Objective To investigate the gene expression profile in rat pancreatic ductal stem cells(PDSCs)when induced to differentiate into insulin-secreting cells(IPCs),with the goal of identifying key genes involved in this differentiation process.Methods The expanded PDSCs were categorized into a normal control(NC)group and an induced(Tre)group.PDSCs continued expansion culture in NC group,and cultured in induction medium for 28 days to facilitate the differentiation of PDSCs into IPCs in Tre group.Dithizone staining was employed to morphologically assess whether the cells exhibited a reddish-brown coloration,indicating a positive result.The immunofluorescence staining method was used to detect the expression of insulin(Ins)and PDX1 in the cells following induction.Additionally,ELISA was conducted to measure the Ins release from IPCs,thereby verifying the responsiveness of the induced cells to glucose-stimulated Ins secretion.Concurrently,cells were collected on induction days 0 and 28 for RNA sequencing(RNA-seq),and differentially expressed genes(DEGs)were analyzed and functionally annotated.The analysis revealed that regulatory factor X3(RFX3)was overexpressed in PDSCs,and the impact of RFX3 upregulation on differentiation induction was subsequently verified.Results Compared with NC group,DTZ staining was positive,PDX1 and Ins proteins were expressed,and an increased release of Ins in response to sugar stimulation was demonstrated in the Tre group.RNA-seq analysis identified 4270 DEGs,and functional enrichment analysis utilizing the Gene Ontology and Kyoto Encyclopedia of Genes and Genomes databases revealed associations with Ins response,positive regulation of Ins secretion,pancreatic endocrine cell development,and overall pancreatic development.Additionally,functionally related genes such as ALDHA2,CREB5,EIF6,FOXO1,RFX3,WNT5a,OGT,GPR39,SMAD6,and TRPM2 were identified,indicating involvement in the cell cycle,TGF-β1 signaling pathway,FOXO signaling pathway,and Wnt signaling pathway in the regulation of the differentiation of pancreatic ductal stem cells(PDSCs)into insulin-producing cells(IPCs).Furthermore,the upregulation of RFX3 can inhibit the expression of TGF-β1 within 72 hours,thereby promoted the formation and release of Ins from insulin-positive cells.Conclusions Multiple genes and signaling pathways associated with pancreatic β-cell function collectively regulate the differentiation of rat PDSCs into IPCs.Notably,the upregulation of RFX3 enhances this differentiation process.

Objective To investigate how dietary supplementation with tetrahydrocurcumin(THC)improves kidney injury in type 2 dia-betic mellitus(T2DM)and its mechanism of action using transcriptome sequencing(RNA-seq).Methods C57BL/6J mice were randomly assigned to the control,T2DM,and T2DM+THC groups.After a high-fat meal and streptozotocin injection,the body weights and fasting blood glucose levels of each mouse with T2DM were measured.Hematoxylin and eosin staining,Oil red O staining,and RNA-seq were performed to examine kidney pathology,lipid deposition,and differentially expressed genes,respectively,in the mice.Results Mice in T2DM group exhibited significantly higher fasting blood glucose levels(P＜0.001),renal tubule degeneration,glomeruli enlargement,disordered epithelial cells,and increased kidney lipid deposition after 12 weeks compared with those of the control group.THC adminis-tration alleviated all these conditions(P＜0.001).RNA-seq analysis revealed significant gene expression variations among the control,T2DM,and T2DM+THC groups.THC may protect against T2DM-induced kidney injury and lipid deposition by regulating the cell cycle(apoptosis),P53 signaling pathway,and PPARγ signaling path way,as indicated by Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analyses.In mice with T2DM,THC intervention may upregulate the expression of Cd36,Lpl,PPARγ,and Plin4 genes in renal tissues,while downregulating Ccnb1,Ccnb2,Cdk1,Bub1,and Cdc25c gene expressions.The proteins encoded by the four upregulated genes interact,as do those encoded by the five downregulated genes.Conclusion THC administration improves fasting blood glucose levels,reduces renal damage,and decreases fat deposition in mice with T2DM.The processes may involve decreasing apoptosis,blocking the P53 signaling pathway,and activating the PPARγ signaling pathway.

