Objective To construct a traditional Chinese medicine diagnostic scale suitable for chronic obstructive pulmonary disease(COPD)with lung qi stagnation syndrome,and to verify the reliability and validity of the scale.Methods Preliminary research has identified 16 core symptom items for lung qi stagnation syndrome.Diagnosis and scale collection were conducted on 95 patients using both traditional Chinese and Western medicine,with scores of 0,1,2,and 3 based on the severity of symptoms.By frequency t-test,discrete trend,and Cronbach's alpha coefficient screening items were used to evaluate the internal consistency of the scale,Spearman Brown coefficient was used to evaluate the stability of the scale,and exploratory factor analysis was used to determine the structural validity of the scale.Results Partial items were excluded and the final 11 scale items were confirmed.The overall Cronbach's alpha coefficient of the scale was 0.719,and the overall Spearman Brown coefficient was 0.647;The KMO test value is 0.612>0.5,The significance level of Bartlett's sphericity test is P<0.01;Extracting common factors with feature roots greater than 1,the maximum total variance explained by 64.122%was achieved when extracting four common factors.The common factor loadings for each item were all greater than 0.5,and the variance was all greater than 0.4,indicating good structural validity of the scale.Conclusion This study constructed and validated a traditional Chinese medicine diagnostic scale for COPD with lung qi stagnation syndrome.The scale has good reliability and validity,providing a reliable tool for clinical diagnosis and treatment.

Objective To investigate the protective effect and mechanism of heat acclimation(HA)on electromagnetic field(EMF)induced hippocampus neuron injury in mice.Methods Forty healthy BALB/c male mice(18～22 g,7 weeks old)were randomly divided into 4 groups(n=10):Control group(Con),HA group(34℃,30 d),EMF group(2 450 MHz,20 min/d,4 weeks)and HA+EMF group(HA preconditioning+EMF).Sucrose preference test was performed to evaluate sucrose preference levels of mice in each group.Tail suspension test and forced swimming test were utilized to observe the immobility time.Morris water maze test was conducted to determine the learning and memory capabilities.Pathological changes in the hippocampus were observed with HE staining.Immunohistochemical assay for Iba1(marker of microglia),CD68(marker of pro-inflammatory phenotype)and CD206(marker of anti-inflammatory phenotype)were used to detect the number and activation phenotype of microglia in the hippocampus.ELISA was applied to measure the levels of TNF-α,IL-1β,TGF-β and IL-10 in the hippocampus of each group.Western blotting was performed to determine the protein levels of HSP70 in the hippocampus.Results As compared with the Con group,the EMF group showed a decreased preference for sucrose(P<0.05),prolonged immobile time in the tail suspension test(P<0.01)as well as in the forced swimming test(P<0.01),extented escape latency on the 7th day(P<0.01),and a decreased time of crossing the platform(P<0.05).EMF exposure resulted in that the hippocampal neurons were in disordered arrangement,loose structure and irregular morphology,with swollen cytoplasm and condensed nuclei,swollen and more microglial cells in the hippocampus(P<0.01),and enhanced relative fluorescence intensity of CD68(P<0.01),but not in CD206 fluorescence intensity(P=0.885).All these findings suggested that activated microglia predominantly exhibited a pro-inflammatory M1 phenotype during this phase.In the hippocampus,the levels of TNF-α and IL-1β were significantly increased,while the levels of IL-10 and TGF-β were significantly decreased(P<0.01).HA treatment reversed the conditions induced by EMF exposure,including better preference for sucrose(P<0.01),shorten immobile time in tail suspension test(P<0.05)and forced swimming test(P<0.01),less escape latency on the 7th day(P<0.01),and improved hippocampal cell injuries.Compared with the Con group,there were more microglial cells in the hippocampus in the HA+EMF group,with increased relative fluorescence intensity of M2 phenotype marker CD206(P<0.01)and decreased CD68 fluorescence intensity(P<0.01).HA treatment also significantly decreased the expression of TNF-α and IL-1β levels(P<0.01),increased the expression of IL-10 and TGF-β(P<0.01),and elevated the protein level of HSP70(P<0.01)when compared with the EMF group.Conclusion HA may ameliorate EMF-induced hippocampus neurons injury in mice by altering the phenotype of activated microglia and inhibiting inflammatory responses.

