Objective To investigate the effects of sauchinone(Sch)on the growth and immune escape of liver cancer cells by regulating the Krüppel like factor 5(KLF5)-erythropoietin-producing hepatocellular receptor A2(EphA2)pathway.Methods Huh-7 cells were grouped into liver cancer group,Sch low,medium,and high-dose groups(Sch-L group,Sch-M group,Sch-H group),Sch-H+KLF5 activator negative control group(OE-NC),and Sch-H+KLF5 activator(OE-KLF5)group.5-bromo-2-deoxyuracil(EdU)staining,CCK-8,scratch assay,and Transwll were used to detect the proliferation,migration,and invasion of Huh-7 cells,respectively.Western blot was applied to detect proliferative cell nuclear antigen(PCNA),migration invasion enhancer(MIEN1),matrix metalloproteinase-2(MMP-2),programmed death receptor ligand 1(PD-L1),KLF5,and EphA2 proteins in Huh-7 cells.The Huh-7 cells in above 6 groups were co cultured with activated CD8+T cells in a 96 well plate and named as liver cancer co culture group,Sch-L co culture group,Sch-M co culture group,Sch-H co culture group,Sch-H+OE-NC co culture group,and Sch-H+OE-KLF5 co culture group.The killing rate of CD8+T cells in the co culture system and the levels of interferon-γ(IFN-γ),interleukin-4(IL-4),and tumor necrosis factor-α(TNF-α)in the supernatant were detected.Results Compared with the liver cancer group,the EdU positive rate,OD450 value,cell invasion number,and PCNA,MIEN1,MMP-2,PD-L1,KLF5,and EphA2 proteins in the Sch-L,Sch-M,and Sch-H groups reduced,the migration distance shorten(P<0.05).Compared with the Sch-H group and Sch-H+OE-NC group,the EdU positive rate,OD450 value,cell invasion number,and PCNA,MIEN1,MMP-2,PD-L1,KLF5,and EphA2 proteins in the Sch-H+OE-KLF5 group increased,the migration distance prolonged(P<0.05).Compared with the liver cancer co culture group,the killing rate of CD8+T cells on Huh-7 cells and the levels of IFN-γ,TNF-α,and IL-4 in the supernatant in the Sch-L co culture group,Sch-M co culture group,and Sch-H co culture group increased(P<0.05).Compared with the Sch-H co culture group and the Sch-H+OE-NC co culture group,the killing rate of CD8+T cells on Huh-7 cells and the levels of IFN-γ,TNF-α,and IL-4 in the supernatant in the Sch-H+OE-KLF5 co culture group decreased(P<0.05).Conclusion Sch may inhibit growth and immune escape of liver cancer cells by inhibiting the KLF5-EphA2 pathway.

Pain modulation encompasses a complex neurobiological process, in which the lateral habenula (LHb) plays a crucial role in integrating, regulating and modulating pain signals. It is also involved in pain-related memory functions associated with perception, transmission and regulation of pain. Furthermore, the LHb collaborates with structures such as the spinal dorsal horn, forebrain, and amygdala to form an essential neural circuit that contributes to sensitization, development of tolerance, and adaptation processes related to pain. However, there remains limited understanding regarding the specific roles and interactions among different neuron subtypes within the LHb concerning pain regulation. Additionally, further investigation is warranted to explore functional changes and plasticity within both the LHb and its associated neural circuits in chronic pain models. Future research endeavors should utilize advanced neuroimaging techniques alongside optogenetics and gene editing technologies to elucidate intricate neural circuits, cellular architecture, and molecular mechanisms governing LHb function in pain regulation. In conclusion, this paper aims to comprehensively review existing literature on the involvement of the LHb and its neural circuits in modulating pain, thereby enhancing our understanding of their neurobiological mechanisms while providing novel targets for precise therapeutic strategies aimed at alleviating pain.