ObjectiveTo investigate the target proteins directly bound by neochlorogenic acid （NA） and the molecular mechanisms that ameliorate the proliferation and inflammatory response of psoriatic keratinocytes. MethodsM5-induced HaCaT cells were used as a psoriatic keratinocyte proliferation and inflammatory cell model. The synthesized NA probe （NA-P） and NA prodrug were first evaluated for cell viability using a cell proliferation/cell counting kit-8（CCK-8）. The potency of NA and NA-P was evaluated in the safe concentration range， and the effects of 0-100 μmol·L^-1 NA and probe on M5-induced proliferation of HaCaT cells were detected using CCK-8. The effects of 20， 40， 80 μmol·L^-1 NA and 80 μmol·L^-1 NA-P on the expression of tumor necrosis factor-α （TNF-α）， interleukin-1β （IL-1β）， interleukin-23 （IL-23）， and interleukin-17A （IL-17A） inflammatory factors were detected by enzyme-linked immunosorbent assay （ELISA）， and real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） was used to measure the effects of NA on the mRNA expression of keratin 16 （K16） in HaCaT cells， S100 calcium-binding protein A9 （S100A9）， S100 calcium-binding protein A7 （S100A7）， IL-6， IL-17A， and chemokine 1 （CXCL1）. In vitro fluorescence labeling and competition experiments using NA-P were performed， and target protein angling and analysis using pull-down experiments combined with liquid chromatography-mass spectrometry （Pull-down/LC-MS/MS） were conducted. Target validation was performed using pull-down experiments combined with protein immunoblotting （Pull down-WB）， cellular heat transfer analysis combined with protein immunoblot （CETSA-WB） experiments， and molecular docking. Finally， Real-time PCR was utilized to detect the effects of 20， 40， 80 μmol·L^-1 NA and 80 μmol·L^-1 NA-P on the mRNA expression of IL-1β， nucleotide-binding oligomeric structural domain-like receptor protein 3 （NLRP3）， apoptosis-associated speckled-like protein （ASC）， and cysteine protease-1 （Caspase-1） in HaCaT cells. Protein immunoblot （Western blot） was used to detect the effects of phosphorylated p65 （p-p65）， p65， phosphorylated human nuclear factor-κB inhibitory protein α （p-IκBα）， human nuclear factor κB inhibitory protein α （IκBα）， and heat shock protein 90 （HSP90） expression. ResultsIn the 200 μmol·L^-1 safe concentration range， HaCaT cell proliferation， increased expression of TNF-α， IL-1β， IL-23， and IL-17A inflammatory factors， and increased mRNA expression of K16， S100A9， S100A7， IL-6， IL-17A， and CXCL1 were observed in the M5 group compared with the blank group. Cell proliferation in 5-100 μmol·L^-1 NA and NA-P groups was inhibited， and the expression of TNF-α， IL-1β， IL-23， and IL-17A inflammatory factors was decreased in the NA-L， NA-M， NA-H， and NA-P-H groups. The mRNA expression of K16， S100A9， S100A7， IL-6， IL-17A， and CXCL1 was decreased （P<0.05）. High-confidence targets were screened for HSP90 protein by Pull-down/LC-MS/MS using 200 μmol·L^-1 NA competing with 100 μmol·L^-1 NA-P. Compared with that in the blank group， the mRNA expression of NLRP3， IL-1β， ASC， and Caspase-1， as well as the expression of p-p65/p65， p-IκBα/IκBα， and HSP90 protein， were increased in HaCaT cells in the M5 group （P<0.05）. Compared with that in the M5 group， the mRNA expression of NLRP3， IL-1β， ASC， and Caspase-1 of cells in the NA-L group， the NA-M group， the NA-H group， and the NA-P-H group was decreased （P<0.05）. p-p65/p65 and p-IκBα/IκBα were decreased in the NA-M and NA-H groups （P<0.05）， and there was no change in HSP90 protein. Pull down-WB showed that NA could directly target HSP90 protein， and NA binding to HSP90 protein enhanced its thermal stability. Molecular docking of NA with HSP90 family proteins HSP90AA1， HSP90B1， and HSP90AB1 all resulted in highly stable binding. ConclusionNA can inhibit the proliferation and inflammatory response of psoriatic keratinocytes by a mechanism that may be achieved by targeting HSP90 to modulate the NF-κB/NLRP3 signaling pathway.

