siRNA Transfection Research Articles

Cisplatin induces acute liver injury by triggering caspase-3/GSDME-mediated cell pyroptosis

BackgroundCisplatin triggers Gasdermin E (GSDME) cleavage, causing membrane bubble formation, content release, and inflammation. Caspase-3 activation initiates GSDME cleavage, and thus inhibiting this pathway mitigates cisplatin-induced pyroptosis in hepatocytes. This study aimed to delve into how cisplatin induces liver injury via pyroptosis. MethodsFor animal experiments, C57BL/6J mice were divided into three groups: control, liver injury model group, and Ac-DMLD-CMK (caspase-3 inhibitor) intervention group. The liver histology was evaluated by hematoxylin and eosin staining, immunohistochemistry, immunofluorescence and TUNEL staining. The mRNA and protein levels were detected by real-time polymerase chain reaction (PCR) and Western blot analysis. For in vitro experiments, HL-7702 cells were treated with cisplatin or GSDME siRNA. Cell pyroptosis was determined via cellular morphology, cytotoxicity and viability detection, flow cytometric assay, and Western blot detection for the expression of pyroptosis-related proteins. ResultsCisplatin-induced distinct liver morphological changes, hepatocellular injury, and inflammation in mice, along with elevated serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels and increased pro-inflammatory cytokine expression. Heightened macrophage infiltration and hepatocellular death indicated cisplatin-induced hepatotoxicity. Cisplatin upregulated GSDME activation, along with Bax-mediated caspase-3 cleavage both in vivo and in vitro, implicating caspase-3/GSDME-dependent pyroptosis in liver injury. Treatment with Ac-DMLD-CMK ameliorated cisplatin-induced liver injury, reducing hepatocellular lesions, serum ALT and AST levels, cytokine expression, macrophage infiltration, and hepatocyte death. Ac-DMLD-CMK also attenuated GSDME-dependent pyroptosis post-cisplatin induction, as evidenced by decreased GSDME expression, Bax upregulation, and cleaved caspase-3 activation. For HL-7702 cells, GSDME siRNA transfection reduced GSDME expression, attenuated typical signs of cisplatin-induced pyroptosis, partially restored cell viability, and significantly inhibited cytotoxicity and a decrease in the proportion of propidium iodide-positive cells, indicating protection against cisplatin-induced hepatocyte pyroptosis. ConclusionOur study underscores the role of the caspase-3/GSDME signaling pathway in mediating cisplatin-induced hepatotoxicity, particularly in cases of excessive or cumulative cisplatin exposure. These findings suggest that targeting GSDME could represent a promising therapeutic approach to mitigate cisplatin-induced liver damage.

Opposing Functions of Maspin Are Regulated by Its Subcellular Localization in Lung Squamous Cell Carcinoma Cells.

Mammary serine protease inhibitor (maspin) is a tumor suppressor protein downregulated during carcinogenesis and cancer progression; cytoplasmic-only maspin expression is an independent, unfavorable prognostic indicator in patients with lung squamous cell carcinoma (LUSC). We hypothesized that the cytoplasmic-only localization of maspin has tumor-promoting functions in LUSC. The subcellular localization of maspin and the invasive capability of LUSC cell lines were investigated using RNA sequencing (RNA-seq), Western blotting, and siRNA transfection. Maspin mRNA and protein expression were suppressed in LK-2 and RERF-LC-AI cells. Cell invasion significantly increased in response to siRNA-mediated maspin knockdown in KNS-62 cells expressing both nuclear and cytoplasmic maspin. In LK-2 cells, both nuclear and cytoplasmic maspin were re-expressed, and cell invasion and migration were significantly decreased. In contrast, re-expressed maspin in RERF-LC-AI cells was detected only in the cytoplasm (cytMaspin), and cell invasion and migration were significantly promoted. RNA-seq and downstream analyses revealed that increased cytMaspin expression downregulated the genes associated with cell adhesion and activated PYK2 and SRC, which play important roles in cancer progression. Our study demonstrates a novel biological function of cytMaspin in enhancing the invasive capabilities of LUSC cells. Understanding cytoplasm-to-nuclear maspin translocation dysregulation may develop novel therapeutic approaches to improve the prognosis of patients with LUSC.

Ropivacaine Administration Suppressed A549 Lung Adenocarcinoma Cell Proliferation and Migration via ACE2 Upregulation and Inhibition of the Wnt1 Pathway.

Previous studies have suggested that perioperative anesthesia could have direct impacts on cancer cell biology. The present study investigated the effects of ropivacaine administration on lung adenocarcinoma cells. Ropivacaine was administered to A549 cells at concentrations of 0.1, 1, and 6 mM for 2 h. Angiotensin-converting enzyme 2 (ACE2) small interfering RNA (siRNA) transfection was performed 6 h prior to ropivacaine administration. Cell proliferation and migration were assessed with cell counting kit 8 (CCK-8) and a wound healing assay at 0 and 24 h after anesthesia exposure. PCR arrays were performed, followed by PCR validation. Ropivacaine administration inhibited A549 cell proliferation and migration in a concentration-dependent manner, with ACE2 upregulation and HIF1α (hypoxia-inducible factor 1α) downregulation. The anticancer effect of ropivacaine was canceled out via ACE2 siRNA transfection. PCR arrays showed specific gene change patterns in the ropivacaine and respective ACE2-knockdown groups. EGFR (epidermal growth factor receptor), BAX (Bcl-2-associated X protein) and BCL2 (B-cell/CLL lymphoma 2) were suppressed with ropivacaine administration; these effects were reversed via ACE2 siRNA induction. Ropivacaine administration inhibited A549 cell biology in conjunction with ACE2 upregulation via the inhibition of the Wnt1 (wingless/Integrated 1) pathway.

