Objective To investigate the effect of hypoxia micro?environment on anoikis in human high?metastatic cell line 95D of giant cell carci?noma of lung. Methods Suspension technology was used to culture 95D to establish the model of anoikis?resistant 95D cells. Hypoxic culture was conducted in the experimental group,and regular culture was conducted in the control group. The effect of hypoxia on proliferation of anoikis?resis?tant 95D was investigated by MTT and the apoptosis in the two groups were detected by flow cytometer. The invasive ability of the cells was assessed by Transwell test. The effect of hypoxia on the expression of HIF?1α,VEGF and MMP?2 in anoikis?resistant 95D was detected by Western blot. Re?sults The growth rate of the anoikis?resistant 95D cells treated with hypoxia was 52.9%,and the apoptosis rate of these cells was higher than that in the non?hypoxic group(40.4%vs 21.7%,P<0.05). The treatment of hypoxia down?regulated the invasive ability,the number of migration cells un?der hypoxia was higher than that in the control group,with statistical significance(40.1±6.7 vs 12.5±7.9,P<0.05). The up?regulation of HIF?1α, the down?regulation of VEGF and MMP?2 were observed in the group of hypoxia. Conclusion During anoikis of human high?metastatic lung can?cer cell line 95D,hypoxia inhibited the survival ability and the metastasis ability of anoikis?resistant cells,which,however,might be the early mani?festation of hypoxia.

Objective To establish mouse models of intracerebral hemorrhage using autologous arterial blood,to study the physiological property of hippocampal neurons,brain edema changes and learning ability in the mouse models after intracerebral hemorrhage.Methods Eighty male C57/BL6 mice were randomly divided into intracerebral hemorrhage group and control group (n=40); 20 μL arterial blood from the tail arteries or normal saline were injected into the caudate nucleus of intracerebral hemorrhage group and control group by stereotactic technique,respectively.One,three,five and seven d after injection,the neurological impairment was scored; the behavioral changes of the mice in the Morris water maze (navigation test and space exploration experiment) were observed; brain edema was measured by wet and dry weight method and electrophysiological differences of hippocampal neurons were recorded by whole-cell patch-clamp technique and computer software.Results As compared with those in the control group,significantly increased neurological deficit scores one,three,five and seven d after injection,statistically decreased residence time in the platform on the fifth d of training,obviously increased water content around the brain edema one,three,five and seven d after injection,and significantly decreased resting membrane potential and input resistance in the hippocampal CA1 pyramidal cells five d after injection of mice in the intracerebral hemorrhage group were noted (P＜0.05).Conclusion The hippocampus-dependent spatial leaming ability of intracerebral hemorrhage mice is decreased,and the permeability of potassium channels is enhanced.

Objective To explore the efficacy and safety of perforator flap eyelid reconstruction surgery for eyelid repair and reconstruction in patients with eyelid tumors after surgery.Methods Totally 80 patients(80 eyes)who re-ceived eyelid tumor surgery and planned to undergo the perforator flap eyelid reconstruction surgery in our hospital from January 2012 to December 2022 were included.All of them have early-stage and mid-stage eyelid tumors with no orbital or systemic metastasis.A perforator flap eyelid reconstruction surgery was conducted to treat the eyelid defects.The differ-ence in height and length of the palpebral fissure,aesthetic function scores and levels of depression and anxiety of patients before and after repair were compared;the clinical efficacy,adverse reactions and prognosis of patients were evaluated.Results Among the 80 eyes,there were 35 eyes with significant effect,35 eyes with effective impact,and 10 eyes with no effect,with a total effective rate of 87.5％.The height and length of palpebral fissure of patients after repair were(1.49±0.47)mm and(1.43±0.55)mm,respectively,which were significantly lower than those before repair[(2.55± 0.35)mm and(2.38±0.49)mm](both P＜0.01).The aesthetic function score before repair was 0.91±0.23,and that after repair was 1.87±0.19,with a significant difference(P＜0.01).The scores of the Self-rating Anxiety Scale and Self-rating Depression Scale after repair were 40.14±6.54 and 39.45±7.65,respectively,which were significantly lower than those before repair(59.56±8.23 and 57.93±8.19)(both P＜0.01).Among the 80 patients,the total incidence of compli-cations was 27.5％(22/80).All patients were followed up for 6-36 months,with an average follow-up time of(12.39± 6.17)months.No tumor recurrence was found during the follow-up.All composite flaps survived without infection,necro-sis or displacement.Conclusion The perforator flap eyelid reconstruction surgery has a good and safe therapeutic effect on repairing full-layer eyelid defects in patients who underwent eyelid tumor surgery.

