Analysis of global gene expression using RNA-sequencing reveals novel mechanism of Yanghe Pingchuan decoction in the treatment of asthma.

BACKGROUND: Yanghe Pingchuan decoction (YPD) has been used for asthma treatment for many years in China. We sought to understand the mechanism of YPD, and find more potential targets for YPD-based treatment of asthma. METHODS: An ovalbumin-induced asthma model in rats was created. Staining (hematoxylin and eosin, Masson) was used to evaluate the treatment effect of YPD. RNA-sequencing was carried out to analyze global gene expression, and differentially expressed genes (DEGs) were identified. Analysis of the functional enrichment of genes was done using the Gene Ontology database (GO). Analysis of signaling-pathway enrichment of genes was done using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. Real-time reverse transcription-quantitative polymerase chain reaction was undertaken to measure expression of DEGs. RESULTS: Pathology showed that YPD had an improvement effect on rats with asthma. RNA-sequencing showed that YPD led to upregulated and downregulated expression of many genes. The YPD-based control of asthma pathogenesis may be related to calcium ion (Ca2+) binding, inorganic cation transmembrane transporter activity, microtubule motor activity, and control of canonical signaling (e.g., peroxisome proliferator-activated receptor, calcium, cyclic adenosine monophosphate). Enrichment analyses suggested that asthma pathogenesis may be related to Ca2 + binding and contraction of vascular smooth muscle. A validation experiment showed that YPD could reduce the Ca2 + concentration by inhibiting the Angiopoietin-II (Ang-II)/Phospholipase (PLA)/calmodulin (CaM0 signaling axis. CONCLUSION: Control of asthma pathogenesis by YPD may be related to inhibition of the Ang-II/PLA/CaM signaling axis, reduction of the Ca2+ concentration, and relaxation of airway smooth muscle (ASM).

Hugan Qingzhi tablets attenuates endoplasmic reticulum stress in nonalcoholic fatty liver disease rats by regulating PERK and ATF6 pathways.

BACKGROUND: Endoplasmic reticulum (ER) stress, promoting lipid metabolism disorders and steatohepatitis, contributes significantly to the pathogenesis of nonalcoholic fatty liver disease (NAFLD). Hugan Qingzhi tablets (HQT) has a definite effect in the clinical treatment of NAFLD patients, but its mechanism is still unclear. This study aims to investigate the effects of HQT on ER stress in the liver tissues of NAFLD rats and explore the underlying mechanism. METHODS: The NAFLD rat model was managed with high-fat diet (HFD) for 12weeks. HQT was administrated in a daily basis to the HFD groups. Biochemical markers, pro-inflammatory cytokines, liver histology were assayed to evaluate HQT effects in HFD-induced NAFLD rats. Furthermore, the expression of ER stress-related signal molecules including glucose regulating protein 78 (GRP78), protein kinase RNA-like endoplasmic reticulum kinase (PERK), p-PERK, eukaryotic translation initiation factor 2α (EIF2α), p-EIF2α, activating transcription factor 4 (ATF4), acetyl-coenzyme A-carboxylase (ACC), activating transcription factor (ATF6), and nuclear factor-kappa B-p65 (NF-κB-p65) were detected by western blot and/or qRT-PCR. RESULTS: The histopathological characteristics and biochemical data indicated that HQT exhibited protective effects on HFD-induced NAFLD rats. Furthermore, it caused significant reduction in the expression of ERS markers, such as GRP78, PERK, p-PERK, and ATF6, and subsequently downregulated the expression of EIF2α, p-EIF2α ATF4, ACC, and NF-κB-p65. CONCLUSIONS: The results suggested that HQT has protective effect against hepatic steatosis and inflammation in NAFLD rats by attenuating ER stress, and the potential mechanism is through inhibition of PERK and ATF6 pathways.

The increase of Nrf2 m6A modification induced by FTO downregulation promotes hippocampal neuron injury and aggravates the progression of epilepsy in a rat model.