GPX4 Inhibition Enhances the Antitumor Effect of PARP Inhibitor on Homologous Recombination Proficient Ovarian Cancer Cells.

Poly (ADP-ribose) polymerase inhibitors (PARPi) are now widely used in BRCA1/2 mutation or homologous recombination (HR) deficiency ovarian cancer but have limited efficacy in HR-proficient patients. GPX4 is a key regulator of ferroptosis and has been proven to be associated with multiple drug sensitivities. As a molecule that regulates the sensitivity of multiple drugs, the relationship between GPX4 and the efficacy of PARPi in HR-proficient ovarian cancer has not been elucidated. In this study, siRNA transfection was used to regulate the expression of GPX4. The effect of GPX4 inhibition on HR-proficient ovarian cancer was determined by CCK-8 assay and flow cytometry. Immunofluorescence and comet assays were used to reflect DNA dam-age. ROS production was measured using DCFH-DA and flow cytometry. The combination index of PARP inhibitors and RSL3 was calculated using CompuSyn software based on Chou-Talalay methodology. GPX4 inhibition confers HR-proficient ovarian cancer cells sensitive to PARPi due to ROS generation and oxidative stress caused by DNA double-strand breakage. The combina-tion of olaparib and niraparib with GPX4 inhibitor RSL3 also showed a synergistic effect. Combining GPX4 inhibition with PARP inhibitors resulted in a notable increase in DNA damage, ultimately causing the death of cancer cells with proficient HR pathways. Our findings may provide new therapeutic options for HR-proficient patients to benefit from PARP inhibitors and improve outcomes.

Ac-HSP20 regulates autophagy and promotes the encystation of Acanthamoeba castellanii by inhibiting the PI3K/AKT/mTOR signaling pathway

BackgroundThe encystation of Acanthamoeba castellanii has important ecological and medical significance. Blocking encystation is the key to preventing transmission and curing infections caused by A. castellanii. The formation of autophagosomes is one of the most important changes that occur during the encystation of Acanthamoeba. Our previous studies have shown that the heat shock protein 20 of A. castellanii (Ac-HSP20) is involved in its encystation. This study aimed to determine the role and mechanism of Ac-HSP20 in regulating autophagy involved in the encystation of A. castellanii.MethodsImmunofluorescence assay, western blotting and transmission electron microscopy were used to analyze the dynamic changes in autophagy during the initiation and continuation of encystation. The knockdown of Ac-HSP20 was performed to clarify its regulation of encystation and autophagy and to elucidate the molecular mechanism by which Ac-HSP20 participates in autophagy to promote cyst maturation.ResultsThe encystation rates and autophagosomes were significantly decreased by treatment with the autophagy inhibitor 3-MA. The autophagy marker LC3B and autophagic lysosomes increased with the induced duration of encystation and reached the maximum at 48 h. The encystation rate, LC3B expression and autophagosomes decreased when Ac-HSP20 was knocked down by siRNA transfection. In addition, the expression levels of Ac-HSP20 and LC3B increased and the expressions of p-AKT and p-mTOR decreased after 48 h of encystation without knockdown. However, the expressions of p-AKT and p-mTOR increased while the expression of LC3B decreased under the knockdown of Ac-HSP20. Furthermore, the protein expression of LC3B increased when the PI3K/AKT/mTOR signaling pathway was inhibited but decreased when the pathway was activated.ConclusionsThe results demonstrated that autophagy is positively correlated with the encystation of A. castellanii, and Ac-HSP20 regulates autophagy to maintain the homeostasis of A. castellanii by inhibiting the PI3K /AKT /mTOR signaling pathway, thus promoting the maturation and stability of encystation.Graphical

Age-related mitophagy regulates orthodontic tooth movement by affecting PDLSCs mitochondrial function and RANKL/OPG.

A thorough comprehension of age-related variances in orthodontic tooth movement (OTM) and bone remodeling response to mechanical force holds significant implications for enhancing orthodontic treatment. Mitophagy plays a crucial role in bone metabolism and various age-related diseases. However, the impact of mitophagy on the bone remodeling process during OTM remains elusive. Using adolescent (6 weeks old) and adult (12 months old) rats, we established OTM models and observed that orthodontic force increased the expression of the mitophagy proteins PTEN-induced putative kinase 1 (PINK1) and Parkin, as well as the number of tartrate-resistant acid phosphatase-positive osteoclasts and osteocalcin-positive osteoblasts. These biological changes were found to be age-related. Invitro, compression force loading promoted PINK1/Parkin-dependent mitophagy in periodontal ligament stem cells (PDLSCs) derived from adolescents (12-16 years old) and adults (25-35 years old). Furthermore, adult PDLSCs exhibited lower levels of mitophagy, impaired mitochondrial function, and a decreased ratio of RANKL/OPG compared to young PDLSCs after compression. Transfection of siRNA confirmed that inhibition of mitophagy in PDLSC resulted in decreased mitochondrial function and reduced RANKL/OPG ratio. Application of mitophagy inducer Urolithin A enhanced bone remodeling and accelerated OTM in rats, while the mitophagy inhibitor Mdivi-1 had the opposite effect. These findings indicate that force-stimulated PDLSC mitophagy contributes to alveolar bone remodeling during OTM, and age-related impairment of mitophagy negatively impacts the PDLSC response to mechanical stimulus. Our findings enhance the understanding of mitochondrial mechanotransduction and offer new targets to tackle current clinical challenges in orthodontic therapy.

