UTX inhibition suppresses proliferation and promotes apoptosis in patient-derived glioblastoma stem cells by modulating periostin expression.

Glioblastoma stem cells (GSCs) exert a crucial influence on glioblastoma (GBM) development, progression, resistance to therapy, and recurrence, making them an attractive target for drug discovery. UTX, a histone H3K27 demethylase, participates in regulating multiple cancer types. However, its functional role in GSCs remains insufficiently explored. This study aims to investigate the role and regulatory mechanism of UTX on GSCs. Analysis of TCGA data revealed heightened UTX expression in glioma, inversely correlating with overall survival. Inhibiting UTX suppressed GBM cell growth and induced apoptosis. Subsequently, we cultured primary GSCs from three patients, observing that UTX inhibition suppressed cell proliferation and induced apoptosis. RNA-seq was performed to analyze the gene expression changes after silencing UTX in GSCs. The results indicated that UTX-mediated genes were strongly correlated with GBM progression and regulatory tumor microenvironment. The transwell co-cultured experiment showed that silencing UTX in the transwell chamber GSCs inhibited the well plate cell proliferation. Protein-protein interaction analysis revealed that periostin (POSTN) played a role in the UTX-mediated transcriptional regulatory network. Replenishing POSTN reversed the effects of UTX inhibition on GSC proliferation and apoptosis. Our study demonstrated that UTX inhibition hindered POSTN expression by enhancing the H3K27me2/3 level, eventually resulting in inhibiting proliferation and promoting apoptosis of patient-derived GSCs. Our findings may provide a novel and effective strategy for the treatment of GBM.

miR-155 facilitates the synergistic replication between avian leukosis virus subgroup J and reticuloendotheliosis virus by targeting a dual pathway.

IMPORTANCE: The synergy of two oncogenic retroviruses is an essential phenomenon in nature. The synergistic replication of ALV-J and REV in poultry flocks increases immunosuppression and pathogenicity, extends the tumor spectrum, and accelerates viral evolution, causing substantial economic losses to the poultry industry. However, the mechanism of synergistic replication between ALV-J and REV is still incompletely elusive. We observed that microRNA-155 targets a dual pathway, PRKCI-MAPK8 and TIMP3-MMP2, interacting with the U3 region of ALV-J and REV, enabling synergistic replication. This work gives us new targets to modulate ALV-J and REV's synergistic replication, guiding future research on the mechanism.

Therapeutic stapled peptides: Efficacy and molecular targets.

Peptide stapling, by employing a stable, preformed alpha-helical conformation, results in the production of peptides with improved membrane permeability and enhanced proteolytic stability, compared to the original peptides, and provides an effective solution to accelerate the rapid development of peptide drugs. Various reviews present peptide stapling chemistries, anchoring residues and one- or two-component cyclization, however, therapeutic stapled peptides have not been systematically summarized, especially focusing on various disease-related targets. This review highlights the latest advances in therapeutic peptide drug development facilitated by the application of stapling technology, including different stapling techniques, synthetic accessibility, applicability to biological targets, potential for solving biological problems, as well as the current status of development. Stapled peptides as therapeutic drug candidates have been classified and analysed mainly by receptor- and ligand-based stapled peptide design against various diseases, including cancer, infectious diseases, inflammation, and diabetes. This review is expected to provide a comprehensive reference for the rational design of stapled peptides for different diseases and targets to facilitate the development of therapeutic peptides with enhanced pharmacokinetic and biological properties.

GPR15-C10ORF99 functional pairing initiates colonic Treg homing in amniotes.

