ObjectiveTo analyze the "compatibility" relationship of sugars and glycosides and the heat-clearing and blood-cooling effect of the roots of four varieties of Rehmannia glutinosa and provide a basis for research on the pharmacodynamic material basis and quality control of R. glutinosa. MethodsThe content of sugars and glycosides in the roots of four varieties of R. glutinosa was determined during the growth period. The principal component analysis （PCA）， orthogonal partial least squares-discriminant analysis （OPLS-DA）， and the "compatibility" relationship of active components were employed to screen out the differential samples. A rat model of bleeding due to blood heat was used to verify the pharmacodynamic differences and the potential active components of differential samples. ResultsThe content and proportion characteristics of various components in roots of the four varieties of R. glutinosa during the expansion stage and the maturity stage had obvious differences. The proportion of phenylethanoid glycosides at the maturity stage was higher than that at the expansion stage. The R. glutinosa variety 85-5 had special quality characteristics among the tested varieties. All the samples alleviated the symptoms in the rat model. The effect of clearing heat and cooling blood was different between the maturity stage and the expansion stage， as well as between 85-5 samples at the maturity stage and other samples. The effect of clearing heat and cooling blood of R. glutinosa roots was the result of the combined action of multiple components in R. glutinosa roots and might be related to the high proportions of polysaccharides， iridoid glycosides， and phenylethanoid glycosides. ConclusionThe growth stage and variety affect the quality of R. glutinosa roots. The effect of clearing heat and cooling blood of R. glutinosa roots was related to the content and proportions of various components. The study can provide a basis for the basic research on the active components and quality control of R. glutinosa.

ObjectiveTo analyze the "compatibility" relationship of sugars and glycosides and the heat-clearing and blood-cooling effect of the roots of four varieties of Rehmannia glutinosa and provide a basis for research on the pharmacodynamic material basis and quality control of R. glutinosa. MethodsThe content of sugars and glycosides in the roots of four varieties of R. glutinosa was determined during the growth period. The principal component analysis （PCA）， orthogonal partial least squares-discriminant analysis （OPLS-DA）， and the "compatibility" relationship of active components were employed to screen out the differential samples. A rat model of bleeding due to blood heat was used to verify the pharmacodynamic differences and the potential active components of differential samples. ResultsThe content and proportion characteristics of various components in roots of the four varieties of R. glutinosa during the expansion stage and the maturity stage had obvious differences. The proportion of phenylethanoid glycosides at the maturity stage was higher than that at the expansion stage. The R. glutinosa variety 85-5 had special quality characteristics among the tested varieties. All the samples alleviated the symptoms in the rat model. The effect of clearing heat and cooling blood was different between the maturity stage and the expansion stage， as well as between 85-5 samples at the maturity stage and other samples. The effect of clearing heat and cooling blood of R. glutinosa roots was the result of the combined action of multiple components in R. glutinosa roots and might be related to the high proportions of polysaccharides， iridoid glycosides， and phenylethanoid glycosides. ConclusionThe growth stage and variety affect the quality of R. glutinosa roots. The effect of clearing heat and cooling blood of R. glutinosa roots was related to the content and proportions of various components. The study can provide a basis for the basic research on the active components and quality control of R. glutinosa.