Objective To construct a traditional Chinese medicine diagnostic scale suitable for chronic obstructive pulmonary disease(COPD)with lung qi stagnation syndrome,and to verify the reliability and validity of the scale.Methods Preliminary research has identified 16 core symptom items for lung qi stagnation syndrome.Diagnosis and scale collection were conducted on 95 patients using both traditional Chinese and Western medicine,with scores of 0,1,2,and 3 based on the severity of symptoms.By frequency t-test,discrete trend,and Cronbach's alpha coefficient screening items were used to evaluate the internal consistency of the scale,Spearman Brown coefficient was used to evaluate the stability of the scale,and exploratory factor analysis was used to determine the structural validity of the scale.Results Partial items were excluded and the final 11 scale items were confirmed.The overall Cronbach's alpha coefficient of the scale was 0.719,and the overall Spearman Brown coefficient was 0.647;The KMO test value is 0.612>0.5,The significance level of Bartlett's sphericity test is P<0.01;Extracting common factors with feature roots greater than 1,the maximum total variance explained by 64.122%was achieved when extracting four common factors.The common factor loadings for each item were all greater than 0.5,and the variance was all greater than 0.4,indicating good structural validity of the scale.Conclusion This study constructed and validated a traditional Chinese medicine diagnostic scale for COPD with lung qi stagnation syndrome.The scale has good reliability and validity,providing a reliable tool for clinical diagnosis and treatment.

Objective To investigate the effect of microglial derived extracellular vesicles on neuronal damage in the context of heat stress.Methods After BV2 microglial cells were exposed to heat stress,the supernatant was collected and subjected to ultracentrifugation at different speeds to obtain large and small vesicles,respectively.Nano Particle Tracking and Zeta Potential Distribution Analyzer was used to measure and analyze the size distribution of the large vesicles and small vesicles.Western blotting was used to detect the expression of specific vesicle surface markers,TSG101,CD63 and flotillin-1.Microglial extracellular vesicles were labeled with PKH67 dye and then co-cultured with N2a cells to examine the uptake by capacity the neurons.After large and small vesicles derived from microglia after heat stress stimulation were co-cultured with N2a cells,respectively,CCK-8 assay,lactate dehydrogenase (LDH)assay,Trypan blue staining and TUNEL assay were employed to evaluate heat stress induced neuronal damage.Results The small vesicles were in a particle size of 30～120 nm,and highly expressed TSG101 and CD63,whereas the large vesicles,in a size of 90～1000 nm,highly expressed flotillin-1.The BV2-derived extracellular vesicles could be taken up by N2a cells and were proved to be involved in the modulation of N2a cell injury caused by heat stress.CCK-8 assay showed that both large and small vesicles of microglial cells inhibited the viability of N2a cells after heat exposure (P<0.05).The results of LDH assay,Trypan blue staining and TUNEL assay showed that both large (P<0.05)and small vesicles (P<0.01)significantly enhanced the LDH release,blue stain intensity and apoptosis of N2a cells after heat exposure,and the release,intensity and apoptosis were stronger in the cells treated with small vesicles than those group of large vesicles.Conclusion Microglia aggravate heat stress-induced neuronal damage through releasing extracellular vesicles.

Background: s/Aims: Transmembrane 6 superfamily member 2 (TM6SF2) E167K variant is closely associated with the occurrence and development of metabolic dysfunction-associated steatotic liver disease (MASLD). However, the role and mechanism of TM6SF2 E167K variant during MASLD progression are not yet fully understood. Methods: The Tm6sf2167K knock-in (KI) mice were subjected to high-fat diet (HFD). Hepatic lipid levels of Tm6sf2167K KI mice were detected by lipidomics analysis. Thin-layer chromatography (TLC) was used to measure the newly synthesized triglyceride (TG) and phosphatidylcholine (PC). Results: The TM6SF2 E167K variant significantly aggravated hepatic steatosis and injury in HFD-induced mice. Decreased polyunsaturated PC level and increased polyunsaturated TG level were found in liver tissue of HFDinduced Tm6sf2167K KI mice. Mechanistic studies demonstrated that the TM6SF2 E167K variant increased the interaction between TM6SF2 and PNPLA3, and impaired PNPLA3-mediated transfer of polyunsaturated fatty acids (PUFAs) from TG to PC. The TM6SF2 E167K variant increased the level of fatty acid-induced malondialdehyde and reactive oxygen species, and decreased fatty acid-downregulated cell membrane fluidity. Additionally, the TM6SF2 E167K variant decreased the level of hepatic PC containing C18:3, and dietary supplementation of PC containing C18:3 significantly attenuated the TM6SF2 E167K-induced hepatic steatosis and injury in HFD-fed mice. Conclusions The TM6SF2 E167K variant could promote its interaction with PNPLA3 and inhibit PNPLA3-mediated transfer of PUFAs from TG to PC, resulting in the hepatic steatosis and injury during MASLD progression. PC containing C18:3 could act as a potential therapeutic supplement for MASLD patients carrying the TM6SF2 E167K variant.