BACKGROUND:Intertrochanteric femoral fractures are the main type of fragility fracture in the elderly,and proximal femoral nail antirotation is the preferred surgical option,but the factors associated with postoperative internal fixation failure are controversial.OBJECTIVE:A new"three-column"classification of intertrochanteric femoral fractures was proposed by evaluating patients'imaging data preoperatively and analyzing its interaction with postoperative internal fixation failure.A risk prediction model was developed and validated by using numerical algorithms,which facilitates clinicians to identify and intervene in high-risk patients preoperatively.METHODS:Patients with intertrochanteric femur fractures in Sanshui Branch of Foshan Hospital of Traditional Chinese Medicine between June 2012 and June 2022 were selected.The patients were divided into the internal fixation failure group and the internal fixation maintenance group according to whether they had internal fixation failure after surgery.According to the preoperative radiographs,the proximal femur was divided into three columns:the medial column,the lateral column,and the middle column.Each column had different subgroups.The relationship between the morphological characteristics of the"three columns"and the failure of proximal femoral nail antirotation internal fixation was analyzed,and the independent risk factors for internal fixation failure were screened out by single and then multifactorial logistic regression analyses.A risk prediction model was constructed according to the independent risk factors using R language software.The Bootstrap method was used to resample 1 000 times.The area under the curve,calibration curve,and clinical decision curve were used to evaluate the differentiation,calibration ability,and clinical application value of the model.The Youden index was used to determine the optimal risk cut-off value of the prediction model,according to which the patients were divided into high and low risk groups.The stability and extensibility of the model were evaluated according to the accuracy of its risk prediction ability.RESULTS AND CONCLUSION:(1)The four independent risk factors for postoperative internal fixation failure after surgery were predicted using the"three-column"typing system:medial column(comminuted fracture of the lesser trochanter and femoral talar)[odds ratio=5.385,95％CI(1.961,14.782),P=0.001],medial column(chimney type)[odds ratio=2.893,95％CI(1.167,7.173),P=0.022],lateral column(lateral wall thickness＜20.5 mm)[odds ratio=2.804,95％CI(1.078,7.297),P=0.035]and lateral column(lateral wall fracture)[odds ratio=4.278,95％CI(1.670,10.959),P=0.012].(2)The constructed risk prediction model showed good discrimination and accuracy[area under the receiver operating characteristic curve=0.852,95％CI(0.837,0.922)].The calibration curve showed good agreement between the model-predicted risk and the actual risk of occurrence.(3)The clinical decision curve suggested that the model had good clinical applicability when the risk threshold probability was in the range of 0.2-0.82.The risk probability of 28％was the optimal threshold for risk stratification of the model,and the predictive performance of the model was better in patients with different risk groups.(4)The"three-column"typing system constructs a predictive model to calculate the risk probability of postoperative internal fixation failure in patients with intertrochanteric femoral fractures.This method is accurate,simple,and easy to apply clinically,and can be used as a digital tool to guide personalized clinical treatment.

Objective:To investigate impacts of paeoniflorin on inflammation and nuclear transcription factor-κB(NF-κB)/nucleotide-binding oligomeric domain-like receptor protein 3(NLRP3)signal pathway in knee osteoarthritis(KOA)model rats.Meth-ods:SD rats were randomly grouped into model group,glucosamine group,paeoniflorin low-dose group,paeoniflorin high-dose group,paeoniflorin high-dose+phorbol ester(PMA)(NF-κB activator)group,with 10 rats in each group,another 10 rats were regarded as control group,and only knee joint capsule was cut,after treatment with glucosamine,paeoniflorin and PMA,joint pain symptoms of rats were detected by mechanical stimulation method and thermal radiation method;knee joint width,joint swelling and synovial thick-ness were measured;HE staining was applied to detect joint tissue structure and morphology of rats in each group;levels of inflamma-tory factors IL-1β,monocyte chemoattractant protein 1(MCP-1)and IL-9 in joint fluid and serum of rats were detected by ELISA;and Western blot was applied to detect expressions of NF-κB/NLRP3 signal pathway related proteins in rat joint tissue.Results:Com-pared with control group,joint tissue structure of model group was significantly damaged,mechanical pain threshold and thermal sen-sitivity pain threshold were significantly lower(P<0.05),knee joint width,joint swelling and synovial thickness,levels of IL-1β,MCP-1 and IL-9 in joint fluid and serum,and expression of p-NF-κB p65/NF-κB p65 and NLRP3 proteins in joint tissue were higher(P<0.05).Compared with model group,joint tissue damage of rats in glucosamine group,paeoniflorin low-dose group and paeoniflorin high-dose group was reduced,mechanical pain threshold and thermal sensitivity pain threshold were higher(P<0.05),knee joint width,joint swelling and synovial thickness,levels of IL-1β,MCP-1 and IL-9 in joint fluid and serum,and expressions of p-NF-κB p65/NF-κB p65 and NLRP3 proteins in joint tissue were lower(P<0.05);joint tissue injury of rats in paeoniflorin high-dose group was further reduced compared with paeoniflorin low-dose group,mechanical pain threshold and thermal sensitivity pain threshold were further higher(P<0.05),knee joint width,joint swelling and synovial thickness,levels of IL-1β,MCP-1 and IL-9 in joint fluid and serum,and expression of p-NF-κB p65/NF-κB p65 and NLRP3 proteins in joint tissue were further lower(P<0.05).Compared with paeoniflorin high-dose group,joint tissue damage of rats in paeoniflorin high-dose+PMA group was increased,mechanical pain threshold and thermal sensitivity pain threshold were lower(P<0.05),knee joint width,joint swelling and synovial thickness,levels of IL-1β,MCP-1 and IL-9 in joint fluid and serum,and expression of p-NF-κB p65/NF-κB p65 and NLRP3 proteins in joint tissue were higher(P<0.05);there was no significant change in all indexes of rats in glucosamine group(P>0.05).Conclusion:Paeoniflorin can play an anti-inflammatory role by down-regulating the NF-κB/NLRP3 signal pathway,thus alleviating joint injury and joint pain symptoms of KOA rats.

