Plantaginis semen ameliorates diabetic kidney disease via targeting the sphingosine kinase 1/sphingosine-1-phosphate pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Plantaginis Semen (PS) is widely utilized as a common herb in several Asian countries, particularly China, due to its diuretic, anti-hypertensive, anti-hyperlipidemic, and anti-hyperglycemic properties. Furthermore, it is acknowledged for its ability to mitigate renal complications associated with metabolic syndrome. Despite its extensive usage, there is limited systematic literature elucidating its therapeutic mechanisms, thus emphasizing the necessity for comprehensive investigations in this field. AIM: This study aims to comprehensively evaluate the therapeutical potential of PS in treating diabetic kidney disease (DKD) and to elucidate the underlying mechanisms through in vivo and in vitro models. METHODS: The main composition of PS were characterized using the UPLC-QTOF-MS method. For the in vivo investigation, a mouse model mediated by streptozocin (STZ) associated with a high-fat diet (HFD) and unilateral renal excision was established. The mice were split into 6 groups (n = 8): control group (CON group), DKD group, low-dose of Plantago asiatica L. seed extract group (PASE-L group, 3 g/kg/d), medium-dose of PASE group (PASE-M, 6 g/kg/d), high-dose of PASE group (PASE-H, 9 g/kg/d), and positive drug group (valsartan, VAS group, 12 mg/kg/d). After 8 weeks of treatment, the damage induced by DKD was evaluated by using relevant parameters of urine and blood. Furthermore, indicators of inflammation and factors associated with the SphK1-S1P signaling pathway were investigated. For the in vitro study, the cell line HBZY-1 was stimulated by high glucose (HG), they were then co-cultured with different concentrations of PASE, and the corresponding associated inflammatory and sphingosine kinase 1/sphingosine-1-phosphate (SphK1-S1P) factors were examined. RESULTS: A total of 59 major components in PS were identified, including flavonoids, iridoids, phenylethanol glycosides, guanidine derivatives, and fatty acids. In the mouse model, PS was found to significantly improve body weight, decrease fasting blood glucose (FBG) levels, increased glucose tolerance and insulin tolerance, improved kidney-related markers compared to the DKD group, pathological changes in the kidneys also improved dramatically. These effects showed a dose-dependent relationship, with higher PASE concentrations yielding significantly better outcomes than lower concentrations. However, the effects of the low PASE concentration were not evident for some indicators. In the cellular model, the high dose of PASE suppressed high glucose (HG) stimulated renal mesangial cell proliferation, suppressed inflammatory factors and NF-κB, and decreased the levels of fibrillin-1(FN-1) and collagen IV(ColIV). CONCLUSION: Our results indicate that PS exerts favorable therapeutic effects on DKD, with the possible mechanisms including the inhibition of inflammatory pathways, suppression of mRNA levels and protein expressions of SphK1 and S1P, consequently leading to reduced overexpression of FN-1 and ColIV, thereby warranting further exploration.

NAT10 mediated ac4C acetylation driven m6A modification via involvement of YTHDC1-LDHA/PFKM regulates glycolysis and promotes osteosarcoma.

The dynamic changes of RNA N6-methyladenosine (m6A) during cancer progression participate in various cellular processes. However, less is known about a possible direct connection between upstream regulator and m6A modification, and therefore affects oncogenic progression. Here, we have identified that a key enzyme in N4-acetylcytidine (ac4C) acetylation NAT10 is highly expressed in human osteosarcoma tissues, and its knockdown enhanced m6A contents and significantly suppressed osteosarcoma cell growth, migration and invasion. Further results revealed that NAT10 silence inhibits mRNA stability and translation of m6A reader protein YTHDC1, and displayed an increase in glucose uptake, a decrease in lactate production and pyruvate content. YTHDC1 recognizes differential m6A sites on key enzymes of glycolysis phosphofructokinase (PFKM) and lactate dehydrogenase A (LDHA) mRNAs, which suppress glycolysis pathway by increasing mRNA stability of them in an m6A methylation-dependent manner. YTHDC1 partially abrogated the inhibitory effect caused by NAT10 knockdown in tumor models in vivo, lentiviral overexpression of YTHDC1 partially restored the reduced stability of YTHDC1 caused by lentiviral depleting NAT10 at the cellular level. Altogether, we found ac4C driven RNA m6A modification can positively regulate the glycolysis of cancer cells and reveals a previously unrecognized signaling axis of NAT10/ac4C-YTHDC1/m6A-LDHA/PFKM in osteosarcoma. Video Abstract.

