Radiation-induced thrombocytopenia (RIT) faces a perplexing challenge in the clinical treatment of cancer patients, and current therapeutic approaches are inadequate in the clinical settings. In this research, oxymatrine, a new molecule capable of healing RIT was screened out, and the underlying regulatory mechanism associated with magakaryocyte (MK) differentiation and thrombopoiesis was demonstrated. The capacity of oxymatrine to induce MK differentiation was verified in K-562 and Meg-01 cells in vitro. The ability to induce thrombopoiesis was subsequently demonstrated in Tg (cd41:enhanced green fluorescent protein (eGFP)) zebrafish and RIT model mice. In addition, we carried out network pharmacological prediction, drug affinity responsive target stability assay (DARTS) and cellular thermal shift assay (CETSA) analyses to explore the potential targets of oxymatrine. Moreover, the pathway underlying the effects of oxymatrine was determined by Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses, Western blot (WB), and immunofluorescence. Oxymatrine markedly promoted MK differentiation and maturation in vitro. Moreover, oxymatrine induced thrombopoiesis in Tg (cd41:eGFP) zebrafish and accelerated thrombopoiesis and platelet function recovery in RIT model mice. Mechanistically, oxymatrine directly binds to toll-like receptor 2 (TLR2) and further regulates the downstream pathway stimulator of interferon genes (STING)/nuclear factor-kappaB (NF-κB), which can be blocked by C29 and C-176, which are specific inhibitors of TLR2 and STING, respectively. Taken together, we demonstrated that oxymatrine, a novel TLR2 agonist, plays a critical role in accelerating MK differentiation and thrombopoiesis via the STING/NF-κB axis, suggesting that oxymatrine is a promising candidate for RIT therapy.

Radiation-induced thrombocytopenia(RIT)faces a perplexing challenge in the clinical treatment of cancer patients,and current therapeutic approaches are inadequate in the clinical settings.In this research,oxy-matrine,a new molecule capable of healing RIT was screened out,and the underlying regulatory mecha-nism associated with magakaryocyte(MK)differentiation and thrombopoiesis was demonstrated.The capacity of oxymatrine to induce MK differentiation was verified in K-562 and Meg-01 cells in vitro.The ability to induce thrombopoiesis was subsequently demonstrated in Tg(cd41:enhanced green fluorescent protein(eGFP))zebrafish and RIT model mice.In addition,we carried out network pharmacological pre-diction,drug affinity responsive target stability assay(DARTS)and cellular thermal shift assay(CETSA)analyses to explore the potential targets of oxymatrine.Moreover,the pathway underlying the effects of oxymatrine was determined by Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analyses,Western blot(WB),and immunofluorescence.Oxymatrine markedly promoted MK differentiation and maturation in vitro.Moreover,oxymatrine induced thrombopoiesis in Tg(cd41:eGFP)zebrafish and accelerated thrombopoiesis and platelet function recovery in RIT model mice.Mechanistically,oxymatrine directly binds to toll-like receptor 2(TLR2)and further regulates the downstream pathway stimulator of interferon genes(STING)/nuclear factor-kappaB(NF-κB),which can be blocked by C29 and C-176,which are specific inhibitors of TLR2 and STING,respectively.Taken together,we demonstrated that oxymatrine,a novel TLR2 agonist,plays a critical role in accelerating MK differentiation and thrombopoiesis via the STING/NF-κB axis,suggesting that oxymatrine is a promising candidate for RIT therapy.

