Baicalin alleviates angiotensin II-induced cardiomyocyte apoptosis and autophagy and modulates the AMPK/mTOR pathway.

As a main extraction compound from Scutellaria baicalensis Georgi, Baicalin exhibits various biological activities. However, the underlying mechanism of Baicalin on hypertension-induced heart injury remains unclear. In vivo, mice were infused with angiotensin II (Ang II; 500 ng/kg/min) or saline using osmotic pumps, followed by intragastrically administrated with Baicalin (5 mg/kg/day) for 4 weeks. In vitro, H9C2 cells were stimulated with Ang II (1 µM) and treated with Baicalin (12.5, 25 and 50 µM). Baicalin treatment significantly attenuated the decrease in left ventricular ejection fraction and left ventricular fractional shortening, increase in left ventricular mass, left ventricular systolic volume and left ventricular diastolic volume of Ang II infused mice. Moreover, Baicalin treatment reversed 314 differentially expressed transcripts in the cardiac tissues of Ang II infused mice, and enriched multiple enriched signalling pathways (including apoptosis, autophagy, AMPK/mTOR signalling pathway). Consistently, Baicalin treatment significantly alleviated Ang II-induced cell apoptosis in vivo and in vitro. Baicalin treatment reversed the up-regulation of Bax, cleaved-caspase 3, cleaved-caspase 9, and the down-regulation of Bcl-2. Meanwhile, Baicalin treatment alleviated Ang II-induced increase of autophagosomes, restored autophagic flux, and down-regulated LC3II, Beclin 1, as well as up-regulated SQSTM1/p62 expression. Furthermore, autophagy inhibitor 3-methyladenine treatment alleviated the increase of autophagosomes and the up-regulation of Beclin 1, LC3II, Bax, cleaved-caspase 3, cleaved-caspase 9, down-regulation of SQSTM1/p62 and Bcl-2 expression after Ang II treated, which similar to co-treatment with Baicalin. Baicalin treatment reduced the ratio of p-AMPK/AMPK, while increased the ratio of p-mTOR/mTOR. Baicalin alleviated Ang II-induced cardiomyocyte apoptosis and autophagy, which might be related to the inhibition of the AMPK/mTOR pathway.

Bioreactor-based stem cell therapy for liver fibrosis.

Stem cell therapy, achieved using mesenchymal stem cells (MSCs), has been highlighted for the treatment of liver fibrosis. Infusion into the circulatory system is a traditional application of MSCs; however, this approach is limited by phenotypic drift, stem cell senescence, and vascular embolism. Maintaining the therapeutic phenotype of MSCs while avoiding adverse infusion-related reactions is the key to developing next-generation stem cell therapy technologies. Here, we propose a bioreactor-based MSCs therapy to avoid cell infusion. In this scheme, 5% liver fibrosis serum was used to induce the therapeutic phenotype of MSCs, and a fluid bioreactor carrying a co-culture system of hepatocytes and MSCs was constructed to produce the therapeutic medium. In a rat model of liver fibrosis, the therapeutic medium derived from the bioreactor significantly alleviated liver fibrosis. Therapeutic mechanisms include immune regulation, inhibition of hepatic stellate cell activation, establishment of hepatocyte homeostasis, and recovery of liver stem cell subsets. Overall, the bioreactor-based stem cell therapy (scheme) described here represents a promising new strategy for the treatment of liver fibrosis and will be beneficial for the development of 'cell-free' stem cell therapy.

Pretreatment of UC-MSCs with IFN-α2 improves treatment of liver fibrosis by recruiting neutrophils.