Regulatory T lymphocyte (Treg) homing reactions mediated by G protein-coupled receptor (GPCR)-ligand interactions play a central role in maintaining intestinal immune homeostasis by restraining inappropriate immune responses in the gastrointestinal tract. However, the origin of Treg homing to the colon remains mysterious. Here, we report that the C10ORF99 peptide (also known as CPR15L and AP57), a cognate ligand of GPR15 that controls Treg homing to the colon, originates from a duplication of the flanking CDHR1 gene and is functionally paired with GPR15 in amniotes. Evolutionary analysis and experimental data indicate that the GPR15-C10ORF99 pair is functionally conserved to mediate colonic Treg homing in amniotes and their expression patterns are positively correlated with herbivore diet in the colon. With the first herbivorous diet in early amniotes, a new biological process (herbivorous diet short-chain fatty acid-C10ORF99/GPR15-induced Treg homing colon immune homeostasis) emerged, and we propose an evolutionary model whereby GPR15-C10ORF99 functional pairing has initiated the first colonic Treg homing reaction in amniotes. Our findings also highlight that GPCR-ligand pairing leads to physiological adaptation during vertebrate evolution.

All-hydrocarbon stapling enables improvement of antimicrobial activity and proteolytic stability of peptide Figainin 2.

Figainin 2 is a cationic, hydrophobic, α-helical host-defense peptide with 28 residues, which was isolated from the skin secretions of the Chaco tree frog. It shows potent inhibitory activity against both Gram-negative and Gram-positive pathogens and has garnered considerable interest in developing novel classes of natural antibacterial agents. However, as a linear peptide, conformational flexibility and poor proteolytic stability hindered its development as antibacterial agent. To alleviate its susceptibility to proteolytic degradation and improve its antibacterial activity, a series of hydrocarbon-stable analogs of Figainin 2 were synthesized and evaluated for their secondary structure, protease stability, antimicrobial, and hemolytic activities. Among them, F2-12 showed significant improvement in protease resistance and antimicrobial activity compared to that of the template peptide. This study provides a promising strategy for the development of antimicrobial drugs.

Interaction between Avian Leukosis Virus Subgroup J Surface Protein and Doublecortin-Like Kinase 1 Accelerates Cell Proliferation and Epithelial-Mesenchymal Transition.

Avian leukosis virus subgroup J (ALV-J) induces myelocytomas, which can metastasize to multiple organs in diseased chickens. Although metastasis is the primary cause of death in such cases, the mechanism for it remains unclear. Here, we found that interaction between ALV-J surface protein (SU) and doublecortin-like kinase 1 (DCLK1) promotes epithelial-mesenchymal transition (EMT) and cell proliferation. We found that ALV-J can activate EMT in infected cells. Subsequently, proteomics analysis revealed that DCLK1, a well-established putative tumor stem cell marker, which is highly expressed in ALV-J-infected DF-1 cells and chickens, might be a potential factor mediating EMT. Furthermore, using immunofluorescence and immunoprecipitation, we verified that SU interacts with DCLK1. Functional studies suggested that overexpression of DCLK1 increased viral replication and promoted cell proliferation by accelerating the progression of cells from the G0/G1 phase to the S phase of cell cycle, whereas RNA interference of DCLK1 reduced viral replication and arrested cell proliferation by retarding cell cycle progression from the late G1 phase into the S phase in ALV-J-infected cells. Moreover, we demonstrate that the increased accumulation of DCLK1 promotes EMT by increasing the expression of N-cadherin, vimentin, MMP2, and transcription factor Snail1 and decreasing the expression of epithelial marker E-cadherin. These results suggest that ALV-J SU interacts with DCLK1, and accelerates cell proliferation, leading to increased viral replication and ultimately activating EMT, which paves the way for tumor metastasis. IMPORTANCE Tumor metastasis is a major challenge in cancer research, because of its systemic nature and the resistance of disseminated tumor cells to existing therapeutic agents. It is estimated that >90% of mortality from cancer is attributable to metastases. We found that ALV-J can activate EMT, which plays a critical role in cancer metastasis. Subsequently, we identified a tumor stem cell marker, DCLK1, in ALV-J infected cells, which interacts with surface protein (SU) of ALV-J to promote virus replication, activate EMT, and accelerate cell proliferation enabling ALV-J to obtain metastatic ability. Understanding the process of participation of ALV-J in EMT and the route of metastasis will help elucidate the mechanism of virus-induced tumor metastasis and help identify promising molecular targets and key obstacles for ALV-J control and clinical technology development.

Differential MC5R loss in whales and manatees reveals convergent evolution to the marine environment.