Objective To investigate the effects of sauchinone(Sch)on the growth and immune escape of liver cancer cells by regulating the Krüppel like factor 5(KLF5)-erythropoietin-producing hepatocellular receptor A2(EphA2)pathway.Methods Huh-7 cells were grouped into liver cancer group,Sch low,medium,and high-dose groups(Sch-L group,Sch-M group,Sch-H group),Sch-H+KLF5 activator negative control group(OE-NC),and Sch-H+KLF5 activator(OE-KLF5)group.5-bromo-2-deoxyuracil(EdU)staining,CCK-8,scratch assay,and Transwll were used to detect the proliferation,migration,and invasion of Huh-7 cells,respectively.Western blot was applied to detect proliferative cell nuclear antigen(PCNA),migration invasion enhancer(MIEN1),matrix metalloproteinase-2(MMP-2),programmed death receptor ligand 1(PD-L1),KLF5,and EphA2 proteins in Huh-7 cells.The Huh-7 cells in above 6 groups were co cultured with activated CD8+T cells in a 96 well plate and named as liver cancer co culture group,Sch-L co culture group,Sch-M co culture group,Sch-H co culture group,Sch-H+OE-NC co culture group,and Sch-H+OE-KLF5 co culture group.The killing rate of CD8+T cells in the co culture system and the levels of interferon-γ(IFN-γ),interleukin-4(IL-4),and tumor necrosis factor-α(TNF-α)in the supernatant were detected.Results Compared with the liver cancer group,the EdU positive rate,OD450 value,cell invasion number,and PCNA,MIEN1,MMP-2,PD-L1,KLF5,and EphA2 proteins in the Sch-L,Sch-M,and Sch-H groups reduced,the migration distance shorten(P<0.05).Compared with the Sch-H group and Sch-H+OE-NC group,the EdU positive rate,OD450 value,cell invasion number,and PCNA,MIEN1,MMP-2,PD-L1,KLF5,and EphA2 proteins in the Sch-H+OE-KLF5 group increased,the migration distance prolonged(P<0.05).Compared with the liver cancer co culture group,the killing rate of CD8+T cells on Huh-7 cells and the levels of IFN-γ,TNF-α,and IL-4 in the supernatant in the Sch-L co culture group,Sch-M co culture group,and Sch-H co culture group increased(P<0.05).Compared with the Sch-H co culture group and the Sch-H+OE-NC co culture group,the killing rate of CD8+T cells on Huh-7 cells and the levels of IFN-γ,TNF-α,and IL-4 in the supernatant in the Sch-H+OE-KLF5 co culture group decreased(P<0.05).Conclusion Sch may inhibit growth and immune escape of liver cancer cells by inhibiting the KLF5-EphA2 pathway.