Epilepsy is a common chronic neurological disorder characterized by widespread neuronal death. The purpose of this study was to investigate the role of nuclear factor erythroid 2-related factor 2 (Nrf2) m6A methylation in epilepsy. To create epileptic models, the rats were given Lithium chloride and pilocarpine, and isolated primary rat hippocampal neurons were cultured in an Mg2+ -free medium. The frequency of seizures was recorded in the epilepsy group of rats. The functional tests included TUNEL, MTT, and flow cytometry. Mechanistically, RNA degradation assay, RNA immunoprecipitation, and methylated RNA immunoprecipitation were performed. In epileptic models, Nrf2 and fat mass and obesity-associated (FTO) levels were downregulated, whereas YT521-B homology (YTH) domain family protein 2 (YTHDF2) was upregulated. Additionally, in epileptic models, there was a rise in the m6A methylation level of Nrf2 mRNA. Overexpressing FTO increased cell viability and reduced apoptosis, but Nrf2 interference reversed these effects. Meanwhile, FTO overexpression decreased the m6A methylation of Nrf2 mRNA. Moreover, YTHDF2 bound to Nrf2 mRNA and decreased its stability. Furthermore, FTO overexpression reduced seizure frequency in rats and inhibited hippocampal neuron apoptosis via lowering the m6A methylation level of Nrf2 mRNA. Overexpressing FTO reduced m6A methylation of Nrf2 mRNA, increased cell viability, suppressed apoptosis, and slowed the progression of epileptic diseases, which is linked to YTHDF2 binding to m6A-modified Nrf2 and promoting its degradation, as well as downregulating Nrf2 expression in hippocampal neurons.

A modified Fangji Huangqi decoction ameliorates pulmonary artery hypertension via phosphatidylinositide 3-kinases/protein kinase B-mediated regulation of proliferation and apoptosis of smooth muscle cells in vitro and in vivo.

ETHNOPHARMACOLOGICAL RELEVANCE: Pulmonary artery hypertension (PAH) is a progressive and fatal lung disease of multifactorial etiology, which arouses an enhanced interest in PAH disease therapy. Modified Fangji Huangqi decoction (MFJHQ), a traditional Chinese medicine (TCM) formula, has a crucial role in the treatment of PAH. However, the pharmacological roles and mechanisms of MFJHQ on PAH remain unknown. AIM OF THE STUDY: To investigate the effects and potential mechanism of MFJHQ on pulmonary vascular remodeling in PAH. MATERIAL AND METHODS: Ultra-performance liquid chromatography (UPLC) was employed to quantitate the principal components in MFJHQ. Rats were treated with MFJHQ by gavage for final 2 weeks in monocrotaline (MCT)-induced PAH rats. RNA-sequencing and network pharmacology analysis were performed to explore the potential mechanism. The primary rat pulmonary artery smooth muscle cells (PASMCs) were utilized to evaluate the regulatory effect of MFJHQ in vitro. RESULTS: Seven active components from MFJHQ were quantitated by UPLC. In rats with MCT-induced PAH, MFJHQ treatment significantly improved hemodynamic parameters, right ventricular hypertrophy index, lung function, and attenuated pulmonary vascular remodeling. Mechanistically, we further confirmed that MFJHQ inhibits MCT-induced phosphatidylinositide 3-kinases/protein kinase B (PI3K/Akt) pathway predicated by network pharmacology and RNA-sequencing analysis to reduce the proliferation of pulmonary arteries and promote pulmonary artery apoptosis in lung tissues. Additionally, MFJHQ hindered the proliferation and migration, and accelerated apoptosis in PDGF-BB-induced PASMCs in vitro, which can be enhanced by the presence of the PI3K inhibitor LY294002. CONCLUSIONS: Our results indicated that MFJHQ inhibited MCT-induced pulmonary vascular remodeling by decreasing proliferation and migration of PASMCs and promoting PASMC apoptosis through PI3K/Akt pathway, which provides a novel treatment option for PAH with multi-targeting mechanisms inspired by TCM theory.

Effects of Resveratrol on Pulmonary Fibrosis via TGF-ß/Smad/ERK Signaling Pathway.

Pulmonary fibrosis (PF) is a progressive pulmonary disease with no effective treatment and high mortality. Resveratrol has shown promising benefits in the treatment of PF. However, the probable efficacy and underlying mechanism of resveratrol in PF treatment remain unclear. This study investigates the intervention effects and potential mechanisms underpinning the treatment of PF with resveratrol. The histopathological analysis of lung tissues in PF rats showed that resveratrol improved collagen deposition and reduced inflammation. Resveratrol decreased the levels of collagen, glutathione, superoxide dismutase, myeloperoxidase, and hydroxyproline, lowered total anti-oxidant capacity, and suppressed the migration of TGF-[Formula: see text]1 and LPS-induced 3T6 fibroblasts. With resveratrol intervention, the protein and RNA expressions of TGF-[Formula: see text]1, a-SMA, Smad3/4, p-Smad3/4, CTGF, and p-ERK1/2 were markedly downregulated. Similarly, the protein and RNA expression levels of Col-1 and Col-3 were significantly downregulated. However, Smad7 and ERK1/2 were evidently upregulated. The protein and mRNA expression levels of TGF-[Formula: see text], Smad, and p-ERK correlated positively with the lung index, while the protein and mRNA expression levels of ERK correlated negatively with the lung index. These results reveal that resveratrol may have therapeutic effects on PF by reducing collagen deposition, oxidation, and inflammation. The mechanism is associated with the regulation of the TGF-[Formula: see text]/Smad/ERK signaling pathway.