Melanocortin 5 receptor (MC5R), which is expressed in the terminally differentiated sebaceous gland, is a G protein-coupled receptor (GPCR). MC5R exists mostly in mammals but is completely lost in whales; only the relic of MC5R can be detected in manatees, and phenotypically, they have lost sebaceous glands. Interestingly, whales and manatees are both aquatic mammals but have no immediate common ancestors. The loss of MC5R and sebaceous glands in whales and manatees is likely to be a result of convergent evolution. Here, we find that MC5R in whales and manatees are lost by two different mechanisms. Homologous recombination of MC5R in manatees and the insertion of reverse transcriptase in whales lead to the gene loss, respectively. On one hand, in manatees, there are two "TTATC" sequences flanking MC5R, and homologous recombination of the segments between the two "TTATC" sequences resulted in the partial loss of the sequence of MC5R. On the other hand, in whales, reverse transcriptase inserts between MC2R and RNMT on the chromosome led to the loss of MC5R. Based on these two different mechanisms for gene loss in whales and manatees, we finally concluded that MC5R loss might be the result of convergent evolution to the marine environment, and we explored the impact on biological function that is significant to environmental adaptation.

Efficient Pathogen Capture and Sensing Promoted by Dynamic Deformable Nanointerfaces.

The M13 bacteriophage (M13 phage) has emerged as an attractive bionanomaterial due to its chemistry/gene modifiable feature and unique structures. Herein, a dynamic deformable nanointerface is fabricated taking advantage of the unique feature of the M13 phage for ultrasensitive detection of pathogens. PIII proteins at the tip of the M13 phage are genetically modified to display 6His peptide for site-specific anchoring onto Ni-NTA microbeads, whereas pVIII proteins along the side of the M13 phage are orderly arranged with thousands of aptamers and their complementary strands (c-apt). The flexible M13 nanofibers with rich recognition sites act as octopus tentacles, resulting in a 19-fold improvement in the capture affinity toward the target. The competitive binding of the target pathogen releases c-apts and initiates rolling circle amplification (RCA). The sway motion of M13 nanofibers accelerates the diffusion of c-apts, thus promoting RCA efficiency. Benefiting from the strengthened capture ability toward the target and the accelerated RCA process, three-orders of magnitude improvement in the sensitivity is achieved, with a detection limit of 8 cfu mL-1 for Staphylococcus aureus. The promoted capture ability and assay performance highlights the essential role of the deformable feature of the engineered interface. This may provide inspiration for the construction of more efficient reaction interfaces.

TMT-based proteomic analysis reveals integrins involved in the synergistic infection of reticuloendotheliosis virus and avian leukosis virus subgroup J.

BACKGROUND: Co-infection with the avian leukosis virus subgroup J (ALV-J) and the reticuloendotheliosis virus (REV) increases mutual viral replication, causing a more serious pathogenic effect by accelerating the progression of neoplasia and extending the tumor spectrum. However, the molecular mechanism underlying the synergistic replication of ALV-J and REV remains unclear. RESULTS: Here, we performed this study to compare the differentially expressed proteins among CEF cells infected with ALV-J, REV or both at the optimal synergistic infection time using TMT-based quantitative proteomics. We identified a total of 719 (292 upregulated and 427 downregulated) and 64 (35 upregulated and 29 downregulated) proteins by comparing co-infecting both viruses with monoinfecting ALV-J and REV, respectively. GO annotation and KEGG pathway analysis showed the differentially expressed proteins participated in virus-vector interaction, biological adhesion and immune response pathways in the synergistic actions of ALV-J and REV at the protein levels. Among the differentially expressed proteins, a large number of integrins were inhibited or increased in the co-infection group. Further, eight integrins, including ITGα1, ITGα3, ITGα5, ITGα6, ITGα8, ITGα9, ITGα11 and ITGß3, were validated in CEF cells by qRT-PCR or western blot. CONCLUSIONS: These findings proved that integrins may be key regulators in the mechanism of synergistic infection of REV and ALV-J, which will provide more insight into the pathogenesis of synergism of REV and ALV-J at protein level.

