ObjectiveTo investigate the regulatory effect of overexpressed protein tyrosine phosphatase non-receptor type 2 （Ptpn2） on the inflammatory response of mouse alveolar macrophages （MH-S） induced by SiO₂. MethodsCells with overexpressed Ptpn2 were constructed and induced by SiO₂. The experimental groups were divided into four groups： the negative control group with an empty vector （NC）， the overexpressed Ptpn2 group （P）， the negative control group with an empty vector + SiO₂ induction （NS）， and the overexpressed Ptpn2 + SiO₂ induction group （PS）. Isobaric tags for relative and absolute quantification （iTRAQ） combined with liquid chromatography-tandem mass spectrometry （LC-MS/MS） were used to screen differential proteins， followed by Gene Ontology （GO） and Kyoto Encyclopedia of Genes and Genomes （KEGG） database analyses. Immunofluorescence staining was used to detect the expressions of Tumor necrosis factor （TNF） α， Gasdermin D （GSDMD）， and Transforming growth factor （TGF）-β1. Western blot was used to detect the protein expression levels of PTPN2， Toll-like receptor 4 （TLR4）， tumor necrosis factor-α （TNF-α）， nucleotide-binding oligomerization domain-like receptor protein 3 （NLRP3）， and proteins related to the TGF-β1 signaling pathway in the cells of each group. ResultsiTRAQ results identified 144 differential proteins among the four groups. GO analysis showed that in biological processes （BP）， these differential proteins were mainly enriched in IκB kinase/nuclear factor-κB （NF-κB） signaling， cell activation and signal transduction involved in immune responses， and regulation of receptor signaling pathways by signal transducer and activator of transcription （STAT）， etc. KEGG analysis revealed that the differential proteins were mainly enriched in Toll-like receptor signaling pathway， NF-κB signaling pathway， NOD-like receptor signaling pathway， TGF-β signaling pathway， and TNF signaling pathway. The results of immunofluorescence staining showed that compared with the NC group， the expressions of TNF α， GSDMD， and TGF-β1 in the cells of the NS group increased （P < 0.05）； compared to the NS group， the expression of the aforementioned proteins in the PS group decreased in cellular proteins（P < 0.05）. The results of Western blot showed that compared with the NC group， the protein expression levels of PTPN2， p-NF-κB，MyD88，TLR4，NLRP3，GSDMD，Caspase-1，IL-1β， TGF-βR1， TGF-βR，p-Smad2/3 in the NS group were significantly upregulated （P < 0.05）； compared with the NS group， the expression levels of the aforementioned proteins in the PS group were significantly downregulated （P < 0.05）. ConclusionOverexpression of Ptpn2 can inhibit the protein expressions of TLR4-TNF-α signaling， NLRP3 signaling， and TGF-β1 signaling closely related to inflammatory response in SiO₂-mediated MH-S macrophages.

Objective:To evaluate the feasibility of a six-step suspension technique for endoscopic lateral neck dissection (LND) through the chest-breast approach in patients with papillary thyroid carcinoma (PTC).Methods:This is a retrospective case series study.Clinical data of 81 PTC patients who underwent endoscopic LND via the chest-breast approach using the six-step suspension method at the Department of Thyroid Surgery, Shenzhen People′s Hospital were collected from January 2022 to October 2024. The cohort consisted of 15 male and 66 female patients, with age of (35.2±10.2)years (range:8.5 to 65.0 years). Key variables, including LND duration, total operative time, postoperative hospital stay, details of lymph node metastasis, postoperative complications, and follow-up data were recorded and analyzed.Results:The duration of LND was (131.8±42.2)minutes (range: 65 to 275 minutes), and the total operative time was (195.5±49.6)minutes (range: 110 to 390 minutes). The postoperative hospital stay was (4.8±1.5)days(range:3 to 15 days). The number of dissected lateral cervical lymph nodes was 32.7±10.1 (range: 11 to 54). The maximum tumor diameter was (16.1±10.1)mm(range:2 to 30 mm), while the maximum size of metastatic lymph nodes was (16.7±6.2)mm(range:7 to 30 mm). The positivity rate was 24.7% (841/3 410) in the lateral cervical+central lymph node and 16.1% (427/2 646) in the lateral cervical lymph node. Postoperative lymphatic leakage occurred in 2 patients, both of whom were successfully treated conservatively. No other significant complications were reported. During the postoperative follow-up period, which lasted for (18.3±7.4) months (range: 1.1 to 34.4 months), the mean postoperative serum thyroglobulin (Tg) level ( M(IQR)) was 0.05 (0.50) μg/L (range: 0.01 to 7.90 μg/L), with 86.4% of patients showing a Tg ≤1.00 μg/L. Through imaging evaluations, no evidence of residual disease or recurrence was detected. Conclusion:Endoscopic LND via the chest-breast approach, utilizing the six-step suspension method, maybe a feasible and effective technique with promising clinical outcomes.