Urine-derived podocytes from steroid resistant nephrotic syndrome patients as a model for renal-progenitor derived extracellular vesicles effect and drug screening

BackgroundPersonalized disease models are crucial for evaluating how diseased cells respond to treatments, especially in case of innovative biological therapeutics. Extracellular vesicles (EVs), nanosized vesicles released by cells for intercellular communication, have gained therapeutic interest due to their ability to reprogram target cells. We here utilized urinary podocytes obtained from children affected by steroid-resistant nephrotic syndrome with characterized genetic mutations as a model to test the therapeutic potential of EVs derived from kidney progenitor cells (nKPCs).MethodsEVs were isolated from nKPCs derived from the urine of a preterm neonate. Three lines of urinary podocytes obtained from nephrotic patients’ urine and a line of Alport syndrome patient podocytes were characterized and used to assess albumin permeability in response to nKPC-EVs or various drugs. RNA sequencing was conducted to identify commonly modulated pathways after nKPC-EV treatment. siRNA transfection was used to demonstrate the involvement of SUMO1 and SENP2 in the modulation of permeability.ResultsTreatment with the nKPC-EVs significantly reduced permeability across all the steroid-resistant patients-derived and Alport syndrome-derived podocytes. At variance, podocytes appeared unresponsive to standard pharmacological treatments, with the exception of one line, in alignment with the patient’s clinical response at 48 months. By RNA sequencing, only two genes were commonly upregulated in nKPC-EV-treated genetically altered podocytes: small ubiquitin-related modifier 1 (SUMO1) and Sentrin-specific protease 2 (SENP2). SUMO1 and SENP2 downregulation increased podocyte permeability confirming the role of the SUMOylation pathway.ConclusionsnKPCs emerge as a promising non-invasive source of EVs with potential therapeutic effects on podocytes with genetic dysfunction, through modulation of SUMOylation, an important pathway for the stability of podocyte slit diaphragm proteins. Our findings also suggest the feasibility of developing a non-invasive in vitro model for screening regenerative compounds on patient-derived podocytes.

Icariin alleviates the injury of Sertoli cell junction function by upregulating PKR pathway via ERα/c-fos signaling in aged mice

Ethnopharmacological relevenaceSertoli cells are vital to maintain spermatogenesis and their function decline during aging. Epimedium has the effects of tonifying kidney-yang, strengthening bones and muscles, and expelling wind and dampness, and is commonly used in the treatment of kidney-yang deficiency, impotence and spermatorrhea. Icariin is the main active ingredients from Epimedium exhibiting delaying aging effects and improving male reproductive dysfunction. Whereas, it remains poorly understood how icariin alleviates age-associated decline in testicular function by protecting against the damage of junction function of Sertoli cells. Aim of the studyThis study aimed to evaluate the improvement effect of icariin on Sertoli cell junction function damage and explore the underlying mechanisms. Materials and methodsMale C57BL/6 mice and mouse Sertoli cell line TM4 cells were utilized to assess the improvement effect of icariin on aging-associated Sertoli cell junction function injury. H&E staining, transmission electron microscopy, qPCR, Western blot, molecular docking, siRNA transfection, and immunofluorescence were performed in this study. ResultsDietary administration of icariin remarkly attenuated age-associated deterioration in spermatogenic function as evidenced by elevated testicular weight and index, sperm concentration and sperm viability. In addition, icariin protected Sertoli cell junction function from age-associated damage as proven by increased Sertoli cell numbers, improved tight junction ultrastructure, and upregulated junction-related proteins (ZO-1, Occludin and β-Catenin). Moreover, icariin significantly upregulated ERα/c-fos signaling and PKR pathway in testicular Sertoli cells. Similarly, in vitro studies revealed that deletion of ERα, c-fos or PKR abolished the improvement effects of icariin on Sertoli cell junction function damage. ConclusionsIcariin effectively mitigates age-associated decline in testicular function by diminished Sertoli cell junction function damage through upregulating PKR pathway via ERα/c-fos signaling. Therefore, attenuating Sertoli cell junction function injury by the upregulation of PKR pathway via ERα/c-fos signaling probably indicates an effective target for the prevention and treatment of testicular spermatogenic function with aging.