Decarboxylative Addition of Propiolic Acids with Indoles to Synthesize Bis(indolyl)methane Derivatives with a Pd(II)/LA Catalyst.

Exploring new protocols for efficient organic synthesis is crucial for pharmaceutical developments. The present work introduces a Pd(II)/LA-catalyzed (LA: Lewis acid) decarboxylative addition reaction for the synthesis of bis(indolyl)methane derivatives. The presence of Lewis acid such as Sc(OTf)3 triggered Pd(II)-catalyzed decarboxylative addition of propiolic acids with indoles to offer the bis(indolyl)methane derivatives in moderate to good yields, whereas neither Pd(II) nor Lewis acid alone was active for this synthesis. The catalytic efficiency of Pd(OAc)2 was highly dependent on the Lewis acidity of the added Lewis acid, that is, a stronger Lewis acid provided a higher yield of the bis(indolyl)methane derivatives. Meanwhile, this Pd(II)/LA-catalyzed decarboxylative addition reaction showed good tolerance toward versatile electron-rich or -deficient substituents on the indole skeleton and on the benzyl ring of propiolic acids. The studies on the in situ 1H NMR kinetics of this Pd(II)/Sc(III) catalysis disclosed the formation of a transient vinyl-Pd(II)/Sc(III) intermediate generated by the pyrrole addition to the alkynyl-Pd(II)/Sc(III) species after decarboxylation, which was scarcely observed before.

Palladium(II)/Lewis Acid-Catalyzed Oxidative Olefination/Annulation of N-Methoxybenzamides: Identifying the Active Intermediates through NMR Characterizations.

Although Pd(II)-catalyzed C-H activation in arenes has been widely successful in organic synthesis with many palladacycle compounds isolated as the intermediates in ligand-directed C-H activation, direct identification of the reaction intermediates such as the π-complex prior to the C-H activation is still not successful because of their instability. In the present study, we introduce a Pd(II)/LA (LA: Lewis acid)-catalyzed oxidative olefination/annulation reaction between N-methoxybenzamides and acrylates with oxygen as the oxidant source, in which two intermediates, including an unsymmetrical η6-complex and a palladacycle species without the proton releasing to the environment, were identified through NMR characterizations. The in situ formation of the heterobimetallic Pd(II)/LA species such as Pd(II)/Sc(III) may have enhanced the electrophilic properties of the Pd2+ cation, thus improving the stability of the π-complex, herein, an unsymmetrical η6-complex, and improving its catalytic efficiency. The observed insensitive electronic effect preferred the concerted metalation-deprotonation (CMD) mechanism for this C-H activation, and the detected palladacycle intermediate without the proton releasing to the environment offered an experimental clue to support the proposed CMD mechanism.

Trends of incidence and prognosis of gastric neuroendocrine neoplasms: a study based on SEER and our multicenter research.

BACKGROUND: To investigate the recent epidemiological trends of gastric neuroendocrine neoplasms (GNENs) and establish a new tool to estimate the prognosis of gastric neuroendocrine carcinoma (GNEC) and gastric neuroendocrine tumor (GNET). METHODS: Nomograms were established based on a retrospective study on patients diagnosed with GNENs from 1975 to 2016 in Surveillance, Epidemiology and End Results database. External validation was performed among 246 GNENs patients in Jiangsu province to verify the discrimination and calibration of the nomograms. RESULTS: The age-adjusted incidence of GNENs has increased from 0.309 to 6.149 per 1,000,000 persons in the past 4 decades. Multivariate analysis indicated independent prognostic factors for both GNEC and GNET including age, distant metastasis and surgical intervention (P < 0.05). In addition, T, N staging and grade were significantly associated with survival of GNEC, while size was a predictor for GNET (P < 0.05). The C-indexes of the nomograms were 0.840 for GNEC and 0.718 for GNET, which were higher than those of the 8th AJCC staging system (0.773 and 0.599). Excellent discrimination was observed in the validation cohorts (C-index of nomogram vs AJCC staging for GNEC: 0.743 vs 0.714; GNET: 0.945 vs 0.927). Survival rates predicted by nomograms were close to the actual survival rates in the calibration plots in both training and validation sets. CONCLUSIONS: The incidence of the GNENs is increasing steadily in the past 40 years. We established more excellent nomograms to predict the prognosis of GNENs than traditional staging system, helping clinicians to make tailored decisions.

