASK1 phosphorylation and the activation of its downstream JNK/p38 signaling pathway under conditions of metabolic dysfunction. CONCLUSIONS: These data demonstrated that hepatocyte GSTM2 is an endogenous suppressor that protects against NASH progression by blocking ASK1 N-terminal dimerization and phosphorylation. Activating GSTM2 holds promise as a therapeutic strategy for NASH. CLINICAL TRIAL NUMBER: IIT-2021-277. LAY SUMMARY: New therapeutic strategies for non-alcoholic steatohepatitis are urgently needed. We identified that the protein GSTM2 exerts a protective effect in response to metabolic stress. Therapies that aim to increase the activity of GSTM2 could hold promise for the treatment of non-alcoholic steatohepatitis.

FTZ attenuates liver steatosis and fibrosis in the minipigs with type 2 diabetes by regulating the AMPK signaling pathway.

Fufang Zhenzhu Tiaozhi formula (FTZ), a preparation of Chinese herbal medicine, has various pharmacological properties, such as hypoglycemic, hypolipidemic, anticoagulant, and anti-inflammatory activities. Hepatocyte apoptosis is a marker of nonalcoholic steatohepatitis (NASH) and contributes to liver injury, fibrosis, and inflammation. Given the multiple effects of FTZ, we investigated whether FTZ can be a therapeutic agent for NASH and its mechanism. In the present study, we observed that FTZ treatment had an obviously favorable influence on hepatic steatosis and fibrosis in the histopathologic features of type 2 diabetes mellitus (T2DM) and coronary heart disease (CHD) with NASH minipigs. In addition, immunohistochemical analysis showed increased expression of the fibrotic marker α-smooth muscle actin (α-SMA), and a TUNEL assay revealed increased apoptotic positive hepatic cells in the liver tissues of the model group. Furthermore, FTZ administration reduced the increased expression of α-SMA, and FTZ inhibited apoptosis by affecting Bcl-2/Bax and cleaved caspase-3 expression. Mechanistically, our data suggested that FTZ treatment attenuated hepatic steatosis and fibrosis via the adenosine monophosphate-activated protein kinase (AMPK) pathway. In vitro studies showed that FTZ also attenuated intracellular lipid accumulation in HepG2 cells exposed to palmitic acid (PA) and oleic acid (OA). FTZ upregulated the expression levels of P-AMPK and BCL-2 and downregulated BAX. The changes induced by FTZ were reversed by Compound C, an inhibitor of AMPK. In conclusion, FTZ attenuated NASH by ameliorating steatosis and hepatocyte apoptosis, which is attributable to the regulation of the AMPK pathway.

Molecular mechanism of Fufang Zhenzhu Tiaozhi capsule in the treatment of type 2 diabetes mellitus with nonalcoholic fatty liver disease based on network pharmacology and validation in minipigs.