Tea polyphenol nanoparticles enable targeted siRNA delivery and multi-bioactive therapy for abdominal aortic aneurysms

Abdominal aortic aneurysm (AAA) is a life-threatening vascular disease, while there is a lack of pharmaceutical interventions to halt AAA progression presently. To address the multifaceted pathology of AAA, this work develops a novel multifunctional gene delivery system to simultaneously deliver two siRNAs targeting MMP-2 and MMP-9. The system (TPNs-siRNA), formed through the oxidative polymerization and self-assembly of epigallocatechin gallate (EGCG), efficiently encapsulates siRNAs during self-assembly. TPNs-siRNA safeguards siRNAs from biological degradation, facilitates intracellular siRNA transfection, promotes lysosomal escape, and releases siRNAs to silence MMP-2 and MMP-9. Additionally, TPNs, serving as a multi-bioactive material, mitigates oxidative stress and inflammation, fosters M1-to-M2 repolarization of macrophages, and inhibits cell calcification and apoptosis. In experiments with AAA mice, TPNs-siRNA accumulated and persisted in aneurysmal tissue after intravenous delivery, demonstrating that TPNs-siRNA can be significantly distributed in macrophages and VSMCs relevant to AAA pathogenesis. Leveraging the carrier’s intrinsic multi-bioactive properties, the targeted siRNA delivery by TPNs exhibits a synergistic effect for enhanced AAA therapy. Furthermore, TPNs-siRNA is gradually metabolized and excreted from the body, resulting in excellent biocompatibility. Consequently, TPNs emerges as a promising multi-bioactive nanotherapy and a targeted delivery nanocarrier for effective AAA therapy.Graphical

The role and mechanism of β-catenin-mediated skeletal muscle satellite cells in osteoporotic fractures by Jian-Pi-Bu-Shen formula.

Osteoporosis is a metabolic bone disease. β-Catenin is associated with fractures. Jian-Pi-Bu-Shen (JPBS) can promote the healing of osteoporotic fractures (OPF). However, the mechanism of β-catenin-mediated skeletal muscle satellite cells (SMSCs) in OPF by the JPBS is unclear. SMSCs were isolated and divided into five groups. The results showed that the survival rate of SMSCs was significantly higher in the low, medium, and high dose JPBS-containing serum groups after 7 days of incubation. The ALP activity and the number of SMSCs mineralized in the JPBS-containing serum intervention group were elevated. Axin, GSK-3β, β-catenin siRNAs were constructed and transfected into cells. Transfection of siRNAs reduced Axin, GSK-3β, and β-catenin expressions, respectively. β-Catenin-siRNA reversed ALP activity, the number of SMSCs mineralized, and the expression of β-catenin, BMP2, Runx2, COL-I, SP7/Ostrix, Osteocalcin, and BMP-7. Transcriptomic results suggested that the TNF signaling pathway associated with OPF was enriched. SD rats were subjected to the construction of OPF model by removing the ovaries. JPBS decreased the levels of PINP, ALP, CTX, and NTX through β-catenin in OPF rats, while increasing Runx2, β-catenin expressions through β-catenin at the broken end of fractures. Moreover, JPBS decreased BMC, BMD, and BV/TV and improved pathological damage through β-catenin in OPF rats. JPBS decreased the expression of Axin, GSK-3β mRNA, and protein, but increased the expressions of β-catenin, Pax7, COL-II, COL-II, BMP2, and Runx2 through β-catenin in OPF rats. In conclusion, JPBS inhibits Axin/GSK-3β expression, activates the β-catenin signaling, and promotes the osteogenic differentiation of SMSCs.

Cannabidiol (CBD) Inhibits Foam Cell Formation via Regulating Cholesterol Homeostasis and Lipid Metabolism.

The cannabidiol (CBD) in hemp oil has important pharmacological activities. Accumulating evidence suggests that CBD is beneficial in the cardiovascular system and has been applied as a health supplement for atherosclerosis. However, the mechanism remains unclear. This study investigates the impact of CBD on foam cell formation, cholesterol homeostasis, and lipid metabolism in macrophages. CBD elevates the levels of peroxisome proliferator-activated receptor gamma (PPARγ) and its associated targets, such as ATP binding transporter A1/G1 (ABCA1/ABCG1), thus reducing foam cell formation, and increasing cholesterol efflux within macrophages. Notably, the upregulation of ABCA1 and ABCG1 expression induced by CBD is found to be attenuated by both a PPARγ inhibitor and PPARγ small interfering RNA (siRNA). Moreover, transfection of PPARγ siRNA results in a decrease in the inhibitory effect of CBD on foam cell formation and promotion of cholesterol efflux. Through lipidomics analysis, the study finds that CBD significantly reverses the enhancement of ceramide (Cer). Correlation analysis indicates a negative association between Cer level and the expression of ABCA1/ABCG1. This study confirms that CBD can be an effective therapeutic candidate for atherosclerosis treatment by activating PPARγ, up-regulating ABCA1/ABCG1 expression, and down-regulating Cer level.

AEG-1 as a Novel Therapeutic Target in Colon Cancer: A Study from Silencing AEG-1 in BALB/c Mice to Large Data Analysis.