Lewis Acid Promoted Aerobic Oxidative Coupling of Thiols with Phosphonates by Simple Nickel(II) Catalyst: Substrate Scope and Mechanistic Studies.

Exploring new catalysts for efficient organic synthesis is among the most attractive topics in chemistry. Here, using Ni(OAc)2/LA as catalyst (LA: Lewis acid), a novel catalyst strategy was developed for oxidative coupling of thiols and phosphonates to phosphorothioates with oxygen oxidant. The present study discloses that when Ni(OAc)2 alone was employed as the catalyst, the reaction proceeded very sluggishly with low yield, whereas adding non-redox-active metal ions such as Y3+ to Ni(OAc)2 dramatically promoted its catalytic efficiency. The promotional effect is highly Lewis acidity dependent on the added Lewis acid, and generally, a stronger Lewis acid provided a better promotional effect. The stopped-flow kinetics confirmed that adding Y(OTf)3 can obviously accelerate the activation of thiols by Ni(II) and next accelerate its reaction with phosphonate to generate the phosphorothioate product. ESI-MS characterizations of the catalyst disclosed the formation of the heterobimetallic Ni(II)/Y(III) species in the catalyst solution. Additionally, this Ni(II)/LA catalyst can be applied in the synthesis of a series of phosphorothioate compounds including several commercial bioactive compounds. This catalyst strategy has clearly supported that Lewis acid can significantly improve the catalytic efficiency of these traditional metal ions in organic synthesis, thus opening up new opportunities in their catalyst design.

Reticuloendotheliosis virus and avian leukosis virus subgroup J synergistically increase the accumulation of exosomal miRNAs.

BACKGROUND: Co-infection with avian leukosis virus subgroup J and reticuloendotheliosis virus induces synergistic pathogenic effects and increases mortality. However, the role of exosomal miRNAs in the molecular mechanism of the synergistic infection of the two viruses remains unknown. RESULTS: In this study, exosomal RNAs from CEF cells infected with ALV-J, REV or both at the optimal synergistic infection time were analysed by Illumina RNA deep sequencing. A total of 54 (23 upregulated and 31 downregulated) and 16 (7 upregulated and 9 downregulated) miRNAs were identified by comparing co-infection with two viruses, single-infected ALV-J and REV, respectively. Moreover, five key miRNAs, including miR-184-3p, miR-146a-3p, miR-146a-5p, miR-3538 and miR-155, were validated in both exosomes and CEF cells by qRT-PCR. GO annotation and KEGG pathway analysis of the miRNA target genes showed that the five differentially expressed miRNAs participated in virus-vector interaction, oxidative phosphorylation, energy metabolism and cell growth. CONCLUSIONS: We demonstrated that REV and ALV-J synergistically increased the accumulation of exosomal miRNAs, which sheds light on the synergistic molecular mechanism of ALV-J and REV.

miR-130b-3p/301b-3p negatively regulated Rb1cc1 expression on myogenic differentiation of chicken primary myoblasts.

OBJECTIVES: To explore the roles of miR-130b-3p and miR-301b-3p which may regulate Rb1-inducible coiled-coil 1 (Rb1cc1) expression during myogenic differentiation of chicken primary myoblasts. RESULTS: After 4 days of myogenic differentiation, myotubes appeared and after 6 days the cells fused to each other and expression of MyHC could be detected by immunofluorescence staining. TargetScan and RNAhybrid 2.2 showed miR-130b-3p and miR-301b-3p were well complementary with the target site of Rb1cc1 3'-untranslated region (3'-UTR). Using the dual-luciferase assay, we found miR-130b-3p and miR-301b-3p could inhibit Rb1cc1 expression by binding to its 3'-UTR. Real-time PCR showed Rb1cc1 mRNA expression level was almost reciprocal to that of miR-130b-3p or miR-301b-3p during myogenic differentiation. Furthermore, over-expression of miR-130b-3p or miR-301b-3p down-regulated the expression levels of Rb1cc1, myoblast determination protein, myogenin and myosin heavy chain. CONCLUSIONS: miR-130b-3p or miR-301b-3p negatively regulate Rb1cc1 expression to affect myogenic differentiation.