Objective:To investigate the function and mechanism of exosome Linc00665 in modulating CD8+T cell immunoreactivity to promote radiotherapy resistance in OSCC.Methods:HOEC,SCC9 and SCC9-RR exosomes were extracted and identified,and the expression of Linc00665 was detected by qRT-PCR in cell lines and exosomes.The expression of TNF-α,IFN-γ,perforin and granzyme B in each treatment group was detected by ELISA(PBS,SCC9 exo,SCC9-RR exo).The killing ability of CD8+T cells against SCC9 cells in each treatment group was detected by CCK-8 assay.The targets of Linc00665 were further bioinformatically ana-lyzed and verified by qRT-PCR and Western blot.The expression of Linc00665,miR-28-5p and PD-1 in CD8+T cells was exogenous-ly regulated,the expression of immunoreactive factors in the supernatants of each treatment group was detected by ELISA(NC,sh-Linc00665,miR-28-5p inhibitor,sh-PD-1),and the killing ability of cells in each group was detected by CCK-8 method.Results:The concentrations of TNF-α,IFN-γ,perforin and granzyme B in the supernatants of cell culture in the SCC9-RR exo/CD8+T group were significantly decreased compared with those in the PBS/CD8+T group and the SCC9 exo/CD8+T group(P＜0.05),and the kill-ing ability of the cells in the SCC9-RR exo group was significantly decreased compared with those in the PBS group and the SCC9 exo group(P＜0.05),suggesting that SCC9-RR exo could inhibit the tumor killing ability of CD8+T cells.qRT-PCR results suggested that Linc00665 was highly expressed in the SCC9-RR cell line as well as exosome(P＜0.05).It was further verified by bioinformat-ics analysis that Linc00665 could regulate PD-1 expression via miR-28-5p,thereby modulating CD8+T cell immunoreactivity to pro-mote OSCC radiotherapy resistance.Conclusion:Exosome Linc00665 regulates CD8+T cell immunoreactivity through miR-28-5p/PD-1 axis to promote OSCC radiotherapy resistance.

Objective:To investigate the function and mechanism of exosome Linc00665 in modulating CD8+T cell immunoreactivity to promote radiotherapy resistance in OSCC.Methods:HOEC,SCC9 and SCC9-RR exosomes were extracted and identified,and the expression of Linc00665 was detected by qRT-PCR in cell lines and exosomes.The expression of TNF-α,IFN-γ,perforin and granzyme B in each treatment group was detected by ELISA(PBS,SCC9 exo,SCC9-RR exo).The killing ability of CD8+T cells against SCC9 cells in each treatment group was detected by CCK-8 assay.The targets of Linc00665 were further bioinformatically ana-lyzed and verified by qRT-PCR and Western blot.The expression of Linc00665,miR-28-5p and PD-1 in CD8+T cells was exogenous-ly regulated,the expression of immunoreactive factors in the supernatants of each treatment group was detected by ELISA(NC,sh-Linc00665,miR-28-5p inhibitor,sh-PD-1),and the killing ability of cells in each group was detected by CCK-8 method.Results:The concentrations of TNF-α,IFN-γ,perforin and granzyme B in the supernatants of cell culture in the SCC9-RR exo/CD8+T group were significantly decreased compared with those in the PBS/CD8+T group and the SCC9 exo/CD8+T group(P＜0.05),and the kill-ing ability of the cells in the SCC9-RR exo group was significantly decreased compared with those in the PBS group and the SCC9 exo group(P＜0.05),suggesting that SCC9-RR exo could inhibit the tumor killing ability of CD8+T cells.qRT-PCR results suggested that Linc00665 was highly expressed in the SCC9-RR cell line as well as exosome(P＜0.05).It was further verified by bioinformat-ics analysis that Linc00665 could regulate PD-1 expression via miR-28-5p,thereby modulating CD8+T cell immunoreactivity to pro-mote OSCC radiotherapy resistance.Conclusion:Exosome Linc00665 regulates CD8+T cell immunoreactivity through miR-28-5p/PD-1 axis to promote OSCC radiotherapy resistance.