ETHNOPHARMACOLOGICAL RELEVANCE: Fufang Zhenzhu Tiaozhi formula (FTZ) of which a patented preparation of Chinese herbal medicine has been well documented with significant clinical curative effect for hyperglycemia and hyperlipidemia. Because of the complexity of the chemical constituents of Chinese herbal formulas, the holistic pharmacological mechanism of FTZ acting on type 2 diabetes mellitus (T2DM) and nonalcoholic fatty liver disease (NAFLD) remains unclear. AIM OF THE STUDY: To investigate the pharmacological efficacy and mechanism of FTZ in the treatment of T2DM accompanied by NAFLD. MATERIALS AND METHODS: Network pharmacology and validation in minipigs were used in this study. First, potential bioactive compounds of FTZ were identified by the traditional Chinese medicine system pharmacology technology platform (TCMSP). Then, targets of compounds were gathered using DrugBank, SwissTargetPrediction and TCMSP, while targets for T2DM and NAFLD were collected from CTD (compounds-targets-diseases network) and GeneCards. Common targets were defined as direct therapeutic targets acting on T2DM with NAFLD. In addition, crucial targets were chosen by the protein-protein interaction (PPI) network and contribution to compound-therapeutic targets in T2DM with the NAFLD network. Furthermore, gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) were used to analyze the metabolism-related signaling pathways affected by FTZ. Candidate patterns selected by network pharmacology were tested in the minipigs model of T2DM with NAFLD. Measurements of triglycerides (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), fasting insulin (FINS) and fasting blood glucose (FBG) in the blood and the expression levels of proteins, including PI3K-AKT and HIF-1α, in the livers of the minipigs were followed by the administration of FTZ. RESULTS: A total of 116 active compounds and 82 potential targets related to T2DM and NAFLD were found. Pathway and functional enrichment analysis showed that FTZ mainly regulates metabolism-related pathways, including PI3K-AKT, HIF-1α, TNFα and MAPK. Animal experiments showed that FTZ treatment significantly reduced the serum levels of TG, TC, LDL-C and FBG, increased serum levels of HDL-C, ameliorated systemic insulin resistance (IR), and attenuated liver damage in minipigs with T2DM and NAFLD. FTZ treatment has an obviously favorable influence on hepatic steatosis and liver lipid accumulation in the histopathologic features of HE, Oil red O staining, and electron microscopy. Mechanistically, FTZ improved liver metabolism by increasing the phosphorylation of PI3K-AKT and decreasing the expression of HIF-1α. CONCLUSION: Network pharmacology was supported by experimental studies, which indicated that FTZ has demonstrated therapeutic benefits in T2DM and NAFLD by regulating the PI3K-AKT and HIF-1α signaling pathways.

The traditional Chinese medicine formula Fufang-Zhenzhu-Tiaozhi protects myocardia from injury in diabetic minipigs with coronary heart disease.

BACKGROUND AND PURPOSE: Diabetes mellitus (DM) is a major risk factor for coronary heart disease (CHD). Previous research has reported that the Fufang-Zhenzhu-Tiaozhi (FTZ) formula has obvious effects on the treatment of dyslipidemia and hyperglycemia. In the present study, we intended to establish a convenient DM-CHD model in minipigs and investigated the protective effect of FTZ against myocardial injury and its mechanism. METHODS: The DM-CHD model was established by a high-fat/high-sucrose/high-cholesterol diet (HFSCD) combined with balloon injury in the coronary artery. Subsequently, sixteen Wuzhishan minipigs were assigned to three groups: control group, model group, and FTZ group. The model group and FTZ group were given a HFSCD, while the control group was given a normal diet (ND). FTZ was given with meals in the FTZ group. During this time, biochemical parameters, such as total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein (HDL-C), and fasting blood glucose (FBG), were measured by using testing kits. Insulin (INS) was determined by ELISA, and the homeostasis model assessment index of insulin resistance (HOMA-IR) was calculated to evaluate insulin resistance levels. After FTZ administration, the plasma levels of lactate dehydrogenase (LDH), creatine kinase isoenzyme MB (CK-MB), and cardiac troponin I (cTnI) were measured by using ELISA kits to evaluate myocardial injury. Coronary artery stenosis was analyzed by angiographic and HE staining. Myocardial ischemia was assayed with electrocardiogram (ECG). Moreover, cytokines, including interleukin-6 (IL-6), hypersensitive C-reactive protein (hs-CRP), and tumor necrosis factor-alpha (TNF-α), were measured by ELISA kits to assess inflammation. The myocardial tissue was collected, and the pathological morphology was observed by transmission electron microscopy (TEM), HE staining, and Masson staining. Western blots were used to detect the expression of PI3K, AKT, p-AKT, p-NF-κB, and NF-κB. RESULTS: A DM-CHD model in minipigs with glucose-lipid metabolism disorder, coronary artery incrassation and myocardial damage was successfully established through balloon injury in the coronary artery combined with HFSCD. FTZ effectively inhibited coronary artery incrassation and protected the myocardium against injury in DM-CHD minipigs. FTZ decreased proinflammatory cytokine levels and upregulated the protein expression of the PI3K/Akt pathway in the myocardium. CONCLUSIONS: A novel DM-CHD model in minipigs was successfully established through balloon injury in the coronary artery combined with HFSCD. FTZ has a protective effect against myocardial injury in DM-CHD by inhibiting inflammation and activating the PI3K/AKT signaling pathway.