M7G modification of FTH1 and pri-miR-26a regulates ferroptosis and chemotherapy resistance in osteosarcoma.

Doxorubicin and platinum are widely used in the frontline treatment of osteosarcoma, but resistance to chemotherapy limits its curative effect. Here, we have identified that METTL1 mediated N7-Methyladenosine (m7G) low expressed in osteosarcoma tissues, plays a critical oncogenic role, and enhances osteosarcoma chemosensitivity in osteosarcoma. Mechanistically, AlkAniline-Seq data revealed that Ferritin heavy chain (FTH1), the main component of ferritin, which is crucial for iron homeostasis and the inhibition of lipid peroxidation, is one of the top 10 genes with the most significant change in m7G methylation sites mediated by METTL1 in human osteosarcoma cells. Interestingly, METTL1 significantly increased the expression of FTH1 at the mRNA level but was remarkably suppressed at the protein level. We then identified primary (pri)-miR-26a and pri-miR-98 in the Top 20 m7G-methylated pri-miRNAs with highly conserved species. Further results confirmed that METTL1 enhances cell ferroptosis by targeting FTH1 and primary (pri)-miR-26a, promoting their maturity by enhancing RNA stability dependent on m7G methylation. The increase of mature miR-26a-5p that resulted from METTL1 overexpression could further target FTH1 mRNA and eliminate FTH1 translation efficiency. Moreover, the reduction of FTH1 translation dramatically increases cell ferroptosis and promotes the sensitivity of osteosarcoma cells to chemotherapy drugs. Collectively, our study demonstrates the METTL1/pri-miR-26a/FTH1 axis signaling in osteosarcoma and highlights the functional importance of METTL1 and m7G methylation in the progression and chemotherapy resistance of osteosarcoma, suggesting that reprogramming RNA m7G methylation as a potential and promising strategy for osteosarcoma treatment.

Exosomes secreted from cardiomyocytes suppress the sensitivity of tumor ferroptosis in ischemic heart failure.

Heart failure (HF) patients in general have a higher risk of developing cancer. Several animal studies have indicated that cardiac remodeling and HF remarkably accelerate tumor progression, highlighting a cause-and-effect relationship between these two disease entities. Targeting ferroptosis, a prevailing form of non-apoptotic cell death, has been considered a promising therapeutic strategy for human cancers. Exosomes critically contribute to proximal and distant organ-organ communications and play crucial roles in regulating diseases in a paracrine manner. However, whether exosomes control the sensitivity of cancer to ferroptosis via regulating the cardiomyocyte-tumor cell crosstalk in ischemic HF has not yet been explored. Here, we demonstrate that myocardial infarction (MI) decreased the sensitivity of cancer cells to the canonical ferroptosis activator erastin or imidazole ketone erastin in a mouse model of xenograft tumor. Post-MI plasma exosomes potently blunted the sensitivity of tumor cells to ferroptosis inducers both in vitro in mouse Lewis lung carcinoma cell line LLC and osteosarcoma cell line K7M2 and in vivo with xenograft tumorigenesis model. The expression of miR-22-3p in cardiomyocytes and plasma-exosomes was significantly upregulated in the failing hearts of mice with chronic MI and of HF patients as well. Incubation of tumor cells with the exosomes isolated from post-MI mouse plasma or overexpression of miR-22-3p alone abrogated erastin-induced ferroptotic cell death in vitro. Cardiomyocyte-enriched miR-22-3p was packaged in exosomes and transferred into tumor cells. Inhibition of cardiomyocyte-specific miR-22-3p by AAV9 sponge increased the sensitivity of cancer cells to ferroptosis. ACSL4, a pro-ferroptotic gene, was experimentally established as a target of miR-22-3p in tumor cells. Taken together, our findings uncovered for the first time that MI suppresses erastin-induced ferroptosis through releasing miR-22-3p-enriched exosomes derived from cardiomyocytes. Therefore, targeting exosome-mediated cardiomyocyte/tumor pathological communication may offer a novel approach for the ferroptosis-based antitumor therapy.