The effects of Nardosinone on levodopa intervention in the treatment of Parkinson's disease.

BACKGROUND: The roots and rhizomes of Nardostachys jatamansi DC. are reported to be useful for the treatment of Parkinson's disease (PD). Previous research has also shown that Nardosinone, the main active component isolated from Nardostachys jatamansi DC., exhibits the potential to treat PD. AIM OF THE STUDY: To investigate how the effects of Nardosinone could assist levodopa in the treatment of PD, how this process changes the intestinal flora, and to explore the effective forms of Nardosinone in the intestinal flora. MATERIAL AND METHODS: We used behavioral experiments, and hematoxylin-eosin staining and immunohistochemical staining, to investigate the effects of a combination of Nardosinone and levodopa on rotenone-induced PD rats. In addition, we used LC/MS-MS to determine the levels of levodopa, 5-hydroxytryptamine, dopamine and its metabolite 3, 4-dihydroxyphenylacetic acid, and homovanillic acid, to investigate the effect of the intestinal flora on co-administration in the treatment of PD. LC/MS-MS was also used to detect the metabolites of Nardosinone on the gastrointestinal tract and intestinal flora. RESULTS: The behavioral disorders and neuronal damage associated with PD were significantly improved following the co-administration. Analysis also revealed that the co-administration increased the levels of five neurotransmitters in the striatum, plasma and feces. In vitro experiments further demonstrated that the levels of dopamine and levodopa were increased in the intestinal flora. In total, five metabolites of Nardosinone were identified. CONCLUSION: Our findings indicate that Nardosinone and its metabolites might act as a potential adjutant to enhance the efficacy of levodopa via the intestinal flora, thus expanding the therapeutic potential of the combination of Chinese and Western medicine as a treatment method for PD.

Exploring the influence of microstructure and phospholipid type of liposomes on their interaction with lung.

Liposome is a promising carrier for pulmonary drug delivery and the nano-sized liposomes have been widely investigated in the treatment of lung diseases. However, there still lack the knowledge of micron-sized liposomes for lung delivery, which have more advantages in terms of drug loading and sustained drug release capacity. The micron-sized liposomes can be classified into multilamellar liposome (MLL) and multivesicular liposome (MVL) according to their microstructure, thus, this study focused on exploring how the micron-sized liposomes with different microstructure and phospholipid composition influence their interaction with the lung. The MLL and MVL were prepared from different types of phospholipids (including soya phosphatidylcholine (SPC), egg yolk phosphatidylcholine (EPC), and dipalmitoyl phosphatidylcholine (DPPC)) with geometric diameter around 5 µm, and their in vitro pulmonary cell uptake, in vivo lung retention and organ distribution were investigated. The results showed that the microstructure of liposomes didn't affect pulmonary cellular uptake, in vivo lung retention and organ distribution. MLL and MVL prepared with the same phospholipid had similar cellular uptake in both NR8383 cells and A549 cells, and both of them possessed prolonged lung retention and limited distribution in other organs during 72 h. Notably, the phospholipid type presented remarkable influence on liposomes' interaction with the lung. SPC-based liposomes exhibited higher cellular uptake than the DPPC-based ones in both NR8383 cells and A549 cells, also possessed a better lung retention behavior. In conclusion, this study might provide theoretical knowledge for designing micron-sized liposomes intended for lung delivery.

Screening and verification of hemostatic effective components group of Panax Notoginseng based on spectrum-effect relationships.