A Novel Aurora Kinase Inhibitor Attenuates Leukemic Cell Proliferation Induced by Mesenchymal Stem Cells.

Acute myeloid leukemia (AML) mesenchymal stem cells (MSCs) play an essential role in protecting leukemic cells from chemotherapeutic agents through activating a wide range of adhesion molecules and cytokines. Thus, more attention should be paid to attenuate the protection of leukemic cells by MSCs. By examining the gene expression files of MSCs from healthy donors and AML patients through high-throughput microarrays, we found that interleukin (IL)-6 was an important cytokine secreted by AML MSCs to protect leukemic cells, contributing to disease progression. Strikingly, Aurora A (AURKA) was activated by IL-6, offering a new target to interfere with leukemia. Importantly, a novel AURKA inhibitor, PW21, showed excellent AURKA kinase inhibitory activities and attenuated the interaction of leukemic cells and the microenvironment. PW21 inhibited MSC-induced cell proliferation, colony formation, and migration, and it induced cell apoptosis. Mechanically, PW21 could inhibit IL-6 secreted by MSCs. Moreover, we found that PW21 displayed a strong anti-leukemia effect on non-obese diabetic (NOD)-severe combined immunodeficiency (SCID) and murine MLL-AF9 leukemic models. PW21 significantly prolonged the survival of leukemic mice and eliminated the leukemic progenitor cells. AURKA inhibitor PW21 could provide a new approach for treatment of leukemia through blocking the protection by the leukemic microenvironment in clinical application.

M2b Macrophages Regulate Cardiac Fibroblast Activation and Alleviate Cardiac Fibrosis After Reperfusion Injury.

BACKGROUND: Macrophages play an important role in the development of cardiac fibrosis. However, the roles of different macrophage subtypes in cardiac fibroblast (CF) activation and cardiac fibrosis are unknown.Methods and Results:Bone marrow-derived macrophages (BMDMs) were treated with different stimuli to induce differentiation into M1, M2a, M2b, and M2c macrophage subtypes. CFs were co-cultured with different subtypes of macrophages or cultured with macrophage supernatants. Results revealed that M2b macrophages significantly suppressed the proliferation and migration of CFs, the expression of fibrosis-related proteins (collagen I [COL-1] and α-smooth muscle actin [α-SMA]), and differentiation into cardiac myofibroblasts (MFs). The opposite effects were observed with M2a macrophages. A rat model of cardiac ischemia/reperfusion (I/R) injury was used to determine the effect of M2b macrophages transplantation. After cardiac I/R injury, transplantation of M2b macrophages improved cardiac function and reduced cardiac fibrosis. The effect of macrophage subtypes on p-ERK, ERK, p-p38, and p38 phosphorylation was examined by Western blotting. The results showed that M2b macrophages significantly inhibited the mitogen-activated protein kinase (MAPK) signaling pathway. CONCLUSIONS: These study results demonstrate for the first time that different subtypes of macrophages have different roles in regulating CF activation. M2b macrophages inhibit CF activation, and thus can be considered anti-fibrotic macrophages. M2a macrophages promote CF activation, and thus are pro-fibrotic macrophages.

M2b macrophage polarization and its roles in diseases.