ObjectiveTo investigate the target proteins directly bound by neochlorogenic acid （NA） and the molecular mechanisms that ameliorate the proliferation and inflammatory response of psoriatic keratinocytes. MethodsM5-induced HaCaT cells were used as a psoriatic keratinocyte proliferation and inflammatory cell model. The synthesized NA probe （NA-P） and NA prodrug were first evaluated for cell viability using a cell proliferation/cell counting kit-8（CCK-8）. The potency of NA and NA-P was evaluated in the safe concentration range， and the effects of 0-100 μmol·L^-1 NA and probe on M5-induced proliferation of HaCaT cells were detected using CCK-8. The effects of 20， 40， 80 μmol·L^-1 NA and 80 μmol·L^-1 NA-P on the expression of tumor necrosis factor-α （TNF-α）， interleukin-1β （IL-1β）， interleukin-23 （IL-23）， and interleukin-17A （IL-17A） inflammatory factors were detected by enzyme-linked immunosorbent assay （ELISA）， and real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） was used to measure the effects of NA on the mRNA expression of keratin 16 （K16） in HaCaT cells， S100 calcium-binding protein A9 （S100A9）， S100 calcium-binding protein A7 （S100A7）， IL-6， IL-17A， and chemokine 1 （CXCL1）. In vitro fluorescence labeling and competition experiments using NA-P were performed， and target protein angling and analysis using pull-down experiments combined with liquid chromatography-mass spectrometry （Pull-down/LC-MS/MS） were conducted. Target validation was performed using pull-down experiments combined with protein immunoblotting （Pull down-WB）， cellular heat transfer analysis combined with protein immunoblot （CETSA-WB） experiments， and molecular docking. Finally， Real-time PCR was utilized to detect the effects of 20， 40， 80 μmol·L^-1 NA and 80 μmol·L^-1 NA-P on the mRNA expression of IL-1β， nucleotide-binding oligomeric structural domain-like receptor protein 3 （NLRP3）， apoptosis-associated speckled-like protein （ASC）， and cysteine protease-1 （Caspase-1） in HaCaT cells. Protein immunoblot （Western blot） was used to detect the effects of phosphorylated p65 （p-p65）， p65， phosphorylated human nuclear factor-κB inhibitory protein α （p-IκBα）， human nuclear factor κB inhibitory protein α （IκBα）， and heat shock protein 90 （HSP90） expression. ResultsIn the 200 μmol·L^-1 safe concentration range， HaCaT cell proliferation， increased expression of TNF-α， IL-1β， IL-23， and IL-17A inflammatory factors， and increased mRNA expression of K16， S100A9， S100A7， IL-6， IL-17A， and CXCL1 were observed in the M5 group compared with the blank group. Cell proliferation in 5-100 μmol·L^-1 NA and NA-P groups was inhibited， and the expression of TNF-α， IL-1β， IL-23， and IL-17A inflammatory factors was decreased in the NA-L， NA-M， NA-H， and NA-P-H groups. The mRNA expression of K16， S100A9， S100A7， IL-6， IL-17A， and CXCL1 was decreased （P<0.05）. High-confidence targets were screened for HSP90 protein by Pull-down/LC-MS/MS using 200 μmol·L^-1 NA competing with 100 μmol·L^-1 NA-P. Compared with that in the blank group， the mRNA expression of NLRP3， IL-1β， ASC， and Caspase-1， as well as the expression of p-p65/p65， p-IκBα/IκBα， and HSP90 protein， were increased in HaCaT cells in the M5 group （P<0.05）. Compared with that in the M5 group， the mRNA expression of NLRP3， IL-1β， ASC， and Caspase-1 of cells in the NA-L group， the NA-M group， the NA-H group， and the NA-P-H group was decreased （P<0.05）. p-p65/p65 and p-IκBα/IκBα were decreased in the NA-M and NA-H groups （P<0.05）， and there was no change in HSP90 protein. Pull down-WB showed that NA could directly target HSP90 protein， and NA binding to HSP90 protein enhanced its thermal stability. Molecular docking of NA with HSP90 family proteins HSP90AA1， HSP90B1， and HSP90AB1 all resulted in highly stable binding. ConclusionNA can inhibit the proliferation and inflammatory response of psoriatic keratinocytes by a mechanism that may be achieved by targeting HSP90 to modulate the NF-κB/NLRP3 signaling pathway.