Astrocyte elevated gene-1 (AEG-1) is overexpressed in various malignancies. Exostosin-1 (EXT-1), a tumor suppressor, is an intermediate for malignant tumors. Understanding the mechanism behind the interaction between AEG-1 and EXT-1 may provide insights into colon cancer metastasis. AOM/DSS was used to induce tumor in BALB/c mice. Using an in vivo-jetPEI transfection reagent, transient transfection of AEG-1 and EXT-1 siRNAs were achieved. Histological scoring, immunohistochemical staining, and gene expression studies were performed from excised tissues. Data from the Cancer Genomic Atlas and GEO databases were obtained to identify the expression status of AEG-1 and itsassociation with the survival. In BALB/c mice, the AOM+DSS treated mice developed necrotic, inflammatory and dysplastic changes in the colon with definite clinical symptoms such as loss of goblet cells, colon shortening, and collagen deposition. Administration of AEG-1 siRNA resulted in a substantial decrease in the disease activity index. Mice treated with EXT-1 siRNA showed diffusely reduced goblet cells. In vivo investigations revealed that PTCH-1 activity was influenced by upstream gene AEG-1, which in turn may affect EXT-1 activity. Data from The Cancer Genomic Atlas and GEO databases confirmed the upregulation of AEG-1 and downregulation of EXT-1 in cancer patients. This study revealed that AEG-1 silencing might alter EXT-1 expression indirectly through PTCH-1, influencing cell-ECM interactions, and decreasing dysplastic changes, proliferation and invasion.

Calycosin activates Nrf2/Keap1 signaling to ameliorate hydrogen peroxide-induced spinal cord neuron death and mitochondrial dysfunction.

Oxidative stress is a hallmark of secondary injury of spinal cord injuries. Controlling oxidative stress is crucial for mitigating secondary injury and promotingfunctional recovery after spinal cord injuries. Calycosin is an O-methylated isoflavone with antioxidant activity. To evaluate the effect of calycosin on spinal cord neurons under oxidative stress and clarify the molecular mechanism underlying the effect, we tested the neuroprotective activity of calycosin in a primary spinal cord neuron culture model. We found that calycosin protected neurons from H2O2-induced neuronal death in a dose-dependent manner. Further experiments revealed that calycosin decreased H2O2-induced mitochondrial fragmentation and mitochondrial membrane potential loss, and subsequently reduced H2O2-triggered release of mitochondrial cytochrome c into the cytoplasm. In addition, calycosin inhibited H2O2-induced reactive oxygen species generation and activation of NF-κB signaling in spinal cord neurons. Furthermore, the expression of several antioxidant enzymes such as HO-1, NQO1, GCLC, GCLM, TrxR1, and Trx1 was significantly promoted by calycosin. More importantly, we revealed that the Nrf2/Keap1 signal is crucial for the effect of calycosin, because calycosin increased the amount of nuclear Nrf2 while decreasing the amount of cytoplasmic Nrf2. Nrf2 knockdown with siRNA transfection abolished the neuroprotective effect of calycosin. Taken together, this study disclosed a novel mechanism by which calycosin combats oxidative stress. Our study thus sheds light on the potential clinical application of calycosin in SCI treatment.

Fetal growth restriction and placental defects in obese mice are associated with impaired decidualisation: the role of increased leptin signalling modulators SOCS3 and PTPN2

Decidualisation of the endometrium is a key event in early pregnancy, which enables embryo implantation. Importantly, the molecular processes impairing decidualisation in obese mothers are yet to be characterised. We hypothesise that impaired decidualisation in obese mice is mediated by the upregulation of leptin modulators, the suppressor of cytokine signalling 3 (SOCS3) and the protein tyrosine phosphatase non-receptor type 2 (PTPN2), together with the disruption of progesterone (P4)-signal transducer and activator of transcription (STAT3) signalling. After feeding mice with chow diet (CD) or high-fat diet (HFD) for 16 weeks, we confirmed the downregulation of P4 and oestradiol (E2) steroid receptors in decidua from embryonic day (E) 6.5 and decreased proliferation of stromal cells from HFD. In vitro decidualised mouse endometrial stromal cells (MESCs) and E6.5 deciduas from the HFD showed decreased expression of decidualisation markers, followed by the upregulation of SOCS3 and PTPN2 and decreased phosphorylation of STAT3. In vivo and in vitro leptin treatment of mice and MESCs mimicked the results observed in the obese model. The downregulation of Socs3 and Ptpn2 after siRNA transfection of MESCs from HFD mice restored the expression level of decidualisation markers. Finally, DIO mice placentas from E18.5 showed decreased labyrinth development and vascularisation and fetal growth restricted embryos. The present study revealed major defects in decidualisation in obese mice, characterised by altered uterine response to E2 and P4 steroid signalling. Importantly, altered hormonal response was associated with increased expression of leptin signalling modulators SOCS3 and PTPN2. Elevated levels of SOCS3 and PTPN2 were shown to molecularly affect decidualisation in obese mice, potentially disrupting the STAT3-PR regulatory molecular hub.

Melittin Alleviates Oxidative Stress Injury in Schwann Cells by Targeting Interleukin-1 Receptor Type 1 to Downregulate Nuclear Factor Kappa B-Mediated Inflammatory Response In Vitro.