Macrophages play an important role in a wide variety of physiologic and pathologic processes. Plasticity and functional polarization are hallmarks of macrophages. Macrophages commonly exist in two distinct subsets: classically activated macrophages (M1) and alternatively activated macrophages (M2). M2b, a subtype of M2 macrophages, has attracted increasing attention over the past decade due to its strong immune-regulated and anti-inflammatory effects. A wide variety of stimuli and multiple factors modulate M2b macrophage polarization in vitro and in vivo. M2b macrophages possess both protective and pathogenic roles in various diseases. Understanding the mechanisms of M2b macrophage activation and the modulation of their polarization might provide a great perspective for the design of novel therapeutic strategies. The purpose of this review is to discuss current knowledge of M2b macrophage polarization, the roles of M2b macrophages in a variety of diseases and the stimuli to modulate M2b macrophage polarization.

M2b macrophages reduce early reperfusion injury after myocardial ischemia in mice: A predominant role of inhibiting apoptosis via A20.

BACKGROUND: Monocytes or macrophages have been assessed as potential therapeutics to ameliorate myocardial ischemic diseases, but the results have been controversial. As regulatory macrophages, M2b macrophages could have enhanced protective effects. We tested the hypothesis that transplantation of M2b macrophages could ameliorate myocardial ischemia/reperfusion (I/R) injury. The potential mechanisms involved in it were investigated. METHODS: M2b macrophages were polarized by lipopolysaccharide (LPS) and the immune complex (IC) from bone marrow-derived macrophages (BMDMs) of C57BL/6 mice. They were identified based on surface marker expression and cytokine production. Myocardial I/R injury models were established with the same strain of mice. Once the ischemic area was identified, either 1×105 M2b macrophages (MT group) or the same volume of normal saline (CK group) was injected into the ischemic zone. Mice in the sham operation (SO) group underwent the operation without ligation of the coronary artery. RESULTS: We found a significant decrease in serum cardiac troponin I (cTnI) level, the infarct area, apoptosis index, and nuclear factor-κB (NF-κB) signaling activation in the MT group after 2h of reperfusion; the changes were induced by I/R. In addition, the injury resulted in significantly up-regulated expression of A20 and continued to be improved by the transplanted M2b macrophages. CONCLUSIONS: The administration of M2b macrophages significantly attenuated myocardial I/R injury. A20 may be part of the protective mechanism through limiting NF-κB signaling-mediated apoptosis.

Platelet Derived Growth Factor Alpha (PDGFRα) Induces the Activation of Cardiac Fibroblasts by Activating c-Kit.

BACKGROUND Enhanced platelet-derived growth factor receptor a (PDGFRα) signaling pathway activity leads to cardiac fibrosis. However, because of the pleiotropic effects of PDGFR signaling, its role in mediating the cardiac fibrotic response remains poorly understood. This study aimed to investigate the regulatory effect of c-Kit in cardiac fibroblasts activated by PDGFRa signaling. MATERIAL AND METHODS A cardiac fibrosis mice model was induced using isoproterenol, and the heart tissues of mice were tested through western blotting and real-time quantitative PCR (RT-qPCR). The cardiac fibroblasts of neonatal mice were treated with PDGF-AA or transfected with small interfering RNAs (siRNAs) specific for the mouse c-Kit gene. The levels of collagen I, collagen III, and alpha-smooth muscle actin (α-SMA) were analyzed using western blotting and RT-qPCR. RESULTS In the heart of the cardiac fibrosis mice model, the activity of c-Kit was enhanced. PDGF-AA treatment accelerated the activity of c-Kit in cardiac fibroblasts. In addition, imatinib inhibited the activity of c-Kit in vivo and in vitro. Moreover, inhibition of c-Kit by siRNAs reduced the expression of α-SMA and collagens in the activated cardiac fibroblasts. Furthermore, PDGFRa directly bound c-Kit in cardiac fibroblasts and stimulated the expression of stem cell factor (SCF). CONCLUSIONS Our data demonstrated that PDGF/PDGFRa induced the activation of cardiac fibroblasts by activating c-Kit. This study indicated that c-Kit could be used as a potential therapeutic target for treatment of cardiac fibrosis.