This study aims to investigate the mechanism of Qingrun Decoction in alleviating hepatic insulin resistance in type 2 diabetes mellitus(T2DM) rats through the reprogramming of amino acid metabolism. A T2DM rat model was established by inducing insulin resistance through a high-fat diet combined with intraperitoneal injection of streptozotocin. The model rats were randomly divided into five groups: model group, high-, medium-, and low-dose Qingrun Decoction groups, and metformin group. A normal control group was also established. The rats in the normal and model groups received 10 mL·kg~(-1) distilled water daily by gavage. The metformin group received 150 mg·kg~(-1) metformin suspension by gavage, and the Qingrun Decoction groups received 11.2, 5.6, and 2.8 g·kg~(-1) Qingrun Decoction by gavage for 8 weeks. Blood lipid levels were measured in different groups of rats. Pathological damage in rat liver tissue was assessed by hematoxylin-eosin(HE) staining and oil red O staining. Transcriptome sequencing and untargeted metabolomics were performed on rat liver and serum samples, integrated with bioinformatics analyses. Key metabolites(branched-chain amino acids, BCAAs), amino acid transporters, amino acid metabolites, critical enzymes for amino acid metabolism, resistin, adiponectin(ADPN), and mammalian target of rapamycin(mTOR) pathway-related molecules were quantified using quantitative real-time polymerase chain reaction(qRT-PCR), Western blot, and enzyme-linked immunosorbent assay(ELISA). The results showed that compared with the normal group, the model group had significantly increased serum levels of total cholesterol(TC), triglycerides(TG), low-density lipoprotein cholesterol(LDL-C), and resistin and significantly decreased ADPN levels. Hepatocytes in the model group exhibited loose arrangement, significant lipid accumulation, fatty degeneration, and pronounced inflammatory cell infiltration. In liver tissue, the mRNA transcriptional levels of solute carrier family 7 member 2(Slc7a2), solute carrier family 38 member 2(Slc38a2), solute carrier family 38 member 4(Slc38a4), and arginase(ARG) were significantly downregulated, while the mRNA transcriptional levels of solute carrier family 1 member 4(Slc1a4), solute carrier family 16 member 1(Slc16a1), and methionine adenosyltransferase(MAT) were upregulated. Furthermore, the mRNA transcription and protein expression levels of branched-chain α-keto acid dehydrogenase E1α(BCKDHA) and DEP domain-containing mTOR-interacting protein(DEPTOR) were downregulated, while mRNA transcription and protein expression levels of mTOR, as well as ribosomal protein S6 kinase 1(S6K1), were upregulated. The levels of BCAAs and S-adenosyl-L-methionine(SAM) were elevated. The serum level of 6-hydroxymelatonin was significantly reduced, while imidazole-4-one-5-propionic acid and N-(5-phospho-D-ribosyl)anthranilic acid levels were significantly increased. Compared with the model group, Qingrun Decoction significantly reduced blood lipid and resistin levels while increasing ADPN levels. Hepatocytes had improved morphology with reduced inflammatory cells, and fatty degeneration and lipid deposition were alleviated. Differentially expressed genes and differential metabolites were mainly enriched in amino acid metabolic pathways. The expression levels of Slc7a2, Slc38a2, Slc38a4, and ARG in the liver tissue were significantly upregulated, while Slc1a4, Slc16a1, and MAT expression levels were significantly downregulated. BCKDHA and DEPTOR expression levels were upregulated, while mTOR and S6K1 expression levels were downregulated. Additionally, the levels of BCAAs and SAM were significantly decreased. The serum level of 6-hydroxymelatonin was increased, while those of imidazole-4-one-5-propionic acid and N-(5-phospho-D-ribosyl)anthranilic acid were decreased. In summary, Qingrun Decoction may improve amino acid metabolism reprogramming, inhibit mTOR pathway activation, alleviate insulin resistance in the liver, and mitigate pathological damage of liver tissue in T2DM rats by downregulating hepatic BCAAs and SAM and regulating key enzymes involved in amino acid metabolism, such as BCKDHA, ARG, and MAT, as well as amino acid metabolites and transporters.
Animals ; Drugs, Chinese Herbal/administration & dosage* ; Rats ; Insulin Resistance ; Diabetes Mellitus, Type 2/genetics* ; Male ; Liver/drug effects* ; Amino Acids/metabolism* ; Rats, Sprague-Dawley ; Humans ; Metabolic Reprogramming

Cataract surgery is one of the most common ophthalmologic procedures. Advances in technology and medical policies have made it more precise. Residual refractive errors and deviation of target diopters are a main cause of dissatisfaction among patients. Refractive enhancement after cataract surgery can correct or eliminate these errors, improving patients' visual quality of life. There are multiple options for correcting residual refractive errors. The best approach depends on factors like the cause of the error, degrees of residual refractive errors, type of intraocular lens, ocular comorbidities, and patient preference. This paper summarizes the incidence and types of residual refractive errors, advancements in refractive enhancement surgeries, and provides practical solutions for clinical practice.