BACKGROUND: The use of umbilical cord mesenchymal stem cells (UC-MSCs) is a burgeoning method for the treatment of liver cirrhosis. However, the secretory phenotype and regulatory ability of UC-MSCs are easily affected by their microenvironment. Ensuring a specific microenvironment to enhance the UC-MSCs phenotype is a potential strategy for improving their therapeutic efficacy. The aim of this study was to explore therapeutic UC-MSCs phenotypes for improving liver fibrosis. METHODS: RNA-sequencing was used to analyze the response pattern of UC-MSCs after exposure to the serum of cirrhotic patients with HBV. Using immunohistochemistry, quantitative polymerase chain reaction, and immunofluorescence techniques, we evaluated the therapeutic effect of UC-MSCs pretreated with interferon alpha 2 (IFN-α2) (pre-MSCs) in an animal model of cirrhosis. Immunoblotting, ELISA, and other techniques were used to analyze the signaling pathways underlying the IFN-induced changes in UC-MSCs. RESULTS: UC-MSCs exposed to the serum of patients with hepatitis B-induced cirrhosis showed an enhanced response to type I IFN. The activated type I IFN signal induced the highest secretion of colony-stimulating factor 3 (CSF-3), interleukin (IL)-8, and chemokine (C-C motif) ligand 20 (CCL20) by the UC-MSCs. Pre-MSCs showed a higher therapeutic efficacy than untreated UC-MSCs in an animal model of liver fibrosis. Immunohistochemical analysis revealed that pre-MSCs could recruit neutrophils resulting in an increase in the secretion of matrix metalloprotease 8 that alleviated fibrosis. When neutrophils in animals were depleted, the therapeutic effect of pre-MSCs on fibrosis was inhibited. IFN-α2 altered the secretory phenotype of UC-MSCs by activating phosphorylated signal transducer and activator of transcription 1 and 2 (p-STAT1 and p-STAT2). CONCLUSIONS: Pre-MSCs exhibited enhanced secretion of CSF-3, IL-8, and CCL20 and recruited neutrophils to alleviate fibrosis. This new strategy can improve cell therapy for liver cirrhosis.

The Dual-Mode Transition of Myofibroblasts Derived from Hepatic Stellate Cells in Liver Fibrosis.

Hepatic stellate cells (HSCs) are the key promoters of liver fibrosis. In response to liver-fibrosis-inducing factors, HSCs express alpha smooth muscle actin (α-SMA) and obtain myofibroblast phenotype. Collagen secretion and high expression of α-SMA with related high cell tension and migration limitation are the main characteristics of myofibroblasts. How these two characteristics define the role of myofibroblasts in the initiation and progression of liver fibrosis is worth exploring. From this perspective, we explored the correlation between α-SMA expression and collagen secretion in myofibroblasts and the characteristics of collagen deposition in liver fibrosis. Based on a reasonable hypothesis and experimental verification, we believe that the myofibroblast with the α-SMAhighcollagenhigh model do not effectively explain the initial stage and progression characteristics of liver fibrosis. Therefore, we propose a myofibroblast dual-mode transition model in fibrotic liver (DMTM model). In the DMTM model, myofibroblasts have dual modes. Myofibroblasts obtain enhanced α-SMA expression, accompanied by collagen expression inhibition in the high-concentration region of TGF-ß. At the edge of the TGF-ß positive region, myofibroblasts convert to a high-migration and high-collagen secretion phenotype. This model reasonably explains collagen deposition and expansion in the initial stage of liver fibrosis.

Quercetin attenuates angiotensin II-induced proliferation of vascular smooth muscle cells and p53 pathway activation in vitro and in vivo.

Quercetin is an essential flavonoid mostly found in herbal plants, fruits, and vegetables, which exhibits anti-hypertension properties. However, its pharmacological impact on angiotensin II (Ang II) induced the increase of blood pressure along with in-depth mechanism needs further exploration. The present study pointed out the anti-hypertensive role of quercetin and its comprehensive fundamental mechanisms. Our data showed that quercetin treatment substantially reduced the increase in blood pressure, pulse wave velocity, and aortic thickness of abdominal aorta in Ang II-infused C57BL/6 mice. RNA sequencing revealed that quercetin treatment reversed 464 differentially expressed transcripts in the abdominal aorta of Ang II-infused mice. Moreover, overlapping KEGG-enriched signaling pathways identified multiple common pathways between the comparison of Ang II versus control and Ang II + quercetin versus Ang II. Likewise, these pathways included cell cycle as well as p53 pathways. Transcriptome was further validated by immunohistochemistry, indicating that quercetin treatment significantly decreased the Ang II-induced expression of proliferating cell nuclear antigen (PCNA), cyclin-dependent kinase-4 (CDK4), and cyclin D1, while increased protein expression of p53, and p21 in abdominal aortic tissues of mice. In vitro, quercetin treatment meaningfully decreased the cell viability, arrested cell cycle at G0/G1 phase, and up-regulated the p53 and p21 proteins expression, as well as down-regulated the protein expression of cell cycle-related markers, for example, CDK4, cyclin D1 in Ang II stimulated vascular smooth muscle cells (VSMCs). This study addresses pharmacologic and mechanistic perspectives of quercetin against Ang-II-induced vascular injury and the increase of blood pressure.