Pain modulation encompasses a complex neurobiological process, in which the lateral habenula (LHb) plays a crucial role in integrating, regulating and modulating pain signals. It is also involved in pain-related memory functions associated with perception, transmission and regulation of pain. Furthermore, the LHb collaborates with structures such as the spinal dorsal horn, forebrain, and amygdala to form an essential neural circuit that contributes to sensitization, development of tolerance, and adaptation processes related to pain. However, there remains limited understanding regarding the specific roles and interactions among different neuron subtypes within the LHb concerning pain regulation. Additionally, further investigation is warranted to explore functional changes and plasticity within both the LHb and its associated neural circuits in chronic pain models. Future research endeavors should utilize advanced neuroimaging techniques alongside optogenetics and gene editing technologies to elucidate intricate neural circuits, cellular architecture, and molecular mechanisms governing LHb function in pain regulation. In conclusion, this paper aims to comprehensively review existing literature on the involvement of the LHb and its neural circuits in modulating pain, thereby enhancing our understanding of their neurobiological mechanisms while providing novel targets for precise therapeutic strategies aimed at alleviating pain.

Objective To evaluate the sensitivity,repeatability,anti-degradation ability,in vivo temporal stability,and tissue specificity of the DNA methylation sequencing panel constructed in our previous study for height inference,so as to provide a reference for forensic application.Methods Sensitivity:different initial template quantities(50 ng,40 ng,30 ng,20 ng)were set for sequencing.Repeatability:DNA from the same sample was sequenced three times.Anti-degradation ability:whole blood was used to make blood stains,and DNA was extracted and sequenced at 0,3,6 and 9 months,respectively.In vivo temporal stability:the blood was collected at 0,3,6,and 9 months for sequencing.Tissue specificities:published data and findings were used to analyze the tissue specificities of CpGs in the panel.Results The sensitivity test showed that the initial template quantities of 20 ng detected all the CpG sites and still obtained accurate prediction results.The results of the three repeated predictions of the same sample are stable,and the differences are mainly due to the randomness of the DNN model,indicating good detection repeatability.A complete methylation profile was obtained for the blood stains left at room temperature for 9 months,and the predicted results showed a small range of fluctuations.The three samples were predicted to fluctuate within a range of 1.5 cm or less over nine months.Tissue-specific analyses showed a high correlation between blood and saliva,but can not apply to other tissues.Conclusion The DNA methylation detection system we developed in our previous study has good sensitivity,repeatability,anti degradation ability,in vivo time stability,as well as strong tissue specificity,making it suitable for height inference of blood samples.This supports the feasibility of using targeted DNA methylation analysis on whole blood samples to infer height in the field of forensic science.

Aim To investigate whether adipose differentiation-related proteins promote macrophage lipid accu-mulation by upregulating acyl-CoA synthetase long-chain family member 3(ACSL3)expression through PI3K/Akt.Methods The experiments were divided into 24 h group,different PLIN2 expression groups,HA-PLIN2+SC97 group and HA-PLIN2+LY294002 group.Western blot was used to detect the protein expression of PLIN2,Akt,p-Akt and ACSL3 in cells,RT-qPCR was used to detect the mRNA level of PLIN2 in cells,and oil red O was used to observe the degree of lipid accumulation in cells.Results The protein expression levels of Akt,p-Akt and ACSL3 in macrophages overex-pressing PLIN2 were significantly increased(P＜0.05),and p-Akt nuclear translocation was increased,with fluorescence labeling of PLIN2 and Akt overlapping.After adding the PI3K/Akt agonist SC97 to macrophages overexpressing PLIN2,the expression level of ACSL3 significantly increased(P＜0.05),and the degree of intracellular lipid accumulation in-creased;After adding the PI3K/Akt inhibitor LY294002 to macrophages overexpressing PLIN2,the expression level of AC-SL3 significantly decreased(P＜0.05),and the degree of intracellular lipid accumulation decreased.Conclusion PLIN2 upregulates ACSL3 expression through PI3K/Akt,thereby promoting macrophage lipid accumulation.