BACKGROUND:Osteosarcoma has a complex pathogenesis and a poor prognosis.While advancements in medical technology have led to some improvements in the 5-year survival rate,substantial progress in its treatment has not yet been achieved. OBJECTIVE:To screen key molecular markers in osteosarcoma,analyze their relationship with osteosarcoma treatment drugs,and explore the potential disease mechanisms of osteosarcoma at the molecular level. METHODS:GSE99671 and GSE284259(miRNA)datasets were obtained from the Gene Expression Omnibus database.Differential gene expression analysis and Weighted Gene Co-expression Network Analysis(WGCNA)on GSE99671 were performed.Functional enrichment analysis was conducted using Gene Ontology and Kyoto Encyclopedia of Genes and Genomes separately for the differentially expressed genes and the module genes with the highest positive correlation to the disease.The intersection of these module genes and differentially expressed genes was taken as key genes.A Protein-Protein Interaction network was constructed,and correlation analysis on the key genes was performed using CytoScape software,and hub genes were identified.Hub genes were externally validated using the GSE28425 dataset and text validation was conducted.The drug sensitivity of hub genes was analyzed using the CellMiner database,with a threshold of absolute value of correlation coefficient|R|>0.3 and P<0.05. RESULTS AND CONCLUSION:(1)Differential gene expression analysis identified 529 differentially expressed genes,comprising 177 upregulated and 352 downregulated genes.WGCNA analysis yielded a total of 592 genes with the highest correlation to osteosarcoma.(2)Gene Ontology enrichment results indicated that the development of osteosarcoma may be associated with extracellular matrix,bone cell differentiation and development,human immune regulation,and collagen synthesis and degradation.Kyoto Encyclopedia of Genes and Genomes enrichment results showed the involvement of pathways such as PI3K-Akt signaling pathway,focal adhesion signaling pathway,and immune response in the onset of osteosarcoma.(3)The intersection analysis revealed a total of 59 key genes.Through Protein-Protein Interaction network analysis,8 hub genes were selected,which were LUM,PLOD1,PLOD2,MMP14,COL11A1,THBS2,LEPRE1,and TGFB1,all of which were upregulated.(4)External validation revealed significantly downregulated miRNAs that regulate the hub genes,with hsa-miR-144-3p and hsa-miR-150-5p showing the most significant downregulation.Text validation results demonstrated that the expression of hub genes was consistent with previous research.(5)Drug sensitivity analysis indicated a negative correlation between the activity of methotrexate,6-mercaptopurine,and pazopanib with the mRNA expression of PLOD1,PLOD2,and MMP14.Moreover,zoledronic acid and lapatinib showed a positive correlation with the mRNA expression of PLOD1,LUM,MMP14,PLOD2,and TGFB1.This suggests that zoledronic acid and lapatinib may be potential therapeutic drugs for osteosarcoma,but further validation is required through additional basic experiments and clinical studies.

Objective: To explore the effect of different concentrations of relaxin-2(RLN-2) on the proliferation and migration abilities of human immortalized keratinocytes(HaCaT cells). Methods: Methods HaCaT cells were cultured in media with different concentrations of RLN-2, and the cells were cultured in media without RLN-2 as the control group.The effect on cell proliferation was assessed by using the CCK-8 reagent, the cell migration ability was evaluated throughin vitrocell scratch assay, the cell cycle was examined by flow cytometry, and the expression levels of cell cycle proteins Cyclin B1 and Cyclin A2 were detected by Western blot. Results: After being cultured for 24 hours under RLN-2 concentration ranging from 10～100 ng/ml, HaCaT cells showed progressively increased proliferation and migration capabilities compared to the control group, with elevated expression levels of cell cycle proteins Cyclin B1 and Cyclin A2 and an increased proportion of cells in S and G2/M phases, peaking at 100 ng/ml. However, HaCaT cells cultured with 200 ng/ml of RLN-2 exhibited reduced proliferation and migration capabilities, decreased expression levels of Cyclin B1 and Cyclin A2, and a lower proportion of cells in S and G2/M phases compared to the 100 ng/ml group. Conclusion RLN-2 can enhance the migration ability of HaCaT cells within an appropriate concentration range and may also promote cell proliferation by increasing the expression of related cell cycle proteins and the proportion of cells in S and G2/M phases.