AIM:To investigate the modulatory role of E3 ubiquitin-protein ligase ITCH in Alzheimer disease(AD)-like pathology through the thioredoxin-interacting protein(TXNIP)-nucleotide-binding oligomerization domain-like receptor protein 3(NLRP3)signaling pathway using both in vivo and in vitro experimental models.METHODS:(1)Ten 5×FAD(AD model)mice and 10 wild-type(WT)mice at 2-,4-and 6-month-old were randomly allocated into AD and WT groups.Amyloid β-protein(Aβ)plaque burden in the brain was detected by thioflavin-S and immunofluorescence staining,with the latter method additionally applied to assess TXNIP protein expression.The protein levels of ITCH and TXNIP were determined by Western blot,while their interaction was verified by co-immunoprecipitation.(2)Mouse mi-croglia BV2 cells stimulated by lipopolysaccharide(LPS)were used to construct neuroinflammation model,and were di-vided into control(CON)group and LPS+ATP treatment group.The BV2 cells stimulated by Aβ were used to construct AD inflammation model.According to the different treatment time,they were divided into CON,and 12,24 and 48 h treatment groups.Western blot was used to evaluate the expression of ITCH,TXNIP,and NLRP3 inflammasome compo-nents(NLRP3 and caspase-1)as well as the downstream IL-1β.Adenovirus-mediated ITCH overexpression(OE-ITCH)in Aβ-stimulated BV2 cells comprised three experimental groups:negative control group,Aβ oligomer stimulation group,and OE-ITCH group,with subsequent immunoblotting of inflammatory mediators.RESULTS:The deposition of Aβ plaques in the cortex and hippocampus of 5×FAD transgenic mice exhibited an age-dependent progression(P<0.01).Compared with WT mice,the levels of TXNIP protein increased synchronously,and the levels of ubiquitin ligase ITCH was significantly down-regulated(P<0.05).Co-immunoprecipitation confirmed the interaction between ITCH and TXNIP proteins in the brain of 2-and 4-month-old 5×FAD mice,which exhibited marked attenuation by 4 months of age.In BV2 microglial models,Aβ/LPS stimulation provoked significant ITCH suppression,concurrently up-regulating TXNIP,core NLRP3 inflammasome components(NLRP3 and caspase-1),and downstream IL-1β(P<0.05).Overexpression of ITCH significantly inhibited Aβ-induced activation of TXNIP and NLRP3 and therelated inflammatory factors in BV2 cells.CONCLUSION:The results of in vitro and in vivo experiments showed that ITCH protein exerts effects against AD-like pathology by inhibiting the expression of TXNIP-NLRP3 signaling pathway.

Recent studies have revealed a significant association between Porphyromonas gingivalis(P.gingivalis)infection and poor prognosis in esophageal squamous cell carcinoma(ESCC).Although cer-tain circular RNAs(circRNA)have been shown to suppress ESCC tumorigenesis and progression,their regulatory mechanisms in P.gingivalis infection-associated ESCC remain elusive.In this study,RT-qPCR analysis demonstrated that P.gingivalis infection downregulated hsa_circ_0057552 expression in ESCC cells and tissues in a time-and dose-dependent manner.Actinomycin D assays further confirmed that P.gingivalis infection reduced the RNA stability of hsa_circ_0057552 in ESCC cells(P＜0.05).Functional assays in vitro and a subcutaneous tumor xenograft model in vivo revealed that hsa_circ_0057552 overexpression significantly inhibited ESCC cell proliferation,migration,invasion,and tumor growth(P＜0.05).Additionally,PCR array screening combined with RT-qPCR and Western blotting in-dicated that P.gingivalis infection markedly upregulated human antigen R(HuR)expression at both RNA and protein levels(P＜0.05).Mechanistic investigations demonstrated that HuR knockdown signifi-cantly increased hsa_circ_0057552 expression(P＜0.01),whereas hsa_circ_0057552 overexpression had no regulatory effect on HuR.Finally,si-HuR treatment reversed the inhibitory effect of P.gingivalis on hsa_circ_0057552 transcription.This study demonstrated that P.gingivalis may promote the progression of ESCC through a novel mechanism involving the regulation of HuR/hsa_circ_0057552,thereby identif-ying a novel therapeutic target and molecular marker for P.gingivalis-associated ESCC.