In ancient China, bee venom was widely used to treat various diseases.Although using bee venomis not currently a mainstream medical method, some have applied it to treat certain conditions,includingidiopathic facial paralysis(IFP). Recently, melittin(Mel), the main active component of bee venom, has been shown strong anti-inflammatory and analgesic effects. However, how bee venom improves neurological dysfunction in facial paralysis remainsunknown. This study aimed to investigate the anti-neurotraumatic effect of Mel on Schwann cells(SCs), the main cells of the neuron sheath, injured by oxidative stress. A model of hypoxicSCswas established,and CCK-8 assay, siRNA transfection, enzyme-linked immunosorbent assay, quantitative reverse transcription-polymerase chain reaction, western blot, immunofluorescence, and cell ultrastructure analyses were conducted to investigate the mitigation of hypoxia-induced damage to SCs in vitro, revealing the effects of Melon oxidative stress injury in SCs. The overexpression of HIF-1αin CoCl2-induced SCs (p < 0.05) indicated the establishment of an SCs hypoxia model. The proliferation and regeneration process of the hypoxic SCsenhanced in the Mel-treated group compared to the CoCl2group has been proventhrough the CCK-8 experiment (p < 0.0001) and S-100 mRNA expression detection (p < 0.0001). Theincreased level of reactive oxygen species (ROS) (p < 0.001) and decreased superoxide dismutase (SOD) levels (p < 0.05) in the CoCl2-induced SCs indicatedthat Mel can alleviate the oxidative stress damage to SCs induced by CoCl2. Mel alleviated oxidative stress and inflammation in hypoxic SCs by reducing pro-inflammatory cytokines IL-1β (p < 0.0001) and TNF-α (p < 0.0001). In addition, Mel augmented cellular vitality and regulated indicators related to oxygen metabolism, cell repair, neurometabolism, and vascular endothelial formation after hypoxia, such as C-JUN (p < 0.05), glial cell line-derived neurotrophic factor (GDNF; p < 0.001), vascular endothelial growth factor(VEGF; p < 0.05), hypoxia-inducible factor 1-alpha (HIF-1α; p < 0.05), interleukin-1 receptor type 1 (IL-1R1; p < 0.05), enolase1(ENO1; p < 0.05), aldose reductase(AR; p < 0.01), SOD (p < 0.05), nerve growth factor(NGF; p < 0.05), and inducible nitric oxide synthase(iNOS; p < 0.05). In terms of its mechanism, Mel inhibited the expression of proteins associated with the NF-κB pathway such as IKK (p < 0.01), p65 (p < 0.05), p60 (p < 0.001), IRAK1 (p < 0.05), and increased IKB-α (p < 0.0001). Moreover, knocking out of IL-1R1 in the si-IL-1R1 group enhanced the therapeutic effect of Mel compared to the Mel-treated group (all of which p < 0.05). This research provided evidence of the substantial involvement of IL-1R1 in oxidative stress damage caused by hypoxia in SCsand proved that Mel alleviated oxidative stress injury in SCs by targeting IL-1R1 to downregulate the NF-κB-mediated inflammatory response. Mel could potentially serve as an innovative therapeutic approach for the treatment of IFP.

Lipid nanoparticle-mediated hepatocyte delivery of siRNA and silibinin in metabolic dysfunction-associated steatotic liver disease

Lipid nanoparticle-mediated co-delivery of siRNA and small molecule holds a great potential to treat metabolic dysfunction-associated steatotic liver disease (MASLD). However, targeted delivery of therapeutics to hepatocytes remains challenging. Taking the advantage of rising low density lipoprotein receptor/very-low density lipoprotein receptor (LDLR/VLDR) levels in MASLD, the biological fate of dinonylamine-ethylene glycol chlorophosphate-1-nonanol (DNNA-COP-NA) based lipid nanoparticles (LNPs) was oriented to liver tissues via apolipoprotein E (ApoE)-LDLR/VLDLR pathway. We then adopted a three-round screening strategy to optimize the formulation with both high potency and selectivity to deliver siRNA-HIF-1α (siHIF1α) and silibinin (SLB) payloads to hepatocytes. The optimized SLB/siHIF1α-LNPs mediates great siRNA delivery and transfection of hepatocytes. In high fat diet (HFD)- and carbon tetrachloride (CCl4)-induced mouse models of MASLD, SLB/siHIF1α-LNPs enabled the silencing of hypoxia inducible factor-1α (HIF-1α), a therapeutic target primarily expressed by hepatocytes, leading to significantly reduced inflammation and liver fibrosis synergized with SLB. Moreover, it is demonstrated the hepatocyte-targeting delivery of SLB/siHIF1α-LNPs has the potential to restore the immune homeostasis by modulating the population of Tregs and cytotoxic T cells in spleen. This proof-of-concept study enable siRNA and small molecule co-delivery to hepatocytes through intrinsic variation of targeting receptors for MASLD therapy.

Bacillus coagulans restores pathogen-induced intestinal dysfunction via acetate-FFAR2-NF-κB-MLCK-MLC axis in Apostichopus japonicus.