Objective:To investigate the effects of botulinum toxin type A (BTX-A) and magnesium sulfate on the survival rate of random-pattern skin flaps (RSF) with different length-to-width ratios.Methods:Using a random number table method, 45 SD rats were divided into three groups: the saline group (Group A), the BTX-A group (Group B), and the magnesium sulfate group (Group C), with 15 rats in each group. Each group was further subdivided into three subgroups based on different length-to-width ratios of RSF (1∶1, 2∶1, 3∶1), with 5 rats in each subgroup. The preparation of the RSF involved using the midline of the rat’s back as the axis and the level 1 cm below the iliac crest line as the base, extending towards the head. The skin tissue was incised to the dorsal fascia layer, separating the subcutaneous tissue at the superficial layer of the deep fascia, while severing the blood vessels and their branches on both sides and at the base. After hemostasis, the flap was sutured in place. Immediately after surgery, 0.2 ml of saline, BTX-A (25 U/ml), or magnesium sulfate solution (50 mg/ml) was injected into the proximal, middle, and distal ends of the flap. On the seventh day post-surgery, the gross appearance of the flap was assessed, and the survival rate was calculated. The surviving flap tissue underwent hematoxylin and eosin (HE) staining to evaluate microvascular density (MVD) and the degree of vasodilation (including vessel outer diameter, inner diameter, and wall thickness). Immunohistochemistry was used to detect the expression level of vascular endothelial growth factor (VEGF). Statistical analysis was performed using GraphPad Prism 9.0.1 software, with data expressed as Mean ± SD. One-way ANOVA was used for multiple group comparisons, and Tukey’s test was used for pairwise comparisons.Results:On the seventh day post-surgery, flaps with a length-to-width ratio of 1∶1 healed well in all subgroups. In the case of flaps with a 2∶1 ratio, Group A exhibited partial necrosis at the distal end, characterized by blackened, non-elastic scabs and exudate. Groups B and C generally healed well. For flaps with a 3∶1 ratio, Group A exhibited extensive necrosis at both the middle and distal ends, with similar blackened, non-elastic scabs, non-bleeding cut sections, and exudate. Groups B and C showed only partial blackening at the distal end, with most areas healing effectively. The survival rates of flaps with a 1∶1 ratio did not show significant differences among the three groups ( P>0.05). Compared to Group A, Groups B and C had significantly higher survival rates for flaps with 2∶1 and 3∶1 ratios ( P<0.01), with no significant difference between Groups B and C ( P>0.05). HE staining indicated that as the length-to-width ratios increased, tissue edema and inflammatory cell infiltration also increased in all groups. Groups B and C had significantly reduced inflammatory changes compared to Group A, with a greater number of newly formed microvessels observed. Quantitative analysis revealed that MVD in Groups B and C was significantly higher than in Group A, regardless of the flap ratio ( P<0.05), with no significant difference between Groups B and C ( P>0.05). Vasodilation analysis showed that the outer diameter and wall thickness of vessels in Groups B and C were significantly greater than those in Group A ( P<0.05), with no significant difference between Groups B and C ( P>0.05). Immunohistochemical staining revealed that VEGF expression levels in Groups B and C were higher than in Group A, regardless of the flap ratio ( P<0.01). In flaps with a 1∶1 ratio, VEGF expression was higher in Group C than in Group B ( P<0.05), with no significant difference between the two groups for other flap ratios ( P>0.05). Conclusion:In RSF with length-to-width ratios of 2∶1 and 3∶1, subcutaneous injections of BTX-A or magnesium sulfate after replantation can promote the expansion and formation of blood vessels in the flap, increase the expression of VEGF, and improve the survival rate of the RSF.