Objective:To study the influence of methyltransferases like 3(METTL3)on the radiosensitivity of oral squamous cell carcinoma cells(OSCCs).Methods:The apoptosis level of OSCCs CAL27,SCC9 and SCC15 treated with X-ray radiation doses of 2,4 and 8 Gy respectively was compared by flow cytometry,the expression of methylated gene RNA and protein in the cells were examined by qRT-PCR and Western blot.m6A in the cells was quantified by LC/LC-MS method.qRT-PCR and Western blot were used to investigate the expression of methylated gene RNA and protein in the cells.Flow cytometry was used to examine the cell apoptosis level of CAL27 and SCC15 cells treated with METTL3 overexpression and knockdown respectively.The clone forma-tion of CAL27 and SCC15 cells after knockdown and overexpression of target genes followed by radiation treatment was observed by clonogenic assays.Results:The apoptosis rate of all the cell lines increased with the increase dose of radiation at each dose,CAL27 cells showed the highest and SCC15 showed the lowerst apoptosis rate.The RNA and protein expression levels of METTL3 in CAL27 were significantly lower than those of SCC15.m6A quantification showed that the methylation modification in CAL27 cells was lower than that in SCC15.The expression of METTL3 was increased in CAL27 and SCC15 cells after radiation treatment.Knockdown of METTL3 increaced the apoptosis rate and decreased the clonogenesity,overession of METTL3 the decreaced the ap optosis rate and increased the clonogenecity of the cells.Conclusion:Regulation of METTL3 can affect the radiotherapy sensitivity of OSCCs,METTL3 may become a new target for radiosensitization of OSCCs.

Objective To investigate the neuroprotective effect of Tibetan medicine 70 Wei Pearl Pill(RNSP)on 6-hydroxydopamine(6-OHDA)induced Parkinson's disease model rats and its related mechanism.Methods Totally 48 male SD rats were randomly divided into control group,model group,model plus low-dose RNSP group(90 mg/kg),model plus medium-dose RNSP group(180 mg/kg),model plus high-dose RNSP group(360 mg/kg),and model plus madopar group(50 mg/kg),with 8 rats in per group.Except for the control group,the other rats were treated with 6-OHDA single injection into the striatum to establish the PD model.Each group was given intragastric administration after modeling and the control group and model group were given an equal volume of saline once a day for 4 weeks.The rats were subjected to apomorphine-induced rotation,experiments at the 2nd and 4th week after the completion of modeling,and the open field experiment was conducted the next day after the last rotation experiment to observe the animal behavior.After the behavioral experiment,the rats were stained with tyrosine hydroxylase(TH)in the substantia nigra pars compacta by immunohistochemical method and the positive neurons were counted.The protein levels of Bcl-2,BAX,Caspase-3 in substantia nigra and P38,P-P38,ERK,P-ERK,JNK,P-JNK in the striatum were detected by Western blotting.Results Compared with the control group,the rotation frequency and percentage of rotating rats in the model group increased significantly at the 2nd and 4th weeks after modeling;the open field active distance,average speed and times of crossing the grid were significantly decreased;and the rest time was significantly increased.While the number of TH positive neurons in the substantia nigra was significantly decreased,the BAX and Caspase-3 protein levels were increased significantly,Bcl-2 was decreased significantly,the ratios of P-P38/P38,P-JNK/JNK and P-ERK/ERK in the striatum were significantly increased in PD group.Compared with the model group,the rotation frequency and percentage of rotating rats in the low-,medium-and high-dose groups of RNSP had no significant changes after 2 weeks of administration,the rotation frequency in the high-dose group and percentage of rotating rats in the low-and medium-dose RNSP groups significantly decreased after 4 weeks of administration.The open field active distance,average speed,and times of crossing the grid were significantly increased;the rest time was significantly decreased.The number of TH positive neurons in the substantia nigra was significantly increased,the Bax and Caspase-3 protein levels were decreased significantly while the Bcl-2 was significantly increased.The ratios of P-P38/P38,P-JNK/JNK and P-ERK/ERK in the striatum were significantly decreased.Conclusion Tibetan medicine RNSP can improve the motor ability and reduce the loss of DA neurons in PD rats,and its mechanism may be related to inhibiting P38/JNK/ERK signaling pathway and reducing the apoptosis of midbrain neurons.