Skin ulceration syndrome (SUS) is currently the main disease threatening Apostichopus japonicus aquaculture due to its higher mortality rate and infectivity, which is caused by Vibrio splendidus. Our previous studies have demonstrated that SUS is accompanied by intestinal microbiota (IM) dysbiosis, alteration of short-chain fatty acids (SCFAs) content and the damage to the intestinal barrier. However, the mediating effect of IM on intestine dysfunction is largely unknown. Herein, we conducted comprehensive intestinal microbiota transplantation (IMT) to explore the link between IM and SUS development. Furthermore, we isolated and identified a Bacillus coagulans strain with an ability to produce acetic acid from both healthy individual and SUS individual with IM from healthy donors. We found that dysbiotic IM and intestinal barrier function in SUS recipients A. japonicus could be restored by IM from healthy donors. The B. coagulans strain could restore IM community and intestinal barrier function. Consistently, acetate supply also restores intestinal homeostasis of SUS-diseased and V. splendidus-infected A. japonicus. Mechanically, acetate was found to specifically bind to its receptor-free fatty acid receptor 2 (FFAR2) to mediate IM structure community and intestinal barrier function. Knockdown of FFAR2 by transfection of specific FFAR2 siRNA could hamper acetate-mediated intestinal homeostasis in vivo. Furthermore, we confirmed that acetate/FFAR2 could inhibit V. splendidus-activated NF-κB-MLCK-MLC signaling pathway to restore intestinal epithelium integrity and upregulated the expression of ZO-1 and Occludin. Our findings provide the first evidence that B. coagulans restores pathogen-induced intestinal barrier dysfunction via acetate/FFAR2-NF-κB-MLCK-MLC axis, which provides new insights into the control and prevention of SUS outbreak from an ecological perspective.IMPORTANCESkin ulceration syndrome (SUS) as a main disease in Apostichopus japonicus aquaculture has severely restricted the developmental A. japonicus aquaculture industry. Intestinal microbiota (IM) has been studied extensively due to its immunomodulatory properties. Short-chain fatty acids (SCFAs) as an essential signal molecule for microbial regulation of host health also have attracted wide attention. Therefore, it is beneficial to explore the link between IM and SUS for prevention and control of SUS. In the study, the contribution of IM to SUS development has been examined. Additionally, our research further validated the restoration of SCFAs on intestinal barrier dysfunction caused by SUS via isolating SCFAs-producing bacteria. Notably, this restoration might be achieved by inhibition of NF-κB-MLCK-MLC signal pathway, which could be activated by V. splendidus. These findings may have important implications for exploration of the role of IM in SUS occurrence and provide insight into the SUS treatment.

Histone demethylase JMJD3 inhibits alveolar bone loss by regulating macrophage polarization in periodontitis

Objective: To investigate the expression of histone demethylase, Jumonji domain-containing protein 3 (JMJD3), in inflammatory periodontal tissues and its potential mechanism for the regulation of periodontitis. Methods: The results of single-cell sequencing of periodontal tissues published in the Gene Expression Omnibus (GEO) database in 2022 were analyzed. Nine gingival samples each from healthy and inflamed periodontal patients were collected during periodontal surgery or tooth extractions for immunohistochemical staining and real-time fluorescence quantitative PCR (RT-qPCR). Mice periodontitis models were constructed, and the experimental groups were: healthy control+saline group, silk ligation+saline group, silk ligation+GSK-J4(inhibitor of JMJD3) group. Lipopolysaccharide (LPS) derived from Porphyromonas gingivalis (Pg) (Pg-LPS) was used to mimic the periodontal inflammatory microenvironment. The macrophages were treated with small interfering RNA (siRNA) targeting Jmjd3 and the JMJD3 inhibitor GSK-J4. siRNA transfection experiments were grouped into the following: the NC group (negative control sequence transfection group), the siRNA-Jmjd3 group, the NC+LPS group, siRNA-Jmjd3+LPS group. Inhibitor experiments were grouped as dimethyl sulfoxide (DMSO) group, GSK-J4 group, DMSO+LPS group, GSK-J4+LPS group. Western blotting and immunofluorescence staining were used to explore the effects of JMJD3 on macrophage polarization and periodontal inflammation in the in vivo and in vitro settings. Results: RT-qPCR results showed that JMJD3 expression in gingival tissues of periodontitis patients (1.97±0.91) was significantly higher than that in healthy gingival tissues (1.00±0.33) (t=2.45, P=0.048). RT-qPCR results of in vitro experiments showed that either siRNA knockdown of JMJD3 or inhibition of JMJD3 using GSK-J4 promoted M1 polarization and inhibited M2 polarization in macrophages under inflammatory environment: the expression of arginase I (Arg 1) in the NC+LPS group (0.90±0.06) was significantly higher than that in the siRNA-Jmjd3+LPS group (0.61±0.11) (P<0.01); the expression of interleukin (Il)-6, Il-1β, and tumor necrosis factor alpha (Tnf-α) in the NC+LPS group (8.50±0.16, 5.56±0.20, 3.44±0.16) were significantly lower than those in the siRNA-Jmjd3+LPS group (14.63±0.48, 8.55±0.10, 11.72±0.16) (P<0.01). The expression of Arg-1, Ym1, Il-10 in the DMSO+LPS group (0.82±0.01, 0.35±0.16, 1.47±0.11) were significantly higher (P<0.01) than the GSK-J4+LPS group (0.55±0.03, 0.22±0.21, 0.51±0.11); the expression of Il-6, Il-1β, and Tnf-α in the DMSO+LPS group (2.03±0.13, 3.63±0.14, 4.06±0.03) were significantly lower than the GSK-J4+LPS group (2.69±0.16, 15.04±1.15, 4.36±0.10) (P<0.01). The results of the in vivo experiments revealed that inhibition of JMJD3 exacerbated bone loss in experimental periodontitis mice, increased macrophage M1 polarization, and decreased M2 polarization in inflamed periodontal tissues. The buccal cemento-enamel junction (CEJ)-alveolar bone crest (ABC), palatal CEJ-ABC, as well as the ratio of M1/M2 type macrophages were significantly lower in the silk ligation+saline group [(0.26±0.03), (0.24±0.01) mm, 0.35±0.10) than in the silk ligation+GSK-J4 group [(0.34±0.04), (0.30±0.05) mm, 2.50±0.58) (t=3.65, P=0.006; t=2.67, P=0.049; t=7.31, P=0.004; respectively). Conclusions: Single-cell sequencing as well as the in vitro and in vivo experiments verified that JMJD3 expression was upregulated in periodontitis periodontal tissues. JMJD3 may exert a protective role in periodontitis by regulating macrophage polarization, thereby inhibiting alveolar bone destruction associated with the periodontitis.