Aurora A Kinase Inhibitor AKI603 Induces Cellular Senescence in Chronic Myeloid Leukemia Cells Harboring T315I Mutation.

The emergence of resistance to imatinib mediated by mutations in the BCR-ABL has become a major challenge in the treatment of chronic myeloid leukemia (CML). Alternative therapeutic strategies to override imatinib-resistant CML are urgently needed. In this study, we investigated the effect of AKI603, a novel small molecule inhibitor of Aurora kinase A (AurA) to overcome resistance mediated by BCR-ABL-T315I mutation. Our results showed that AKI603 exhibited strong anti-proliferative activity in leukemic cells. AKI603 inhibited cell proliferation and colony formation capacities in imatinib-resistant CML cells by inducing cell cycle arrest with polyploidy accumulation. Surprisingly, inhibition of AurA by AKI603 induced leukemia cell senescence in both BCR-ABL wild type and T315I mutation cells. Furthermore, the induction of senescence was associated with enhancing reactive oxygen species (ROS) level. Moreover, the anti-tumor effect of AKI603 was proved in the BALB/c nude mice KBM5-T315I xenograft model. Taken together, our data demonstrate that the small molecule AurA inhibitor AKI603 may be used to overcome drug resistance induced by BCR-ABL-T315I mutation in CML.

Targeting GLI1 Suppresses Cell Growth and Enhances Chemosensitivity in CD34+ Enriched Acute Myeloid Leukemia Progenitor Cells.

BACKGROUND/AIMS: Resistance of leukemia stem cells (LSCs) to chemotherapy in patients with acute myeloid leukemia (AML) causes relapse of disease. Hedgehog (Hh) signaling plays a critical role in the maintenance and differentiation of cancer stem cells. Yet its role in AML remains controversial. The purpose of the present study is to investigate the role of GLI1, the transcriptional activator of Hh signaling, in AML progenitor cells and to explore the anti-AML effects of GLI small-molecule inhibitor GANT61. METHODS: The expression of GLI1 mRNA and protein were examined in AML progenitor cells and normal cells. The proliferation, colony formation, apoptosis and differentiation of AML progenitor cells were also analyzed in the presence of GANT61. RESULTS: Kasumi-1 and KG1a cells, containing more CD34+ cells, expressed higher level of GLI1 compared to U937 and NB4 cells with fewer CD34+ cells. Consistently, a positive correlation between the protein levels of GLI1 and CD34 was validated in the bone marrow mononuclear cells (BMMC) of AML patients tested. GANT61 inhibited the proliferation and colony formation in AML cell lines. Importantly, GANT61 induced apoptosis in CD34+ enriched Kasumi-1 and KG1a cells, whereas it induced differentiation in U937 and NB4 cells. Furthermore, GANT61 enhanced the cytotoxicity of cytarabine (Ara-c) in primary CD34+ AML cells, indicating that inhibition of GLI1 could be a promising strategy to enhance chemosensitivity. CONCLUSIONS: The present findings suggested that Hh signaling was activated in AML progenitor cells. GLI1 acted as a potential target for AML therapy.

A novel compound against oncogenic Aurora kinase A overcomes imatinib resistance in chronic myeloid leukemia cells.

Drug resistance still represents a major obstacle to successful chronic myeloid leukemia (CML) treatment and novel compounds or strategies to override this challenging problem are urgently required. Here, we evaluated a novel compound AKI603 against oncogenic Aurora kinase A (Aur-A) in imatinib-resistant CML cells. We found that Aur-A was highly activated in imatinib-resistant KBM5-T315I cells. AKI603 significantly inhibited the phosphorylation of Aur-A kinase at Thr288, while had little inhibitory effect on BCR-ABL kinase in both KBM5 and KBM5-T315I cells. AKI603 inhibited cell viability, and induced cell cycle arrest with polyploidy accumulation in KBM5 and KBM5-T315I cells. Moreover, inhibition of Aur-A kinase by AKI603 suppressed colony formation capacity without promoting obvious apoptosis. Importantly, AKI603 promoted cell differentiation in both CML cell types. Thus, our study suggested the potential clinical use of small molecule Aurora kinase inhibitor AKI603 to overcome imatinib resistance in CML treatment.