Objective To explore the protective effect of hydroalcoholic extract of Portulaca oleracea L.(POHA)on ulcerative colitis(UC)and bone loss in mice.Methods The C57BL/6 mice were treated with dextran sulfate sodium(DSS)to establish UC model.A total of 50 mice were randomly assigned to including control group,DSS group,mesalazine(MS)group,low dose of POHA(POHAL)group,or high dose of POHA(POHAH)group.The control group freely drank drinking water,while the DSS,MS,POHAL and POHAH groups drank drinking water containing DSS for 8 weeks.Since the 2nd week,the control group and DSS group were given normal saline by gavage.The MS group was given MS(100 mg/kg)by gavage.The POHAL group and POHAH group were given POHA(1 000 mg/kg and 2 000 mg/kg)by gavage,respectively.Body weight and disease activity index(DAI)were recorded and calculated every 2 d.On the 56th day,the colon weight index,liver index,and spleen index were calculated,and the histological changes of colon were observed.Serum levels of bone metabolism markers and microstructure parameters of femur were detected.Results Compared with the control group,the DSS group showed significantly increased DAI score,colon weight index,liver index,and spleen index(all P＜0.01).The DSS group exhibited significant pathological damage in colon tissues and significantly increased serum levels of osteocalcin,C-terminal peptide of collagen type Ⅰ,and tartrate-resistant acid phosphatase 5b(P＜0.01).The bone loss was significant in the DSS group,manifested by markedly decreased bone mineral density(BMD),bone tissue volume to tissue volume ratio(BV/TV),trabecular bone number(Tb.N),and trabecular bone thickness(Tb.Th),and markedly increased bone surface to bone volume ratio(BS/BV)and trabecular bone separation(Tb.Sp)(P＜0.05 or P＜0.01).Compared with the DSS group,the BMD,BV/TV,Tb.N and Tb.Th of the femur in the MS group and POHAH group of mice were all increased(P＜0.05 or P＜0.01),the BS/BV all decreased(P＜0.05 or P＜0.01),and the Tb.Sp all decreased without significant differences(all P＞0.05).The above bone microstructure parameters in the POHAL group showed no significant differences compared with those in the DSS group(all P＞0.05).Conclusion POHA has protective effect on DSS-induced UC and bone loss,and the mechanism may be related to the inhibition of hyperactive bone metabolism.

Objective To investigate the gene expression profile in rat pancreatic ductal stem cells(PDSCs)when induced to differentiate into insulin-secreting cells(IPCs),with the goal of identifying key genes involved in this differentiation process.Methods The expanded PDSCs were categorized into a normal control(NC)group and an induced(Tre)group.PDSCs continued expansion culture in NC group,and cultured in induction medium for 28 days to facilitate the differentiation of PDSCs into IPCs in Tre group.Dithizone staining was employed to morphologically assess whether the cells exhibited a reddish-brown coloration,indicating a positive result.The immunofluorescence staining method was used to detect the expression of insulin(Ins)and PDX1 in the cells following induction.Additionally,ELISA was conducted to measure the Ins release from IPCs,thereby verifying the responsiveness of the induced cells to glucose-stimulated Ins secretion.Concurrently,cells were collected on induction days 0 and 28 for RNA sequencing(RNA-seq),and differentially expressed genes(DEGs)were analyzed and functionally annotated.The analysis revealed that regulatory factor X3(RFX3)was overexpressed in PDSCs,and the impact of RFX3 upregulation on differentiation induction was subsequently verified.Results Compared with NC group,DTZ staining was positive,PDX1 and Ins proteins were expressed,and an increased release of Ins in response to sugar stimulation was demonstrated in the Tre group.RNA-seq analysis identified 4270 DEGs,and functional enrichment analysis utilizing the Gene Ontology and Kyoto Encyclopedia of Genes and Genomes databases revealed associations with Ins response,positive regulation of Ins secretion,pancreatic endocrine cell development,and overall pancreatic development.Additionally,functionally related genes such as ALDHA2,CREB5,EIF6,FOXO1,RFX3,WNT5a,OGT,GPR39,SMAD6,and TRPM2 were identified,indicating involvement in the cell cycle,TGF-β1 signaling pathway,FOXO signaling pathway,and Wnt signaling pathway in the regulation of the differentiation of pancreatic ductal stem cells(PDSCs)into insulin-producing cells(IPCs).Furthermore,the upregulation of RFX3 can inhibit the expression of TGF-β1 within 72 hours,thereby promoted the formation and release of Ins from insulin-positive cells.Conclusions Multiple genes and signaling pathways associated with pancreatic β-cell function collectively regulate the differentiation of rat PDSCs into IPCs.Notably,the upregulation of RFX3 enhances this differentiation process.