Tumor cell density dependent IL-8 secretion induces the fluctuation of tregs/CD8 + T cells infiltration in hepatocellular carcinoma: one prompt for the existence of density checkpoint.

BACKGROUND: Tumor cell density is a basic pathological feature of solid tumors. Chemotherapy, radiotherapy, and targeted therapy reduce tumor cell density, whereas unrestricted tumor cell proliferation promotes this feature. The impact of tumor cells on the microenvironment following changes in tumor cell density is still unclear. In this study, we focused on the response of key immune cell subsets to tumor cell density in hepatocellular carcinoma (HCC). METHODS: We determined the density of tumor and immune cells in the same area by section staining. We then identified potential mediators using polymerase chain reaction (PCR), enzyme-linked immunofluorescence assay (ELISA), 3D and co-culture, flow cytometry, and lentivirus intervention. The mechanism of lactate promotion was verified using lactate tests, bioinformatics, western blotting, and the above methods. The IL-8/DAPK1/lactate/regulatory T cell (Treg) axis was verified using a mouse liver cancer model. Tumor mutation burden was calculated using maftools in R. RESULTS: We found that the Treg/CD8 + T cell ratio is not consistent with tumor cell density in HCC, and a decreased Treg/CD8 + T cell ratio in the range of 5000-6000 cells/mm2 may elicit the possibility for immunotherapy in an immunosuppressive microenvironment. We showed that IL-8 mediates this immune fluctuation and promotes the infiltration of Tregs through the DAPK1/pyruvate kinase activity/lactate axis in HCC. Based on tumor ploidy and mutation burden data, we discussed the potential significance of immune fluctuation in the homeostasis of HCC mutation burden and proposed a "density checkpoint" and "entropy model" to describe this phenomenon. CONCLUSIONS: In summary, we report the mode of infiltration of Tregs/CD8 + T cells in response to tumor cell density and provide a new theoretical basis for IL-8 as a therapeutic target and the selection of an immunotherapy window in HCC.

Heterogeneous matrix stiffness regulates the cancer stem-like cell phenotype in hepatocellular carcinoma.

BACKGROUND: Solid tumors are stiffer than their surrounding normal tissues; however, their interior stiffness is not uniform. Under certain conditions, cancer cells can acquire stem-like phenotypes. However, it remains unclear how the heterogeneous physical microenvironment affects stemness expression in cancer cells. Here, we aimed to evaluate matrix stiffness heterogeneity in hepatocellular carcinoma (HCC) tissues and to explore the regulation effect of the tumor microenvironment on stem-like phenotypic changes through mechanical transduction. METHODS: First, we used atomic force microscopy (AFM) to evaluate the elastic modulus of HCC tissues. We then used hydrogel with adjustable stiffness to investigate the effect of matrix stiffness on the stem-like phenotype expression of HCC cells. Moreover, cells cultured on hydrogel with different stiffness were subjected to morphology, real-time PCR, western blotting, and immunofluorescence analyses to explore the mechanotransduction pathway. Finally, animal models were used to validate in vitro results. RESULTS: AFM results confirmed the heterogenous matrix stiffness in HCC tissue. Cancer cells adhered to hydrogel with varying stiffness (1.10 ± 0.34 kPa, 4.47 ± 1.19 kPa, and 10.61 kPa) exhibited different cellular and cytoskeleton morphology. Higher matrix stiffness promoted the stem-like phenotype expression and reduced sorafenib-induced apoptosis. In contrast, lower stiffness induced the expression of proliferation-related protein Ki67. Moreover, mechanical signals were transmitted into cells through the integrin-yes-associated protein (YAP) pathway. Higher matrix stiffness did not affect YAP expression, however, reduced the proportion of phosphorylated YAP, promoted YAP nuclear translocation, and regulated gene transcription. Finally, application of ATN-161 (integrin inhibitor) and verteporfin (YAP inhibitor) effectively blocked the stem-like phenotype expression regulated by matrix stiffness. CONCLUSIONS: Our experiments provide new insights into the interaction between matrix stiffness, cancer cell stemness, and heterogeneity, while also providing a novel HCC therapeutic strategy.