Activation of necroptosis pathway in podocyte contributes to the pathogenesis of focal segmental glomerular sclerosis.

BACKGROUND: Focal segmental glomerulosclerosis (FSGS) is characterized by podocyte damage and severe proteinuria. The exact mechanism of podocyte damage and loss remains unclear. Necroptosis, a lytic form of programmed cell death mediated by RIP3 and MLKL, has emerged as an important cell death pattern in multiple tissues and cell types. Necroptosis in FSGS has not been investigated. METHODS: Public GEO data regarding podocyte treated with vehicle or adriamycin (ADR) was identified and analyzed. Cultured human podocytes were used to explore the activation of necroptosis upon ADR stimulation. The expression of necroptosis pathway molecules, p-RIP3 and p-MLKL, was examined in the glomeruli and defoliated urinary podocytes of patients with FSGS. The effect of necroptosis inhibition was assessed in ADR-induced glomerulopathy mice using GSK872. RESULTS: Publicly available RNA-sequencing data analysis showed that both necroptosis and NLRP3 inflammasome pathway were up-regulated in ADR-injured podocyte. Immunofluorescent staining showed increased expression of p-RIP3 and p-MLKL, the active forms of RIP3 and MLKL, in podocytes of FSGS patients and ADR-induced glomerulopathy mice but not in the normal control. GSK872, an RIP3 kinase inhibitor, significantly inhibited the expression of p-RIP3, p-MLKL and activation of NLRP3 in cultured podocytes treated with ADR. GSK872 treatment of mice with ADR-induced nephropathy resulted in the reduced expression of p-RIP3, p-MLKL, NLRP3 and caspase-1 p20. GSK872 also significantly inhibited the expression of p-MLKL in the podocytes of ADR-induced nephropathy, resulting in the attenuation of proteinuria and renal histological lesions. CONCLUSION: Necroptosis pathway might be a valuable target for the treatment of FSGS.

RNA Drugs and RNA Targets for Small Molecules: Principles, Progress, and Challenges.

RNA-based therapies, including RNA molecules as drugs and RNA-targeted small molecules, offer unique opportunities to expand the range of therapeutic targets. Various forms of RNAs may be used to selectively act on proteins, transcripts, and genes that cannot be targeted by conventional small molecules or proteins. Although development of RNA drugs faces unparalleled challenges, many strategies have been developed to improve RNA metabolic stability and intracellular delivery. A number of RNA drugs have been approved for medical use, including aptamers (e.g., pegaptanib) that mechanistically act on protein target and small interfering RNAs (e.g., patisiran and givosiran) and antisense oligonucleotides (e.g., inotersen and golodirsen) that directly interfere with RNA targets. Furthermore, guide RNAs are essential components of novel gene editing modalities, and mRNA therapeutics are under development for protein replacement therapy or vaccination, including those against unprecedented severe acute respiratory syndrome coronavirus pandemic. Moreover, functional RNAs or RNA motifs are highly structured to form binding pockets or clefts that are accessible by small molecules. Many natural, semisynthetic, or synthetic antibiotics (e.g., aminoglycosides, tetracyclines, macrolides, oxazolidinones, and phenicols) can directly bind to ribosomal RNAs to achieve the inhibition of bacterial infections. Therefore, there is growing interest in developing RNA-targeted small-molecule drugs amenable to oral administration, and some (e.g., risdiplam and branaplam) have entered clinical trials. Here, we review the pharmacology of novel RNA drugs and RNA-targeted small-molecule medications, with a focus on recent progresses and strategies. Challenges in the development of novel druggable RNA entities and identification of viable RNA targets and selective small-molecule binders are discussed. SIGNIFICANCE STATEMENT: With the understanding of RNA functions and critical roles in diseases, as well as the development of RNA-related technologies, there is growing interest in developing novel RNA-based therapeutics. This comprehensive review presents pharmacology of both RNA drugs and RNA-targeted small-molecule medications, focusing on novel mechanisms of action, the most recent progress, and existing challenges.

Optimized Biobanking Procedures for Preservation of RNA in Tissue: Comparison of Snap-Freezing and RNAlater-Fixation Methods.