A Plantaginis Semen-Coptidis Rhizoma compound alleviates type 2 diabetic mellitus in mice via modulating AGEs-RAGE pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Plantaginis Semen-Coptidis Rhizoma Compound(CQC) was first recorded in Shengji Zonglu. Clinical and experimental studies have reported that both of Plantaginis Semen and Coptidis Rhizoma exerted the effects of lowering blood glocose and lipid. However, the potential mechanism of CQC on type 2 diabetes (T2DM) remain unclear. AIM OF THE STUDY: The main objective of our investigation was to explore the mechanisms of CQC on T2DM based on network pharmacology and experimental research. MATERIALS AND METHODS: Streptozotocin(STZ)/high fat diet(HFD)-induced T2DM models in mice were established to evaluate the antidiabetic effect of CQC in vivo. We obtained the chemical constituents of Plantago and Coptidis from the TCMSP database and literature sources. Potential targets of CQC were gleaned from the Swiss-Target-Prediction database, and T2DM targets were obtained from Drug-Bank, TTD, and DisGeNet. A protein-protein interaction (PPI) network was constructed in the String database. The David database was used for gene ontology (GO) and KEGG pathway enrichment analyses. We then verified the potential mechanism of CQC that were predicted by network pharmacological analysis in STZ/HFD-induced T2DM mouse model. RESULTS: Our experiments confirmed that CQC improved hyperglycemia and liver injury. We identified 21 components and gleaned 177 targets for CQC treatment of T2DM. The core component-target network included 13 compounds and 66 targets. We further demonstrated that CQC improve T2DM through various pathways, especially the AGEs/RAGE signal pathway. CONCLUSION: Our results indicated that CQC could improve the metabolic disorders of T2DM and it is a promising TCM compound for the treatment of T2DM. The potential mechanism may probably involve the regulation of the AGEs/RAGE signaling pathway.

METTL14 Regulates Osteogenesis of Bone Marrow Mesenchymal Stem Cells via Inducing Autophagy Through m6A/IGF2BPs/Beclin-1 Signal Axis.

The development of osteoporosis is often accompanied by autophagy disturbance, which also causes new osteoblast defects from bone marrow mesenchymal stem cells (BMSCs). However, the underlying molecular mechanisms are still not fully understood. Methyltransferase-like 14 (METTL14) is the main enzyme for N6-methyladenosine (m6A), the most prevalent internal modification in mammalian mRNAs, and it has been implicated in many bioprocesses. Herein, we demonstrate that METTL14 plays a critical role in autophagy induction and hinders osteoporosis process whose expression is decreased both in human osteoporosis bone tissue and ovariectomy (OVX) mice. In vivo, METTL14+/- knockdown mice exhibit elevated bone loss and impaired autophagy similar to the OVX mice, while overexpression of METTL14 significantly promotes bone formation and inhibits the progression of osteoporosis caused by OVX surgery. In vitro, METTL14 overexpression significantly enhances the osteogenic differentiation ability of BMSCs through regulating the expression of beclin-1 depending on m6A modification and inducing autophagy; the opposite is true with METTL14 silencing. Subsequently, m6A-binding proteins IGF2BP1/2/3 recognize m6A-methylated beclin-1 mRNA and promote its translation via mediating RNA stabilization. Furthermore, METTL14 negatively regulates osteoclast differentiation. Collectively, our study reveals the METTL14/IGF2BPs/beclin-1 signal axis in BMSCs osteogenic differentiation and highlights the critical roles of METTL14-mediated m6A modification in osteoporosis.

ALKBH5 suppresses tumor progression via an m6A-dependent epigenetic silencing of pre-miR-181b-1/YAP signaling axis in osteosarcoma.

ALKBH5 is the main enzyme for m6A-based demethylation of RNAs and it has been implicated in many biological and pathophysiological processes. Here, we aimed to explore the potential involvement of ALKBH5 in osteosarcoma and decipher the underlying cellular/molecular mechanisms. We discovered downregulated levels of demethylase ALKBH5 were correlated with increased m6A methylation in osteosarcoma cells/tissues compared with normal osteoblasts cells/tissues. ALKBH5 overexpression significantly suppressed osteosarcoma cell growth, migration, invasion, and trigged cell apoptosis. In contrast, inhibition of ALKBH5 produced the opposite effects. Whereas ALKBH5 silence enhanced m6A methylations of pre-miR-181b-1 and YAP-mRNA exerting oncogenic functions in osteosarcoma. Moreover, upregulation of YAP or downregulation of mature miR-181b-5p displayed a remarkable attenuation of anti-tumor activities caused by ALKBH5. Further results revealed that m6A methylated pre-miR-181b-1 was subsequently recognized by m6A-binding protein YTHDF2 to mediate RNA degradation. However, methylated YAP transcripts were recognized by YTHDF1 to promote its translation. Therefore, ALKBH5-based m6A demethylation suppressed osteosarcoma cancer progression through m6A-based direct/indirect regulation of YAP. Thus, ALKBH5 overexpression might be considered a new approach of replacement therapy for osteosarcoma treatment.