Objective:To study the influence of methyltransferases like 3(METTL3)on the radiosensitivity of oral squamous cell carcinoma cells(OSCCs).Methods:The apoptosis level of OSCCs CAL27,SCC9 and SCC15 treated with X-ray radiation doses of 2,4 and 8 Gy respectively was compared by flow cytometry,the expression of methylated gene RNA and protein in the cells were examined by qRT-PCR and Western blot.m6A in the cells was quantified by LC/LC-MS method.qRT-PCR and Western blot were used to investigate the expression of methylated gene RNA and protein in the cells.Flow cytometry was used to examine the cell apoptosis level of CAL27 and SCC15 cells treated with METTL3 overexpression and knockdown respectively.The clone forma-tion of CAL27 and SCC15 cells after knockdown and overexpression of target genes followed by radiation treatment was observed by clonogenic assays.Results:The apoptosis rate of all the cell lines increased with the increase dose of radiation at each dose,CAL27 cells showed the highest and SCC15 showed the lowerst apoptosis rate.The RNA and protein expression levels of METTL3 in CAL27 were significantly lower than those of SCC15.m6A quantification showed that the methylation modification in CAL27 cells was lower than that in SCC15.The expression of METTL3 was increased in CAL27 and SCC15 cells after radiation treatment.Knockdown of METTL3 increaced the apoptosis rate and decreased the clonogenesity,overession of METTL3 the decreaced the ap optosis rate and increased the clonogenecity of the cells.Conclusion:Regulation of METTL3 can affect the radiotherapy sensitivity of OSCCs,METTL3 may become a new target for radiosensitization of OSCCs.

Objective To investigate the neuroprotective effect of Tibetan medicine 70 Wei Pearl Pill(RNSP)on 6-hydroxydopamine(6-OHDA)induced Parkinson's disease model rats and its related mechanism.Methods Totally 48 male SD rats were randomly divided into control group,model group,model plus low-dose RNSP group(90 mg/kg),model plus medium-dose RNSP group(180 mg/kg),model plus high-dose RNSP group(360 mg/kg),and model plus madopar group(50 mg/kg),with 8 rats in per group.Except for the control group,the other rats were treated with 6-OHDA single injection into the striatum to establish the PD model.Each group was given intragastric administration after modeling and the control group and model group were given an equal volume of saline once a day for 4 weeks.The rats were subjected to apomorphine-induced rotation,experiments at the 2nd and 4th week after the completion of modeling,and the open field experiment was conducted the next day after the last rotation experiment to observe the animal behavior.After the behavioral experiment,the rats were stained with tyrosine hydroxylase(TH)in the substantia nigra pars compacta by immunohistochemical method and the positive neurons were counted.The protein levels of Bcl-2,BAX,Caspase-3 in substantia nigra and P38,P-P38,ERK,P-ERK,JNK,P-JNK in the striatum were detected by Western blotting.Results Compared with the control group,the rotation frequency and percentage of rotating rats in the model group increased significantly at the 2nd and 4th weeks after modeling;the open field active distance,average speed and times of crossing the grid were significantly decreased;and the rest time was significantly increased.While the number of TH positive neurons in the substantia nigra was significantly decreased,the BAX and Caspase-3 protein levels were increased significantly,Bcl-2 was decreased significantly,the ratios of P-P38/P38,P-JNK/JNK and P-ERK/ERK in the striatum were significantly increased in PD group.Compared with the model group,the rotation frequency and percentage of rotating rats in the low-,medium-and high-dose groups of RNSP had no significant changes after 2 weeks of administration,the rotation frequency in the high-dose group and percentage of rotating rats in the low-and medium-dose RNSP groups significantly decreased after 4 weeks of administration.The open field active distance,average speed,and times of crossing the grid were significantly increased;the rest time was significantly decreased.The number of TH positive neurons in the substantia nigra was significantly increased,the Bax and Caspase-3 protein levels were decreased significantly while the Bcl-2 was significantly increased.The ratios of P-P38/P38,P-JNK/JNK and P-ERK/ERK in the striatum were significantly decreased.Conclusion Tibetan medicine RNSP can improve the motor ability and reduce the loss of DA neurons in PD rats,and its mechanism may be related to inhibiting P38/JNK/ERK signaling pathway and reducing the apoptosis of midbrain neurons.