ObjectiveTo investigate the role and mechanism of podoplanin （PDPN） in hepatic stellate cell （HSC） activation and liver fibrosis. MethodsLiver biopsy samples were collected from 75 patients with chronic hepatitis B who attended Department of Infectious Diseases， Putuo Hospital Affiliated to Shanghai University of Traditional Chinese Medicine， for the first time from September 2019 to June 2022， and RT-PCR and immunohistochemistry were used to measure the expression of PDPN in liver tissue of patients in different stages of liver fibrosis. A total of 12 male C57/BL6 mice were randomly divided into control group and model group. The mice in the model group were given intraperitoneal injection of 10% CCl₄， and those in the control group were injected with an equal volume of olive oil， for 6 weeks. HE staining and Sirius Red staining were used to observe liver histopathological changes； primary mouse liver cells were separated to measure the mRNA expression of PDPN in various types of cells； primary mouse HSCs were treated with PDPN protein， followed by treatment with the NF-‍κB inhibitor BAY11-708， to measure the expression of inflammatory factors in HSCs induced by PDPN. The independent-samples t test was used for comparison of normally distributed continuous data between two groups； a one-way analysis of variance was used for comparison between multiple groups， and the least significant difference t-test was used for further comparison between two groups. The Spearman correlation analysis was used to investigate data correlation. ResultsAs for the liver biopsy samples， there was a relatively low mRNA expression level of PDPN in normal liver， and there was a significant increase in the mRNA expression level of PDPN in liver tissue of stage S3 or S4 fibrosis （all P<0.001）. Immunohistochemical staining showed that PDPN was mainly expressed in the fibrous septum and the hepatic sinusoid， and the PDPN-positive area in S4 liver tissue was significantly higher than that in S0 liver tissue （t=8.892， P=0.001）. In normal mice， PDPN was mainly expressed in the hepatic sinusoid， and there was a significant increase in the expression of PDPN in CCl₄ model mice （t=0.95， P<0.001）， mainly in the fibrous septum. RT-PCR showed a significant increase in the mRNA expression of PDPN in the CCl₄ model mice （t=11.25， P=0.002）. Compared with hepatocytes， HSCs， Kupffer cells， and bile duct endothelial cells， hepatic sinusoidal endothelial cells showed a significantly high expression level of PDPN （F=20.56， P<0.001）. Compared with the control group， the primary mouse HSCs treated by PDPN protein for 15 minutes showed significant increases in the mRNA expression levels of the inflammation-related factors TNFα， CCL3， CXCL1， and CXCR1 （all P<0.05）， and there were significant reductions in the levels of these indicators after treatment with BAY11-7082 （all P<0.05）. ConclusionThere is an increase in the expression of PDPN mainly in hepatic sinusoidal endothelial cells during liver fibrosis， and PDPN regulates HSC activation and promotes the progression of liver fibrosis via the NF-κB signaling pathway.

ObjectiveTo investigate the mechanism of action of Xiayuxue decoction in inhibiting nonalcoholic fatty liver disease （NAFLD） induced by high-fat diet in mice by regulating nucleotide binding oligomerization domain like receptor containing pyrin domain protein 6 （NLRP6）. MethodsA total of 15 male C57BL/6 mice were randomly divided into low-fat diet （LFD） group， high-fat diet （HFD） group， and Xiayuxue decoction-HFD group （XYXD group）， with 5 mice in each group. Liver function parameters （alanine aminotransferase ［ALT］ and aspartate aminotransferase ［AST］） and blood lipid metabolic indicators （triglycerides ［TG］ and total cholesterol ［TC］） were measured； HE staining and oil red O staining were performed for liver tissue to observe histomorpholoty and lipid droplet deposition； quantitative real-time PCR was used to measure the expression levels of inflammatory factors （tumor necrosis factor-α ［TNF-α］， interleukin-1β ［IL-1β］， interleukin-18 ［IL-18］， and NLRP6） in liver tissue； Western blot was used to measure the protein expression levels of NLRP6， nuclear factor-kappa B （NF-κB）， and NF-κB p65； immunohistochemistry was used to measure the expression of NLRP6 and CD68. Mouse Raw264.7 cells were treated with palmitic acid （PA）， lipopolysaccharide， and serum containing Xiayuxue decoction to observe inflammation. A one-way analysis of variance was used for comparison of continuous data between multiple groups， and the least significant difference t-test was used for further comparison between two groups. ResultsCompared with the LFD group， the HFD group had significant increases in the serum levels of ALT， AST， TC， and TG （all P<0.05）. Liver histopathological examination showed that the HFD group had marked hepatic steatosis and a signficant increase in NAS score （P<0.05）， and quantitative real-time PCR showed significant increases in the inflammatory factors such as IL1β and IL-18 and a significant reduction in the expression of NLRP6 （all P<0.05）. Immunohistochemistry showed that the expression of NLRP6 showed a similar trend as that of the macrophage marker CD68. Western blot showed that after the downregulation of NLRP6 expression， there was a significant increase in phosphorylated NF-κB p65 （P<0.05）. Compared with the HFD group， Xiayuxue decoction effectively improved liver inflammation， upregulated the expression of NLRP6， and downregulated phosphorylated NF-κB p65 in HFD mice （all P<0.05）. After Raw264.7 cells were treated with PA， NLRP6 was downregulated to promote the progression of inflammation （P<0.05）， and treatment with Xiayuxue decoction could upregulate NLRP6 and inhibit inflammation NF-κB （P<0.05）. ConclusionXiayuxue decoction can effectively improve hepatic steatosis and liver inflammation in a mouse model of NAFLD， possibly by regulating NLRP6/NF-κB to alleviate macrophage activation.

Activation of the heart normally begins in the sinoatrial node (SAN). Electrical impulses spontaneously released by SAN pacemaker cells (SANPCs) trigger the contraction of the heart. However, the cellular nature of SANPCs remains controversial. Here, we report that SANPCs exhibit glutamatergic neuron-like properties. By comparing the single-cell transcriptome of SANPCs with that of cells from primary visual cortex in mouse, we found that SANPCs co-clustered with cortical neurons. Tissue and cellular imaging confirmed that SANPCs contained key elements of glutamatergic neurotransmitter system, expressing genes encoding glutamate synthesis pathway (Gls), ionotropic and metabotropic glutamate receptors (Grina, Gria3, Grm1 and Grm5), and glutamate transporters (Slc17a7). SANPCs highly expressed cell markers of glutamatergic neurons (Snap25 and Slc17a7), whereas Gad1, a marker of GABAergic neurons, was negative. Functional studies revealed that inhibition of glutamate receptors or transporters reduced spontaneous pacing frequency of isolated SAN tissues and spontaneous Ca