Introduction: Personalized treatment, supported by biomarkers, would improve survival of ovarian cancer patients. RNA molecules are potentially important biomarkers. The Danish CancerBiobank provides an infrastructure for handling and storage of biological material, including RNA, from Danish cancer patients. The aim of this study was to investigate the effects of handling-time and fresh-freezing versus RNAlater® fixation on RNA degradation in solid tissue from pelvic mass samples. Materials and Methods: We evaluated RNA quality in surgical tissue from patients with a pelvic mass. Corresponding samples were either fresh-frozen or fixed in RNAlater, at eight different time points after the surgery. Integrity was measured using a bioanalyzer, and the amount and quality were further investigated by quantitative reverse transcription-polymerase chain reaction measuring the expression of housekeeping genes B2M and HPRT1. Results: Our results show that tissue RNA is stable up to at least 180 minutes after the surgery, as the quality was comparable to the quality of RNA handled immediately. Likewise, patient RNA was of acceptable quality after both fresh-frezing and RNAlater fixation, but RNAlater fixation was slightly more effective for RNA preservation. Discussion and Conclusion: Our data suggest that RNA in pelvic mass samples is relatively stable. Knowledge about RNA stability is an important prerequisite for research in RNA biomarkers, where the challenge is to balance the need for careful RNA handling and storage with the need for effective large-scale biobanking in a busy clinical setting where patient treatment is the main priority.

RNA Therapeutics for Tissue Engineering.

RNA has several theoretical advantages as a therapeutic agent, but exploiting RNA in this fashion has faced several major hurdles. Not only is it unstable and difficult to deliver across hydrophobic cell membranes, but extraneous RNA also activates innate immune nucleic acid sensors, which makes it toxic and inflammatory.

Structure and Composition Define Immunorecognition of Nucleic Acid Nanoparticles.

Nucleic acid nanoparticles (NANPs) have evolved as a new class of therapeutics with the potential to detect and treat diseases. Despite tremendous advancements in NANP development, their immunotoxicity, one of the major impediments in clinical translation of traditional therapeutic nucleic acids (TNAs), has never been fully characterized. Here, we describe the first systematically studied immunological recognition of 25 representative RNA and DNA NANPs selected to have different design principles and physicochemical properties. We discover that, unlike traditional TNAs, NANPs used without a delivery carrier are immunoquiescent. We show that interferons (IFNs) are the key cytokines triggered by NANPs after their internalization by phagocytic cells, which agrees with predictions based on the experiences with TNAs. However, in addition to type I IFNs, type III IFNs also serve as reliable biomarkers of NANPs, which is usually not characteristic of TNAs. We show that overall immunostimulation relies on NANP shapes, connectivities, and compositions. We demonstrate that, like with traditional TNAs, plasmacytoid dendritic cells serve as the primary interferon producers among all peripheral blood mononuclear cells treated with NANPs, and scavenger receptor-mediated uptake and endosomal Toll-like receptor signaling are essential for NANP immunorecognition. The TLR involvement, however, is different from that expected for traditional TNA recognition. Based on these results, we suggest that NANP technology may serve as a prototype of auxiliary molecular language for communication with the immune system and the modulation of immune responses.

Control the intracellular NF-κB activity by a sensor consisting of miRNA and decoy.

Many diseases are associated with the abnormal activation of NF-κB and its signaling pathway. NF-κB has become an important target for disease treatment and development of new drugs. Many various NF-κB inhibitors were therefore developed; however, they have difficulties to become clinical drugs due to their adverse side effects resulted from the affected normal physiological functions of this transcription factor. To overcome this limitation, this study construct a transgenic vector that can express an artificial miRNA targeting NF-κB RelA under the control of a NF-κB-specific promoter. The promoter consists of a NF-κB decoy and a minimal promoter. The vector was named as decoy minimal promoter-artificial microRNA (DMP-amiRNA). This study verified that this vector can sense and control the intracellular NF-κB activity upon transfection. Working of the vector forms a perfect feedback loop that realizes the NF-κB self-control. With the vector in cells, the higher NF-κB activity, the higher DMP transcriptional activity, and the more amiR533 expression. DMP-amiRNA can moderately inhibit the intracellular NF-κB activity but exert no significant effect on cell viability. This study therefore develops a new strategy for inhibiting over activity of NF-κB, which should has great potential in clinical application.

Patient-derived xenografts for childhood solid tumors: a valuable tool to test new drugs and personalize treatments

The use of preclinical models is essential in translational cancer research and especially important in pediatric cancer given the low incidence of each particular type of cancer. Cell line cultures have led to significant advances in cancer biology. However, cell lines have adapted to growth in artificial culture conditions, thereby undergoing genetic and phenotypic changes which may hinder the translational application. Tumor grafts developed in mice from patient tumor tissues, generally known as patient-derived xenografts (PDXs), are interesting alternative approaches to reproducing the biology of the original tumor. This review is focused on highlighting the interest of PDX models in pediatric cancer research and supporting strategies of personalized medicine. This review provides: (1) a description of the background of PDX in cancer, (2) the particular case of PDX in pediatric cancer, (3) how PDX can improve personalized medicine strategies, (4) new methods to increase engraftment, and, finally, (5) concluding remarks (AU)

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Nonclinical Safety Testing of RNA Vaccines.