Recent studies have revealed a significant association between Porphyromonas gingivalis(P.gingivalis)infection and poor prognosis in esophageal squamous cell carcinoma(ESCC).Although cer-tain circular RNAs(circRNA)have been shown to suppress ESCC tumorigenesis and progression,their regulatory mechanisms in P.gingivalis infection-associated ESCC remain elusive.In this study,RT-qPCR analysis demonstrated that P.gingivalis infection downregulated hsa_circ_0057552 expression in ESCC cells and tissues in a time-and dose-dependent manner.Actinomycin D assays further confirmed that P.gingivalis infection reduced the RNA stability of hsa_circ_0057552 in ESCC cells(P＜0.05).Functional assays in vitro and a subcutaneous tumor xenograft model in vivo revealed that hsa_circ_0057552 overexpression significantly inhibited ESCC cell proliferation,migration,invasion,and tumor growth(P＜0.05).Additionally,PCR array screening combined with RT-qPCR and Western blotting in-dicated that P.gingivalis infection markedly upregulated human antigen R(HuR)expression at both RNA and protein levels(P＜0.05).Mechanistic investigations demonstrated that HuR knockdown signifi-cantly increased hsa_circ_0057552 expression(P＜0.01),whereas hsa_circ_0057552 overexpression had no regulatory effect on HuR.Finally,si-HuR treatment reversed the inhibitory effect of P.gingivalis on hsa_circ_0057552 transcription.This study demonstrated that P.gingivalis may promote the progression of ESCC through a novel mechanism involving the regulation of HuR/hsa_circ_0057552,thereby identif-ying a novel therapeutic target and molecular marker for P.gingivalis-associated ESCC.

AIM:To investigate the modulatory role of E3 ubiquitin-protein ligase ITCH in Alzheimer disease(AD)-like pathology through the thioredoxin-interacting protein(TXNIP)-nucleotide-binding oligomerization domain-like receptor protein 3(NLRP3)signaling pathway using both in vivo and in vitro experimental models.METHODS:(1)Ten 5×FAD(AD model)mice and 10 wild-type(WT)mice at 2-,4-and 6-month-old were randomly allocated into AD and WT groups.Amyloid β-protein(Aβ)plaque burden in the brain was detected by thioflavin-S and immunofluorescence staining,with the latter method additionally applied to assess TXNIP protein expression.The protein levels of ITCH and TXNIP were determined by Western blot,while their interaction was verified by co-immunoprecipitation.(2)Mouse mi-croglia BV2 cells stimulated by lipopolysaccharide(LPS)were used to construct neuroinflammation model,and were di-vided into control(CON)group and LPS+ATP treatment group.The BV2 cells stimulated by Aβ were used to construct AD inflammation model.According to the different treatment time,they were divided into CON,and 12,24 and 48 h treatment groups.Western blot was used to evaluate the expression of ITCH,TXNIP,and NLRP3 inflammasome compo-nents(NLRP3 and caspase-1)as well as the downstream IL-1β.Adenovirus-mediated ITCH overexpression(OE-ITCH)in Aβ-stimulated BV2 cells comprised three experimental groups:negative control group,Aβ oligomer stimulation group,and OE-ITCH group,with subsequent immunoblotting of inflammatory mediators.RESULTS:The deposition of Aβ plaques in the cortex and hippocampus of 5×FAD transgenic mice exhibited an age-dependent progression(P<0.01).Compared with WT mice,the levels of TXNIP protein increased synchronously,and the levels of ubiquitin ligase ITCH was significantly down-regulated(P<0.05).Co-immunoprecipitation confirmed the interaction between ITCH and TXNIP proteins in the brain of 2-and 4-month-old 5×FAD mice,which exhibited marked attenuation by 4 months of age.In BV2 microglial models,Aβ/LPS stimulation provoked significant ITCH suppression,concurrently up-regulating TXNIP,core NLRP3 inflammasome components(NLRP3 and caspase-1),and downstream IL-1β(P<0.05).Overexpression of ITCH significantly inhibited Aβ-induced activation of TXNIP and NLRP3 and therelated inflammatory factors in BV2 cells.CONCLUSION:The results of in vitro and in vivo experiments showed that ITCH protein exerts effects against AD-like pathology by inhibiting the expression of TXNIP-NLRP3 signaling pathway.