Objective:To investigate the effects of botulinum toxin type A (BTX-A) and magnesium sulfate on the survival rate of random-pattern skin flaps (RSF) with different length-to-width ratios.Methods:Using a random number table method, 45 SD rats were divided into three groups: the saline group (Group A), the BTX-A group (Group B), and the magnesium sulfate group (Group C), with 15 rats in each group. Each group was further subdivided into three subgroups based on different length-to-width ratios of RSF (1∶1, 2∶1, 3∶1), with 5 rats in each subgroup. The preparation of the RSF involved using the midline of the rat’s back as the axis and the level 1 cm below the iliac crest line as the base, extending towards the head. The skin tissue was incised to the dorsal fascia layer, separating the subcutaneous tissue at the superficial layer of the deep fascia, while severing the blood vessels and their branches on both sides and at the base. After hemostasis, the flap was sutured in place. Immediately after surgery, 0.2 ml of saline, BTX-A (25 U/ml), or magnesium sulfate solution (50 mg/ml) was injected into the proximal, middle, and distal ends of the flap. On the seventh day post-surgery, the gross appearance of the flap was assessed, and the survival rate was calculated. The surviving flap tissue underwent hematoxylin and eosin (HE) staining to evaluate microvascular density (MVD) and the degree of vasodilation (including vessel outer diameter, inner diameter, and wall thickness). Immunohistochemistry was used to detect the expression level of vascular endothelial growth factor (VEGF). Statistical analysis was performed using GraphPad Prism 9.0.1 software, with data expressed as Mean ± SD. One-way ANOVA was used for multiple group comparisons, and Tukey’s test was used for pairwise comparisons.Results:On the seventh day post-surgery, flaps with a length-to-width ratio of 1∶1 healed well in all subgroups. In the case of flaps with a 2∶1 ratio, Group A exhibited partial necrosis at the distal end, characterized by blackened, non-elastic scabs and exudate. Groups B and C generally healed well. For flaps with a 3∶1 ratio, Group A exhibited extensive necrosis at both the middle and distal ends, with similar blackened, non-elastic scabs, non-bleeding cut sections, and exudate. Groups B and C showed only partial blackening at the distal end, with most areas healing effectively. The survival rates of flaps with a 1∶1 ratio did not show significant differences among the three groups ( P>0.05). Compared to Group A, Groups B and C had significantly higher survival rates for flaps with 2∶1 and 3∶1 ratios ( P<0.01), with no significant difference between Groups B and C ( P>0.05). HE staining indicated that as the length-to-width ratios increased, tissue edema and inflammatory cell infiltration also increased in all groups. Groups B and C had significantly reduced inflammatory changes compared to Group A, with a greater number of newly formed microvessels observed. Quantitative analysis revealed that MVD in Groups B and C was significantly higher than in Group A, regardless of the flap ratio ( P<0.05), with no significant difference between Groups B and C ( P>0.05). Vasodilation analysis showed that the outer diameter and wall thickness of vessels in Groups B and C were significantly greater than those in Group A ( P<0.05), with no significant difference between Groups B and C ( P>0.05). Immunohistochemical staining revealed that VEGF expression levels in Groups B and C were higher than in Group A, regardless of the flap ratio ( P<0.01). In flaps with a 1∶1 ratio, VEGF expression was higher in Group C than in Group B ( P<0.05), with no significant difference between the two groups for other flap ratios ( P>0.05). Conclusion:In RSF with length-to-width ratios of 2∶1 and 3∶1, subcutaneous injections of BTX-A or magnesium sulfate after replantation can promote the expansion and formation of blood vessels in the flap, increase the expression of VEGF, and improve the survival rate of the RSF.

Objective To investigate the effects of 6-pyruvoyl-tetrahydropterin synthase(PTS)on gastric cancer cells.Methods The expression of PTS protein and mRNA in gastric mucosal epithelial cells GES-1 and gastric cancer cell lines HGC-27 cells,MGC-803 cells,AGS cells and MKN-45 cells were determined by Western blot(WB)and reverse transcription quantitative polymerase chain reaction(RT-qPCR).AGS cells were selected for follow-up experiments.AGS cells were divided into control(NC)group,sh-PTS group,and sh-PTS+BML-284 group.Cell proliferation was measured by CCK-8.Cell migration ability was measured by scratch healing.Cell invasion and migration were measured by Transwell assay.Apoptosis was measured by flow cytometry.The protein and mRNA levels of β-catenin,c-myc,GSK-3β and Wnt5a were detected by WB and RT-qPCR.Results The expression of PTS in gastric mucosal epithelial cells GES-1 was lower than that in gastric cancer cells,and the expression of PTS was the highest in AGS cells.Knocking down PTS could inhibit proliferation,migration and invasion of gastric cancer cells,promote cell apoptosis,decreased β-catenin,c-myc,Wnt5a protein levels,and increased GSK-3β protein levels(P＜0.05).Compared with sh-PTS group,cell proliferation,migration and invasion ability of sh-PTS+BML-284 group were enhanced,apoptosis level was decreased,protein expression of β-catenin,c-myc and Wnt5a was increased,and protein expression of GSK-3β was decreased(P＜0.05).Conclusion PTS can affect proliferation,migration,invasion and apoptosis of gastric cancer cells by mediating Wnt/β-catenin pathway.

Purpose To observe the clinical and CT features of severe immune checkpoint inhibitor-related pneumonitis(CIP)in lung cancer patients.Materials and Methods A total of 174 patients with lung cancer who received immune checkpoint inhibitor(PD-1/PD-L1 inhibitors)in Xi'an International Medical Center Hospital from September 1,2019 to March 31,2022 were retrospectively collected.Clinical and imaging features of patients with severe CIP were analyzed.Results There were 23 patients who met the diagnostic criteria of severe CIP.Among them,22 were male patients,15 were younger(＜65 years old),17 had a history of underlying lung disease,16 had a history of chemoradiotherapy and other treatments,and 21 had a history of combined radiotherapy and chemoradiotherapy.The median time from the initiation of immune checkpoint inhibitor to CIP was 128(74,348)days.19 patients were non-small cell carcinoma.CIP occurred in 16 patients with right lung cancer,15 had tumor central airway invasion,14 had radiographic features of diffuse alveolar injury/acute interstitial pneumonia pattern,and 20 died during follow-up.Conclusion Severe CIP is likely to occur in male lung cancer patients with a history of basic medical history and radiotherapy and chemotherapy.The clinical manifestations are varied,and the main imaging features are diffuse alveolar injury/acute interstitial pneumonia pattern,and the prognosis is poor.