Huc-MSC-derived exosomes modified with the targeting peptide of aHSCs for liver fibrosis therapy.

BACKGROUND: Effective therapeutics to stop or reverse liver fibrosis have not emerged, because these potential agents cannot specifically target activated hepatic stellate cells (aHSCs) or are frequently toxic to parenchymal cells. Human umbilical cord mesenchymal stem cell (Huc-MSC)-derived exosomes show promise in nanomedicine for the treatment of liver fibrosis. However, systemic injection showed that unmodified exosomes were mainly taken up by the mononuclear phagocyte system. The discovery of ligands that selectively bind to a specific target plays a crucial role in clinically relevant diagnostics and therapeutics. Herein, we aimed to identify the targeting peptide of aHSCs by screening a phage-displayed peptide library, and modify Huc-MSC-derived exosomes with the targeting peptide. RESULTS: In this study, we screened a phage-displayed peptide library by biopanning for peptides preferentially bound to HSC-T6 cells. The identified peptide, HSTP1, also exhibited better targeting ability to aHSCs in pathological sections of fibrotic liver tissues. Then, HSTP1 was fused with exosomal enriched membrane protein (Lamp2b) and was displayed on the surface of exosomes through genetic engineering technology. The engineered exosomes (HSTP1-Exos) could be more efficiently internalized by HSC-T6 cells and outperformed both unmodified exosomes (Blank-Exos) and Lamp2b protein overexpressed exosomes (Lamp2b + Exos) in enhancing the ability of exosomes to promote HSC-T6 reversion to a quiescent phenotype. In vivo results showed HSTP1-Exos could specifically target to the aHSC region after intravenous administration, as demonstrated by coimmunofluorescence with the typical aHSCs marker α-SMA, and enhance the therapeutic effect on liver fibrosis. CONCLUSION: These results suggest that HSTP1 is a reliable targeting peptide that can specifically bind to aHSCs and that HSTP1-modified exosomes realize the precise treatment for aHSCs in complex liver tissue. We provide a novel strategy for clinical liver fibrosis therapy.

The role of matrix stiffness in cancer stromal cell fate and targeting therapeutic strategies.

The tumor microenvironment (TME) is a complex macromolecular network filled with a series of stromal cells. It plays an important role in tumorigenesis, development, immune escape, drug resistance, and other processes and has received increasing attention in recent years. Currently, tumor cell-centered treatments are insufficient to eradicate malignancies, and researchers are constantly searching for better treatments. Over the past decade, the TME has been recognized as a rich resource for anti-cancer drug development. As a significant mechanical feature in the microenvironment of solid tumors, matrix stiffness is increased owing to stromal deposition and remodeling. The effect of matrix stiffness on cancer cells has been described in many studies, whereas its effect on cancer stromal cell fate has rarely been summarized. Therefore, this review discusses the relevant content and drug treatment studies targeting matrix stiffness. STATEMENT OF SIGNIFICANCE: Biochemical and biophysical interactions between tumor cells, stromal cells, and the extracellular matrix (ECM) co-create a distinct tumor microenvironment (TME), which impacts disease outcome. In recent years, there has been a greater emphasis on the physical properties of the ECM, with matrix stiffness being one of the most thoroughly investigated. The matrix stiffness of solid tumors is now commonly acknowledged to be greater than that of normal tissues. Cancer-associated fibroblasts (CAFs), tumor-associated macrophages (TAMs), and endothelial cells (ECs) can all respond to matrix stiffness. At the same time, our current understanding of the TME is insufficient, and an in-depth examination of interactions between ECM and cells could lead to the development of more efficient and specialized treatments.

The Potential of Mesenchymal Stem Cells for the Treatment of Cytokine Storm due to COVID-19.