Objective To investigate how dietary supplementation with tetrahydrocurcumin(THC)improves kidney injury in type 2 dia-betic mellitus(T2DM)and its mechanism of action using transcriptome sequencing(RNA-seq).Methods C57BL/6J mice were randomly assigned to the control,T2DM,and T2DM+THC groups.After a high-fat meal and streptozotocin injection,the body weights and fasting blood glucose levels of each mouse with T2DM were measured.Hematoxylin and eosin staining,Oil red O staining,and RNA-seq were performed to examine kidney pathology,lipid deposition,and differentially expressed genes,respectively,in the mice.Results Mice in T2DM group exhibited significantly higher fasting blood glucose levels(P＜0.001),renal tubule degeneration,glomeruli enlargement,disordered epithelial cells,and increased kidney lipid deposition after 12 weeks compared with those of the control group.THC adminis-tration alleviated all these conditions(P＜0.001).RNA-seq analysis revealed significant gene expression variations among the control,T2DM,and T2DM+THC groups.THC may protect against T2DM-induced kidney injury and lipid deposition by regulating the cell cycle(apoptosis),P53 signaling pathway,and PPARγ signaling path way,as indicated by Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analyses.In mice with T2DM,THC intervention may upregulate the expression of Cd36,Lpl,PPARγ,and Plin4 genes in renal tissues,while downregulating Ccnb1,Ccnb2,Cdk1,Bub1,and Cdc25c gene expressions.The proteins encoded by the four upregulated genes interact,as do those encoded by the five downregulated genes.Conclusion THC administration improves fasting blood glucose levels,reduces renal damage,and decreases fat deposition in mice with T2DM.The processes may involve decreasing apoptosis,blocking the P53 signaling pathway,and activating the PPARγ signaling pathway.

Objective To establish the functional constipated mouse model by compound diphenoxylate, and explore the anti-constipation effect of black garlic polysaccharide. Methods Mouse small intestine ink propulsion experiment and mouse defecation experiment were carried out respectively. The mice in each experiment were randomly divided into blank group, model group, positive group and black garlic polysaccharide （0.25, 0.5, 1 g/kg） groups. Mice in blank group and model group were given distilled water, and in positive group were given lactulose oral solution. Compound diphenoxylate （5 mg/kg） was intragastric administrated after 1 week of administration, and small intestine propulsion experiment and defecation experiment were conducted respectively. Results Compared with model group, intestinal propulsion rate of black garlic polysaccharide groups was significantly increased and first dejection time was significantly shorten, and the number, weight and fecal water content increased significantly at 6 h in middle and high dose groups. Conclusion Black garlic polysaccharide could promote intestinal propelling, shorten defecation time and increase fecal water content.

In recent years, the bacteriophage Φ29 (Phi29) DNA polymerase has garnered increasing attention due to its high-fidelity amplification capacity at constant temperatures. To advance the industrial application of this type of isothermal polymerases, this study mined and characterized new enzymes from the microbial metagenome based on the known Phi29 DNA polymerase sequence. The results revealed that a new enzyme, Php29 DNA polymerase, was identified in the microbial metagenome with plants as the hosts. This enzyme exhibited higher strand displacement activity, with a 59.5% similarity to bacteriophage Φ29. Experimental validation demonstrated that the enzyme had 3'→5' exonuclease activity, and its amplification products can serve as substrates for further catalytic reactions. The discovery and validation of Php29 DNA polymerase gives insights into the future industrial application of isothermal polymerases.
DNA-Directed DNA Polymerase/metabolism* ; Bacillus Phages/genetics* ; Metagenome

Objective To explore the role of pseudogene AC106872.1 in maintaining the self-renewal capacity of human embryonic stem cells(hESCs).Methods AC106872.1 was knocked out in hESCs and knockout effi-ciency was validated by PCR and agarose gel electrophoresis.The colony formation of hESCs was assessed through colony formation assays and alkaline phosphatase(AP)staining.The expression level of pluripotency and differ-entiation marker genes was analyzed by qPCR and flow cytometry.RNA sequencing(RNA-seq)was performed to assess transcriptomic changes upon AC106872.1 knockout.Results Knockout of AC106872.1 significantly in-hibited the colony formation of hESCs(P＜0.05).The expression level of pluripotency marker genes was signifi-cantly reduced(P＜0.000 1),while the expression of differentiation marker genes was markedly increased(P＜0.000 1).Conclusions The pseudogene AC 1 06872.1 plays a crucial role in maintaining human embryonic stem cell self-renewal through regulation of pluripotency genes expression.