In this chapter, we first consider the overall goal of nonclinical safety testing during drug development and have a brief overview of its regulatory background. We then discuss some basic requirements of safety/toxicity testing before concentrating on the safety testing of RNA vaccines and developing a sample RNA vaccine safety testing program.

2'Fluoro Modification Differentially Modulates the Ability of RNAs to Activate Pattern Recognition Receptors.

Although the use of RNAs has enormous therapeutic potential, these RNA-based therapies can trigger unwanted inflammatory responses by the activation of pattern recognition receptors (PRRs) and cause harmful side effects. In contrast, the immune activation by therapeutic RNAs can be advantageous for treating cancers. Thus, the immunogenicity of therapeutic RNAs should be deliberately controlled depending on the therapeutic applications of RNAs. In this study, we demonstrated that RNAs containing 2'fluoro (2'F) pyrimidines differentially controlled the activation of PRRs. The activity of RNAs that stimulate toll-like receptors 3 and 7 was abrogated by the incorporation of 2'F pyrimidine. By contrast, incorporation of 2'F pyrimidines enhanced the activity of retinoic acid-inducible gene 1-stimulating RNAs. Furthermore, we found that transfection with RNAs containing 2'F pyrimidine and 5' triphosphate (5'ppp) increased cell death and interferon-ß expression in human cancer cells compared with transfection with 2'hydroxyl 5'ppp RNAs, whereas RNAs containing 2'O-methyl pyrimidine and 5'ppp completely abolished the induction of cell death and cytokine expression in the cells. Our findings suggest that incorporation of 2'F and 2'O-methyl nucleosides is a facile approach to differentially control the ability of therapeutic RNAs to activate or limit immune and inflammatory responses depending on therapeutic applications.

No impact of DvSnf7 RNA on honey bee (Apis mellifera L.) adults and larvae in dietary feeding tests.

The honey bee (Apis mellifera L.) is the most important managed pollinator species worldwide and plays a critical role in the pollination of a diverse range of economically important crops. This species is important to agriculture and historically has been used as a surrogate species for pollinators to evaluate the potential adverse effects for conventional, biological, and microbial pesticides, as well as for genetically engineered plants that produce pesticidal products. As part of the ecological risk assessment of MON 87411 maize, which expresses a double-stranded RNA targeting the Snf7 ortholog (DvSnf7) in western corn rootworm (Diabrotica virgifera virgifera), dietary feeding studies with honey bee larvae and adults were conducted. Based on the mode of action of the DvSnf7 RNA in western corn rootworm, the present studies were designed to be of sufficient duration to evaluate the potential for adverse effects on larval survival and development through emergence and adult survival to a significant portion of the adult stage. Testing was conducted at concentrations of DvSnf7 RNA that greatly exceeded environmentally relevant exposure levels based on expression levels in maize pollen. No adverse effects were observed in either larval or adult honey bees at these high exposure levels, providing a large margin of safety between environmental exposure levels and no-observed-adverse-effect levels.

Results of xenogeneic I-RNA therapy in patients with metastatic renal cell carcinoma.

Six patients with metastatic renal cell carcinoma were treated with five intravenous infusions (every other day) of autologous lymphocytes incubated in vitro with I-RNA extracted from the lymphoid tissue of guinea pigs immunized with the patient's own tumor. No toxicity was evident. One patient showed regression of multiple pulmonary metastases beginning three months after therapy with complete remission by six months. She remained without evidence of disease until 18 months after therapy. Two other patients had more than 50% regression of measurable metastases lasting eight and ten months after therapy. Two patients showed stabilization of previously growing renal cell carcinoma pulmonary metastases. A single patient with renal cell carcinoma metastatic to brain had progressive tumor growth after a single I-RNA treatment. Serial peripheral blood lymphocyte samples obtained from each of the patients during I-RNA therapy demonstrated progressive increase in in vitro cytolysis of allogeneic renal cell carcinoma targets. Boosts in cytolytic effect were shown in all patients during I-RNA treatment regardless of their subsequent clinical course. These results seem to justify a randomized, prospective trial of xenogeneic I-RNA therapy in renal cell carcinoma patients with lesser tumor burden.