ETHNOPHARMACOLOGICAL RELEVANCE: Panax Notoginseng (PN) can disperse blood stasis, hemostasis, and detumescence analgesic, which can be used for hemoptysis, hematemesis and another traumatic bleeding, and it is known as "A miracle hemostatic medicine". Studies show that the chemical composition of PN is relatively comprehensive, however, its hemostatic active ingredients have not been fully clarified. AIM OF STUDY: This study aimed to clarify the hemostatic effective components group (HECG) of PN, provide a foundation for the assessment of PN's quality and its comprehensive development, and for further studies on the pharmacodynamic material basis of other Traditional Chinese Medicines (TCMs). MATERIALS AND METHODS: UPLC-MS was used to establish the fingerprint and identify the common peaks in 44 batches of PN extracts (PNE). In addition, the plasma recalcification time and in vitro coagulation time were measured. For spectrum-effect analysis, bivariate correlation analysis (BCA) and partial least squares regression analysis (PLSR) were used to screen the hemostasis candidate active monomers of PN. The monomers were prepared by combining several preparative chromatography techniques. The efficacy was verified by plasma recalcification time, in vitro coagulation time, and a rat model of gastric hemorrhage. RESULTS: A total of 30 common peaks and hemostatic efficacy indexes of 44 batches of PNE were obtained. A total of 18 components were positively correlated with the comprehensive coagulation index by two statistical methods. Six and eleven monomers were obtained respectively by chromatographic preparation and procurement, and one monomer was eliminated due to preparation difficulty and other reasons. Seven active monomers with direct hemostatic effect and one active monomer with synergistic hemostatic effect were screened through plasma recalcification time, and their combinations were used as candidate HECG for hemostatic effect verification. The results of in vitro experiments showed that plasma recalcification time and in vitro coagulation time were significantly reduced (P < 0.05) in the HECG group, compared to the PNE group. The results of in vivo experiment also indicated that the hemostatic effect of HECG was comparable to that of PNE and PN powder. CONCLUSION: The composition and efficacy of the HECG of PN were screened and verified using the spectral correlation method and in vivo and in vitro efficacy verification; the HECG included Dencichine, Ginsenoside Rg1, Ginsenoside Rd, Ginsenoside Rh1, Ginsenoside F1, Notoginsenoside R1, Notoginsenoside Ft1 and Notoginsenoside Fe. These results laid a foundation for the quality evaluation of PN and provided a reference for the basic research of pharmacodynamic material basis of other TCMs.

METTL3/YTHDF2 m6A axis mediates the progression of diabetic nephropathy through epigenetically suppressing PINK1 and mitophagy.

AIMS: This research aimed to investigate the specific mechanism of methyltransferase like 3 (METTL3) in the progression of diabetic kidney disease (DKD). MATERIALS AND METHODS: The model of diabetic kidney disease was established with HK-2 cells and mice in vitro and in vivo. The N6 methyladenosine (m6A) contents in the cells and tissues were detected with a commercial kit and the m6A levels of PTEN induced putative kinase 1 (PINK2) were detected with a MeRIP kit. The mRNA and protein levels were determined with RT-qPCR and western blot. The ROS, TNF-α, and IL-6 levels were assessed with ELISA. The cell proliferative ability was measured by a CCK-8 assay and cell apoptosis was determined with TUNEL staining. The HE and Masson staining was performed to observe the renal morphology. The RIP assay was conducted to detect the interaction between METTL3/YTHDF2 and PINK1. RESULTS: The m6A content and METTL3 levels were prominently elevated in diabetic kidney disease. METTL3 silencing promoted the cell growth and the expression of LC3 II, PINK1, and Parkin, while inhibiting the cell apoptosis and the expression of LC3 I and p62 in the high glucose (HG) stimulated HK-2 cells. METTL3 silencing also decreased the ROS, TNF-α, and IL-6 levels in diabetic kidney disease. PINK1 silencing neutralized the function of sh-METTL3 in the HG stimulated HK-2 cells. The HE and Masson staining showed that METTL3 silencing alleviated the kidney injury induced by DKD. METTL3 silencing decreased the m6A levels of PINK1, while increased the mRNA levels of PINK1 which depended on YTHDF2. CONCLUSIONS: METTL3 silencing could inhibit the progression of diabetic nephropathy in vivo and in vitro by regulating the m6A modification of PINK1, which depends on YTHDF2. Our research lays the theoretical foundation for the precise treatment of diabetic kidney disease and the development of targeted drugs in the future.