Chemotherapy is an important adjuvant treatment of glioma, while the efficacy is far from satisfactory, due not only to the biological barriers of blood‒brain barrier (BBB) and blood‒tumor barrier (BTB) but also to the intrinsic resistance of glioma cells via multiple survival mechanisms such as up-regulation of P-glycoprotein (P-gp). To address these limitations, we report a bacteria-based drug delivery strategy for BBB/BTB transportation, glioma targeting, and chemo-sensitization. Bacteria selectively colonized into hypoxic tumor region and modulated tumor microenvironment, including macrophages repolarization and neutrophils infiltration. Specifically, tumor migration of neutrophils was employed as hitchhiking delivery of doxorubicin (DOX)-loaded bacterial outer membrane vesicles (OMVs/DOX). By virtue of the surface pathogen-associated molecular patterns derived from native bacteria, OMVs/DOX could be selectively recognized by neutrophils, thus facilitating glioma targeted delivery of drug with significantly enhanced tumor accumulation by 18-fold as compared to the classical passive targeting effect. Moreover, the P-gp expression on tumor cells was silenced by bacteria type III secretion effector to sensitize the efficacy of DOX, resulting in complete tumor eradication with 100% survival of all treated mice. In addition, the colonized bacteria were finally cleared by anti-bacterial activity of DOX to minimize the potential infection risk, and cardiotoxicity of DOX was also avoided, achieving excellent compatibility. This work provides an efficient trans-BBB/BTB drug delivery strategy via cell hitchhiking for enhanced glioma therapy.

ObjectiveTo introduce the Hr gene of spontaneously mutated SHJH^hr mice into BALB/cAShjh inbred mice with clear genetic background,and provide a basis for study on the molecular mechanism of Hr gene mutation-induced abnormal phenotype and the application of this model.Methods Using a backcross-intercross breeding method guided by phenotypic monitoring, mutant genes from SHJH^hr mice bred by spontaneous mutation were introduced into inbred BALB/cAShjh mice by homozygous mutation introgression, and the mice were bred into BALB/cA.Cg.SHJH^hr (abbreviated as C.Cg.SHJH^hr) mice after 10 generations. The genotypes of 90 single nucleotide polymorphism (SNP) detection sites were analyzed in C.Cg.SHJH^hr mice by multiplex PCR library construction followed by next generation sequencing. Then 14 biochemical locus marker genes were detected in C.Cg.SHJH^hr mice according to the method of GB/T 14927.1-2008. Finally, whole genome exon sequencing was utilized to detect the mutated genes in this mouse. ResultsFrom May 2018 to March 2022, a total of 10 generations of backcross-intercross were conducted to complete the construction of the C.Cg.SHJH^hr mouse line. Among the 90 SNPs loci detected, except for rs13484115 and rs13484116, all the other loci had the same genotype as the recipient mice BALB/cAShjh. The results of biochemical marker gene detection showed that all the 14 loci of the mouse were the same as those of the recipient mouse. Whole genome exon sequencing found that the mouse had 109 site mutations compared with the recipient mouse strain, including 71 synonymous mutations, 1 stopgain, 37 missense mutations, and 20 genes involved in protein sequence alterations (including the reported Hr gene). ConclusionC.Cg.SHJH^hr mice were created. Through exon sequencing and genetic analysis, three Hr mutated genes and associated mutated genes that mainly cause phenotypic variations were identified, which provides a basis for expanding the application of C.Cg.SHJH^hr mice in biomedical research.

Objective To measure and analyze biological characteristics and aging phenotype of SHJH^hr mice and provide basic data for the application of the mouse model in aging mechanisms research and antiaging drug development. MethodsWith ICR mice of the same age as control group, the body mass growth data of SHJH^hr mice at the age of 3 to 16 weeks, the reproduction ability of 1 to 4 fetuses and the life cycle of SHJH^hr mice were measured. Blood routine (30 items) and serum biochemical indexes (25 items) of 6-week-old SHJH^hr mice were measured. The venous blood of 8-week-old SHJH^hr mice was collected for flow cytometry analysis to determine the content of immune cells. The aging bone structure of the cancellous bone and bone mineral density of SHJH^hr mice aged 4, 8 and 26 weeks were measured by micro-CT. Histopathological changes of bone and joint of 8-week-old mice were observed. ResultsCompared with ICR mice, the female and male body mass of SHJH^hr mice were significantly lower at the age of 16 weeks (P < 0.05), and the reproductive performance of female mice was low (P < 0.01) or did not have normal reproductive capacity. The shortest survival time of SHJH^hr mice was 57 weeks and the longest was 71 weeks, which was shorter than those of normal ICR mice, showing obvious rapid aging phenomenon. At the same time, some physiological and biochemical indexes of blood and pathological changes of bone and cartilage tissues also showed the accelerated aging and abnormality of animal physiological functions. ConclusionSHJH^hr mice have some biological characteristics of rapid aging as well as some physiological and pathological changes caused by aging.