The outbreak of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has seriously affected public health and social stability. The main route of the transmission is droplet transmission, where the oral cavity is the most important entry point to the body. Due to both the direct harmful effects of SARS-CoV-2 and disordered immune responses, some COVID-19 patients may progress to acute respiratory distress syndrome or even multiple organ failure. Genetic variants of SARS-CoV-2 have been emerging and circulating around the world. Currently, there is no internationally approved precise treatment for COVID-19. Mesenchymal stem cells (MSCs) can traffic and migrate towards the affected tissue, regulate both the innate and acquired immune systems, and participate in the process of healing. Here, we will discuss and investigate the mechanisms of immune disorder in COVID-19 and the therapeutic activity of MSCs, in particular human gingiva mesenchymal stem cells.

Gly-tRF enhances LCSC-like properties and promotes HCC cells migration by targeting NDFIP2.

BACKGROUND: Accumulating evidence demonstrates that tRFs (tRNA-derived small RNA fragments) and tiRNAs (tRNA-derived stress-induced RNA), an emerging category of regulatory RNA molecules derived from transfer RNAs (tRNAs), are dysregulated in in various human cancer types and play crucial roles. However, their roles and mechanisms in hepatocellular carcinoma (HCC) and liver cancer stem cells (LCSCs) are still unknown. METHODS: The expression of glycine tRNA-derived fragment (Gly-tRF) was measured by qRT-PCR. Flow cytometric analysis and sphere formation assays were used to determine the properties of LCSCs. Transwell assays and scratch wound assays were performed to detect HCC cell migration. Western blotting was conducted to evaluate the abundance change of Epithelial-mesenchymal transition (EMT)-related proteins. Dual luciferase reporter assays and signalling pathway analysis were performed to explore the underlying mechanism of Gly-tRF functions. RESULTS: Gly-tRF was highly expressed in HCC cell lines and tumour tissues. Gly-tRF mimic increased the LCSC subpopulation proportion and LCSC-like cell properties. Gly-tRF mimic promoted HCC cell migration and EMT. Loss of Gly-tRF inhibited HCC cell migration and EMT. Mechanistically, Gly-tRF decreased the level of NDFIP2 mRNA by binding to the NDFIP2 mRNA 3' UTR. Importantly, overexpression of NDFIP2 weakened the promotive effects of Gly-tRF on LCSC-like cell sphere formation and HCC cell migration. Signalling pathway analysis showed that Gly-tRF increased the abundance of phosphorylated AKT. CONCLUSIONS: Gly-tRF enhances LCSC-like cell properties and promotes EMT by targeting NDFIP2 and activating the AKT signalling pathway. Gly-tRF plays tumor-promoting role in HCC and may lead to a potential therapeutic target for HCC.

Baicalin attenuates angiotensin II-induced blood pressure elevation and modulates MLCK/p-MLC signaling pathway.

Scutellaria baicalensis Georgi is an extensively used medicinal herb for the treatment of hypertension in traditional Chinese medicine. Baicalin, is an important flavonoid in Scutellaria baicalensis Georgi extracts, which exhibits therapeutic effects on anti-hypertension, but its underlying mechanisms remain to be further explored. Therefore, we investigated the effects and molecular mechanisms of Baicalin on anti-hypertension. In vivo studies revealed that Baicalin treatment significantly attenuated the elevation in blood pressure, the pulse propagation and thickening of the abdominal aortic wall in C57BL/6 mice infused with Angiotensin II (Ang II). Moreover, RNA-sequencing and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses identified 537 differentially expressed transcripts and multiple enriched signaling pathways (including vascular smooth muscle contraction and calcium signaling pathway). Consistently, we found that Baicalin pretreatment significantly alleviated the Ang II induced constriction of abdominal aortic ring, while promoted NE pre-contracted vasodilation of abdominal aortic ring at least partly dependent on L-type calcium channel. In addition, Ang II stimulation significantly increased cell viability and PCNA expression, while were attenuated after Baicalin treatment. Moreover, Baicalin pretreatment attenuated Ang II-induced intracellular Ca2+ release, Angiotensin II type 1 receptor (AT1R) expression and activation of MLCK/p-MLC pathway in vascular smooth muscle cells (VSMCs). The present work further addressed the pharmacological and mechanistic insights on anti-hypertension of Baicalin, which may help better understand the therapeutic effect of Scutellaria baicalensis Georgi on anti-hypertension.