Objective:To evaluate the feasibility of a six-step suspension technique for endoscopic lateral neck dissection (LND) through the chest-breast approach in patients with papillary thyroid carcinoma (PTC).Methods:This is a retrospective case series study.Clinical data of 81 PTC patients who underwent endoscopic LND via the chest-breast approach using the six-step suspension method at the Department of Thyroid Surgery, Shenzhen People′s Hospital were collected from January 2022 to October 2024. The cohort consisted of 15 male and 66 female patients, with age of (35.2±10.2)years (range:8.5 to 65.0 years). Key variables, including LND duration, total operative time, postoperative hospital stay, details of lymph node metastasis, postoperative complications, and follow-up data were recorded and analyzed.Results:The duration of LND was (131.8±42.2)minutes (range: 65 to 275 minutes), and the total operative time was (195.5±49.6)minutes (range: 110 to 390 minutes). The postoperative hospital stay was (4.8±1.5)days(range:3 to 15 days). The number of dissected lateral cervical lymph nodes was 32.7±10.1 (range: 11 to 54). The maximum tumor diameter was (16.1±10.1)mm(range:2 to 30 mm), while the maximum size of metastatic lymph nodes was (16.7±6.2)mm(range:7 to 30 mm). The positivity rate was 24.7% (841/3 410) in the lateral cervical+central lymph node and 16.1% (427/2 646) in the lateral cervical lymph node. Postoperative lymphatic leakage occurred in 2 patients, both of whom were successfully treated conservatively. No other significant complications were reported. During the postoperative follow-up period, which lasted for (18.3±7.4) months (range: 1.1 to 34.4 months), the mean postoperative serum thyroglobulin (Tg) level ( M(IQR)) was 0.05 (0.50) μg/L (range: 0.01 to 7.90 μg/L), with 86.4% of patients showing a Tg ≤1.00 μg/L. Through imaging evaluations, no evidence of residual disease or recurrence was detected. Conclusion:Endoscopic LND via the chest-breast approach, utilizing the six-step suspension method, maybe a feasible and effective technique with promising clinical outcomes.

Astrocytes are the largest glial population in the mammalian brain. However, we have a minimal understanding of astrocyte development, especially fate specification in different regions of the brain. Through lineage tracing of the progenitors of the third ventricle (3V) wall via in-utero electroporation in the embryonic mouse brain, we show the fate specification and migration pattern of astrocytes derived from radial glia along the 3V wall. Unexpectedly, radial glia located in different regions along the 3V wall of the diencephalon produce distinct cell types: radial glia in the upper region produce astrocytes and those in the lower region produce neurons in the diencephalon. With genetic fate mapping analysis, we reveal that the first population of astrocytes appears along the zona incerta in the diencephalon. Astrogenesis occurs at an early time point in the dorsal region relative to that in the ventral region of the developing diencephalon. With transcriptomic analysis of the region-specific 3V wall and lateral ventricle (LV) wall, we identified cohorts of differentially-expressed genes in the dorsal 3V wall compared to the ventral 3V wall and LV wall that may regulate astrogenesis in the dorsal diencephalon. Together, these results demonstrate that the generation of astrocytes shows a spatiotemporal pattern in the developing mouse diencephalon.
Mice ; Animals ; Astrocytes ; Neuroglia/physiology* ; Diencephalon ; Brain ; Neurons ; Mammals