BACKGROUND:Intertrochanteric femoral fractures are the main type of fragility fracture in the elderly,and proximal femoral nail antirotation is the preferred surgical option,but the factors associated with postoperative internal fixation failure are controversial.OBJECTIVE:A new"three-column"classification of intertrochanteric femoral fractures was proposed by evaluating patients'imaging data preoperatively and analyzing its interaction with postoperative internal fixation failure.A risk prediction model was developed and validated by using numerical algorithms,which facilitates clinicians to identify and intervene in high-risk patients preoperatively.METHODS:Patients with intertrochanteric femur fractures in Sanshui Branch of Foshan Hospital of Traditional Chinese Medicine between June 2012 and June 2022 were selected.The patients were divided into the internal fixation failure group and the internal fixation maintenance group according to whether they had internal fixation failure after surgery.According to the preoperative radiographs,the proximal femur was divided into three columns:the medial column,the lateral column,and the middle column.Each column had different subgroups.The relationship between the morphological characteristics of the"three columns"and the failure of proximal femoral nail antirotation internal fixation was analyzed,and the independent risk factors for internal fixation failure were screened out by single and then multifactorial logistic regression analyses.A risk prediction model was constructed according to the independent risk factors using R language software.The Bootstrap method was used to resample 1 000 times.The area under the curve,calibration curve,and clinical decision curve were used to evaluate the differentiation,calibration ability,and clinical application value of the model.The Youden index was used to determine the optimal risk cut-off value of the prediction model,according to which the patients were divided into high and low risk groups.The stability and extensibility of the model were evaluated according to the accuracy of its risk prediction ability.RESULTS AND CONCLUSION:(1)The four independent risk factors for postoperative internal fixation failure after surgery were predicted using the"three-column"typing system:medial column(comminuted fracture of the lesser trochanter and femoral talar)[odds ratio=5.385,95％CI(1.961,14.782),P=0.001],medial column(chimney type)[odds ratio=2.893,95％CI(1.167,7.173),P=0.022],lateral column(lateral wall thickness＜20.5 mm)[odds ratio=2.804,95％CI(1.078,7.297),P=0.035]and lateral column(lateral wall fracture)[odds ratio=4.278,95％CI(1.670,10.959),P=0.012].(2)The constructed risk prediction model showed good discrimination and accuracy[area under the receiver operating characteristic curve=0.852,95％CI(0.837,0.922)].The calibration curve showed good agreement between the model-predicted risk and the actual risk of occurrence.(3)The clinical decision curve suggested that the model had good clinical applicability when the risk threshold probability was in the range of 0.2-0.82.The risk probability of 28％was the optimal threshold for risk stratification of the model,and the predictive performance of the model was better in patients with different risk groups.(4)The"three-column"typing system constructs a predictive model to calculate the risk probability of postoperative internal fixation failure in patients with intertrochanteric femoral fractures.This method is accurate,simple,and easy to apply clinically,and can be used as a digital tool to guide personalized clinical treatment.

Aim To investigate whether adipose differentiation-related proteins promote macrophage lipid accu-mulation by upregulating acyl-CoA synthetase long-chain family member 3(ACSL3)expression through PI3K/Akt.Methods The experiments were divided into 24 h group,different PLIN2 expression groups,HA-PLIN2+SC97 group and HA-PLIN2+LY294002 group.Western blot was used to detect the protein expression of PLIN2,Akt,p-Akt and ACSL3 in cells,RT-qPCR was used to detect the mRNA level of PLIN2 in cells,and oil red O was used to observe the degree of lipid accumulation in cells.Results The protein expression levels of Akt,p-Akt and ACSL3 in macrophages overex-pressing PLIN2 were significantly increased(P＜0.05),and p-Akt nuclear translocation was increased,with fluorescence labeling of PLIN2 and Akt overlapping.After adding the PI3K/Akt agonist SC97 to macrophages overexpressing PLIN2,the expression level of ACSL3 significantly increased(P＜0.05),and the degree of intracellular lipid accumulation in-creased;After adding the PI3K/Akt inhibitor LY294002 to macrophages overexpressing PLIN2,the expression level of AC-SL3 significantly decreased(P＜0.05),and the degree of intracellular lipid accumulation decreased.Conclusion PLIN2 upregulates ACSL3 expression through PI3K/Akt,thereby promoting macrophage lipid accumulation.