Existing studies have shown that Astragalus membranaceus(AM)and its active ingredients astragalus polysaccharides,oninon,and astragalus methyl glycosides can attenuate X-ray radiation-induced injury.However,there are no studies on how isoliquiritigenin(ISL)attenuate the bystander effect of bone marrow mesenchymal stem cells(BMSCs)induced by carbon ion radiation therapy for lung cancer.This study aimed to investigate the AM-derived small molecule ISL to enhance radiotherapy sensitivity by attenuating the carbon ion radiation-induced bystander effect(RIBE)in BMSCs to elucidate its mecha-nism of action.In this study,we established a C57BL/6 mouse lung cancer transplantation tumor model in vivo and a co-culture model of A549 cells and BMSCs in vitro,and the models were successfully treated with carbon ions.In further work,we used flow cytometry,immunofluorescence,Western blot,enzyme-linked immunosorbent assay(ELISA),inhibitor,short hairpin RNA(shRNA),Cell Counting Kit-8(CCK-8),and other methods to illustrate the mechanism.In the next experiments,we found that ISL combined with carbon ion radiotherapy had a significant anti-tumor effect and protected BMSCs from radiation damage.The aim of this study was to investigate the potential of ISL in enhancing the sensitivity of lung cancer cells to radiotherapy and attenuating RIBE in both in vitro and in vivo settings.Traditional Chinese medicine combined with radiation therapy is a promising and innovative treatment for non-small cell lung cancer.These results establish a theoretical foundation for further clinical development of ISL as a potential radiosensitizer option.

AIM:To investigate the effect of vaccarin(VAC)on endothelial dysfunction in type 2 diabetes mellitus(T2DM),and to uncover the underlying mechanisms.METHODS:(1)C57BL/6 mice received intraperitoneal injection of streptozotocin and were fed with a high-fat diet(21.8 kJ/kg,60%of the energy source was fat)to construct a T2DM mouse model.Thirty mice were randomly divided into control,T2DM and T2DM+VAC groups,with 10 mice in each group.The mice in T2DM+VAC group were given 1 mg/kg VAC via oral gavage for 6 weeks,while those in control and T2DM groups were given the same volume of PBS.The mRNA and protein expression levels of BCL2-interacting pro-tein 3(BNIP3),PTEN-induced kinase 1(PINK1)and parkin in the thoracic aorta were detected by RT-qPCR and West-ern blot.(2)Human umbilical vein endothelial cells(HUVECs)were stimulated by high glucose(HG;35 mmol/L glu-cose).Mitochondrial membrane potential,autophagy and mitochondrial superoxide levels were detected using JC-1,acri-dine orange(AO)and MitoSOX staining,respectively.RESULTS:Compared with control group,the mRNA and protein levels of BNIP3,PINK1 and parkin were significantly increased in the thoracic aorta of T2DM mice(P<0.05).Compared with T2DM group,the mRNA and protein levels of BNIP3,PINK1 and parkin in the thoracic aorta were significantly re-duced in T2DM+VAC group(P<0.05).The results of JC-1,AO and MitoSOX staining showed that VAC attenuated the decrease in mitochondrial membrane potential and the increase in autophagy and mitochondrial superoxide levels in HG-in-duced HUVECs.Treatment with VAC also inhibited HG-mediated mitochondrial damage in HUVECs after BNIP3 overex-pression.The effect of miR-570-3p mimic on mitochondrial damage was similar to VAC.RT-qPCR and Western blot showed that both miR-570-3p mimic and VAC significantly reduced the mRNA and protein levels of BNIP3,PINK1 and parkin.In contrast,inhibition of miR-570-3p exhibited the opposite effects.CONCLUSION:Treatment with VAC alle-viated endothelial dysfunction in T2DM by inhibiting HG-induced mitochondrial dysfunction through miR-570-3p/BNIP3.