MARK4 promotes the malignant phenotype of gastric cancer through the MAPK/ERK signaling pathway

BackgroundMicrotubule affinity regulating kinase 4 (MARK4), which is overexpressed in various tumors, is involved in the regulation of cell division, proliferation, migration, and the cell cycle, and has been considered a potential marker for cancer; however, its mechanism of action in gastric cancer (GC) remains unclear. This study aimed to investigate the role of MARK4 in the proliferation, migration, and invasion of GC cell through the MAPK/ERK signaling pathway by targeting MARK4 knockdown. MethodsUsing The Cancer Genome Atlas data and clinical information, MARK4 expression and its relationship with prognosis were analyzed. Possible pathways involving MARK4 were explored using enrichment analysis. Western blotting and real-time quantitative polymerase chain reaction were used to detect MARK4 expression in GC. After targeted transfection of siRNA, the transfection efficiency of the experimental group was detected in AGS and HGC-27 cells. The effects of knockdown MARK4 on the proliferation, migration, and invasion of GC cells were verified using CCK-8, colony formation, wound healing, and transwell assays. Finally, the relationship between MARK4, the MAPK/ERK pathway, and epithelial-mesenchymal transition in GC was verified by western blotting. ResultsMARK4 expression was upregulated in GC and associated with poor prognosis in patients with GC. Enrichment analysis showed that MARK4 was involved in the activation of the MAPK signaling pathway. Western blotting results indicated that MARK4 overexpression promoted the proliferation, migration, and invasion of GC cells through the MAPK/ERK pathway. ConclusionMARK4 expression was upregulated in GC and promoted the proliferation, migration, and invasion of GC cells through the MAPK/ERK pathway.

SIRT1 inhibits apoptosis of human lens epithelial cells through suppressing endoplasmic reticulum stress in vitro and in vivo.

To explore the effect of silent information regulator factor 2-related enzyme 1 (SIRT1) on modulating apoptosis of human lens epithelial cells (HLECs) and alleviating lens opacification of rats through suppressing endoplasmic reticulum (ER) stress. HLECs (SRA01/04) were treated with varying concentrations of tunicamycin (TM) for 24h, and the expression of SIRT1 and C/EBP homologous protein (CHOP) was assessed using real-time quantitative polymerase chain reaction (RT-PCR), Western blotting, and immunofluorescence. Cell morphology and proliferation was evaluated using an inverted microscope and cell counting kit-8 (CCK-8) assay, respectively. In the SRA01/04 cell apoptosis model, which underwent siRNA transfection for SIRT1 knockdown and SRT1720 treatment for its activation, the expression levels of SIRT1, CHOP, glucose regulated protein 78 (GRP78), and activating transcription factor 4 (ATF4) were examined. The potential reversal of SIRT1 knockdown effects by 4-phenyl butyric acid (4-PBA; an ER stress inhibitor) was investigated. In vivo, age-related cataract (ARC) rat models were induced by sodium selenite injection, and the protective role of SIRT1, activated by SRT1720 intraperitoneal injections, was evaluated through morphology observation, hematoxylin and eosin (H&E) staining, Western blotting, and RT-PCR. SIRT1 expression was downregulated in TM-induced SRA01/04 cells. Besides, in SRA01/04 cells, both cell apoptosis and CHOP expression increased with the rising doses of TM. ER stress was stimulated by TM, as evidenced by the increased GRP78 and ATF4 in the SRA01/04 cell apoptosis model. Inhibition of SIRT1 by siRNA knockdown increased ER stress activation, whereas SRT1720 treatment had opposite results. 4-PBA partly reverse the adverse effect of SIRT1 knockdown on apoptosis. In vivo, SRT1720 attenuated the lens opacification and weakened the ER stress activation in ARC rat models. SIRT1 plays a protective role against TM-induced apoptosis in HLECs and slows the progression of cataract in rats by inhibiting ER stress. These findings suggest a novel strategy for cataract treatment focused on targeting ER stress, highlighting the therapeutic potential of SIRT1 modulation in ARC development.