Objective: To detect the expression of miR-373 in osteosarcoma cells and explore its effects on cell proliferation, invasion, and migration. Methods: Human osteosarcoma cell line SJSA-1 and human osteoblast hFOB 1.19 cultured in vitro. Human osteosarcoma cell line SJSA-1 were randomly divided into four groups: blank control group (CK group), negative control group (NC group), miR-373 over-expressed plasmid group (miR-373 mimic group) and miR-373 inhibited plasmid group (miR-373 inhibitor group). Cells were transfected with FuGENE^® HD transfection reagent. After 48 hours of transfection, the relative expression of miR-373 was detected by quantitative real-time polymerase chain reaction (qRT-PCR), cell proliferation by cell counting kit-8 (CCK-8) and cell invasion and migration by Transwell. Results: The relative expression of miR-373 in SJSA-1 cells was significantly higher than that in hFOB-1.19 cells (P<0.05); compared with CK group and NC group, the relative expression of miR-373 in the miR-373 mimic group increased significantly (P<0.05), and the relative expression of miR-373 in the miR-373 inhibitor group was significantly lower than that in the control group (P<0.05); optical density (OD) value of miR-373 mimic group at 24 h, 36 h, and 48 h were significantly higher than that of CK group and NC group (P<0.05), and OD value of miR-373 inhibitor group at 24 h, 36 h, and 48 h were significantly lower than that of CK group and NC group (P<0.05); the number of cell migration in the miR-373 mimic group was significantly higher than that in the CK group and NC group (P<0.05), and the number of cell migration in the miR-373 inhibitor group was significantly lower than that in CK group and NC group (P<0.05); the number of cell migration in the miR-373 mimic group was significantly higher than that in the CK group and NC group (P<0.05), and the number of cell migration in the miR-373 inhibitor group was significantly lower than that in CK group and NC group (P<0.05). Conclusions The expression of miR-373 is up-regulated in osteosarcoma. Overexpression of miR-373 can promote the proliferation, invasion, and migration of osteosarcoma cells, while the low expression of miR-373 can inhibit the proliferation, invasion, and migration of osteosarcoma cells, which may be an important target for the diagnosis and treatment of osteosarcoma.

OBJECTIVE: To observe the effects of on the expression of β-tubulin Ⅲ and glial fibrillary acidic protein (GFAP) and the proliferation and differentiation of murine neural stem cells (NSCs) . METHODS: An immortalized murine NSC line was divided into model control (MC) group, 10% drug-containing serum group (NLXT group), and 10% Naoluoxintong drug-containing serum with inhibitor Y27632 group (Y-27632 group) with corresponding treatments. The activity of the NSCs was detected after the treatments using MTT assay, and the migration of the cells was observed with Transwell assay. The expressions of β-tubulin Ⅲ, GFAP and MAP-2 proteins in the cells were detected with immunoblotting, and the expressions of DCX, NEUN, and β-tubulin Ⅲ were also detected with immunofluorescence assay. RESULTS: Compared with that in MC group, the number of migrated cells in NLXT group and Y-27632 group increased significantly at 1 day and 3 days after induction ( < 0.05). The survival rate and the number of migrated cells in NLXT group and Y-27632 group increased significantly on day 7 ( < 0.01). Compared with those in MC group, the expressions of β-tubulin Ⅲ, MAP2 and GFAP protein in NLXT group and Y-27632 group were significantly increased on days 3 ( < 0.01) and 7 ( < 0.05). The numbers of β-tubulinⅢ/ GFAP, BrdU/DCX, and BrdU/NEUN labeled cells in the NLXT group and Y-27632 group were significantly greater than those in the MC group. CONCLUSIONS promotes the proliferation and differentiation of murine NSCs by regulating the expressions of β-tubulinⅢ/GFAP.
Animals ; Cell Differentiation ; Cell Proliferation ; Glial Fibrillary Acidic Protein ; Mice ; Neural Stem Cells ; Tubulin