Sequence analysis and molecular characterization of Clonorchis sinensis hexokinase, an unusual trimeric 50-kDa glucose-6-phosphate-sensitive allosteric enzyme.

Clonorchiasis, which is induced by the infection of Clonorchis sinensis (C. sinensis), is highly associated with cholangiocarcinoma. Because the available examination, treatment and interrupting transmission provide limited opportunities to prevent infection, it is urgent to develop integrated strategies to prevent and control clonorchiasis. Glycolytic enzymes are crucial molecules for trematode survival and have been targeted for drug development. Hexokinase of C. sinensis (CsHK), the first key regulatory enzyme of the glycolytic pathway, was characterized in this study. The calculated molecular mass (Mr) of CsHK was 50.0 kDa. The obtained recombinant CsHK (rCsHK) was a homotrimer with an Mr of approximately 164 kDa, as determined using native PAGE and gel filtration. The highest activity was obtained with 50 mM glycine-NaOH at pH 10 and 100 mM Tris-HCl at pH 8.5 and 10. The kinetics of rCsHK has a moderate thermal stability. Compared to that of the corresponding negative control, the enzymatic activity was significantly inhibited by praziquantel (PZQ) and anti-rCsHK serum. rCsHK was homotropically and allosterically activated by its substrates, including glucose, mannose, fructose, and ATP. ADP exhibited mixed allosteric effect on rCsHK with respect to ATP, while inorganic pyrophosphate (PPi) displayed net allosteric activation with various allosteric systems. Fructose behaved as a dose-dependent V activator with the substrate glucose. Glucose-6-phosphate (G6P) displayed net allosteric inhibition on rCsHK with respect to ATP or glucose with various allosteric systems in a dose-independent manner. There were differences in both mRNA and protein levels of CsHK among the life stages of adult worm, metacercaria, excysted metacercaria and egg of C. sinensis, suggesting different energy requirements during different development stages. Our study furthers the understanding of the biological functions of CsHK and supports the need to screen for small molecule inhibitors of CsHK to interfere with glycolysis in C. sinensis.

Inhibition of c-Myc overcomes cytotoxic drug resistance in acute myeloid leukemia cells by promoting differentiation.

Nowadays, drug resistance still represents a major obstacle to successful acute myeloid leukemia (AML) treatment and the underlying mechanism is not fully elucidated. Here, we found that high expression of c-Myc was one of the cytogenetic characteristics in the drug-resistant leukemic cells. c-Myc over-expression in leukemic cells induced resistance to chemotherapeutic drugs, enhanced colony formation capacity and inhibited cell differentiation induced by all-trans retinoic acid (ATRA). Meanwhile, inhibition of c-Myc by shRNA or specific c-Myc inhibitor 10058-F4 rescued the sensitivity to cytotoxic drugs, restrained the colony formation ability and promoted differentiation. RT-PCR and western blotting analysis showed that down-regulation of C/EBPß contributed to the poor differentiation state of leukemic cells induced by c-Myc over-expression. Importantly, over-expression of C/EBPß could reverse c-Myc induced drug resistance. In primary AML cells, the c-Myc expression was negatively correlated with C/EBPß. 10058-F4, displayed anti-proliferative activity and increased cellular differentiation with up-regulation of C/EBPß in primary AML cells. Thus, our study indicated that c-Myc could be a novel target to overcome drug resistance, providing a new approach in AML therapy.