Objective To observe the effects of Bushen Huoxue Prescription on the pathway related to necroptosis of nucleus pulposus cells in a model rabbit of intervertebral disc degeneration;To explore its mechanisms in delaying intervertebral disc degeneration.Methods A intervertebral disc degeneration rabbit model was established using the spinal instability method.Totally 40 model rabbits were randomly divided into model group,ibuprofen group and Bushen Huoxue Prescription low-,medium-and high-dosage groups.Additionally,a normal control group and a sham-operation group were set up,with 8 rabbits in each group.Each treatment groups received the corresponding drugs via gavage for two consecutive weeks.HE staining was used to observe morphology of nucleus pulposus tissue,transmission electron microscopy was used to observe ultrastructure in nucleus pulposus cells,immunohistochemical staining was used to assess the expressions of Aggrecan and Collagen Ⅱ in nucleus pulposus tissue,Western blot was used to detect the expressions of p-RIPK1,p-RIPK3 and p-MLKL protein in nucleus pulposus tissue.Results Compared with the sham-operation group,the model group showed a significant decrease in nucleus pulposus cells,disordered cell arrangement,reduced extracellular matrix,interrupted cell membrane continuity under transmission electron microscopy,organelle swelling,nuclear membrane disruption,partial chromatin loss,and positive expression of Aggrecan and Collagen Ⅱ in nucleus pulposus tissue decreased(P<0.01),while the expressions of p-RIPK1,p-RIPK3 and p-MLKL protein significantly increased(P<0.01).Compared with the model group,the treatment groups showed an increased number of nucleus pulposus cells with orderly arrangement and more extracellular matrix,the ultrastructural damage of the cell membrane,organelle and nucleus in nucleus pulposus cells was partially restored under transmission electron microscopy,the positive expressions of Aggrecan and Collagen Ⅱ significantly increased in Bushen Huoxue Prescription medium-and high-dosage groups and the ibuprofen group(P<0.05,P<0.01),while the expressions of p-RIPK1,p-RIPK3 and p-MLKL protein significantly decreased(P<0.05,P<0.01).Conclusion Bushen Huoxue Prescription may delay intervertebral disc degeneration of the model rabbit by inhibiting the expressions of p-RIPK1,p-RIPK3 and p-MLKL protein in nucleus pulposus cells,and promoting the generation of extracellular matrix components Aggrecan and Collagen Ⅱ.

Objective To observe the effects of Bushen Huoxue Prescription on pressure-induced necroptosis in human nucleus pulposus cells and the expressions of RIPK1/RIPK3/MLKL pathway;To explore its potential mechanism in delaying intervertebral disc degeneration.Methods Human primary nucleus pulposus cells were cultured in vitro,and a model of nucleus pulposus cell degeneration was established using continuous load pressure method.After modeling,the nucleus pulposus cells were divided into model group,Bushen Huoxue Prescription group and inhibitor group,blank serum,Bushen Huoxue Prescription containing serum and necroptotic apoptosis inhibitor(Nec-1)intervention were administered,respectively.Normal group nucleus pulposus cells were cultured routinely.AO/EB fluorescence dual staining method was used for detecting cell apoptosis,flow cytometry was used to detect the necroptosis rate of nucleus pulposus cells,Western blot was used to detect the protein expressions of p-receptor interacting protein kinase(RIPK)1,p-RIPK3 and p-mixed lineage kinase domain like protein(MLKL),RT-qPCR was used to detect the mRNA expressions of RIPK1,RIPK3 and MLKL.Results Compared with the normal group,the model group showed more red fluorescence under AO/EB staining of nucleus pulposus cells,which were round and condensed,the necroptosis rate of nucleus pulposus cells increased(P<0.05),the protein expressions of p-RIPK1,p-RIPK3 and p-MLKL increased(P<0.05,P<0.01),while there was no significant difference in RIPK1 mRNA expression(P>0.05),and RIPK3 and MLKL mRNA expression increased(P<0.01).Compared with the model group,Bushen Huoxue Prescription group and the inhibitor group had less red condensed chromatin in the nucleus pulposus cells,Bushen Huoxue Prescription group had a lower rate of necroptosis(P<0.05),while the inhibitor group showed a decreasing trend in necroptosis rate(P>0.05),the protein expressions of p-RIPK1,p-RIPK3 and p-MLKL decreased in Bushen Huoxue Prescription group and the inhibitor group(P<0.05,P<0.01),while there was no significant difference in RIPK1 mRNA expression(P>0.05),and RIPK3 and MLKL mRNA expressions decreased(P<0.01).Conclusion Bushen Huoxue Prescription can alleviate pressure-induced damage to nucleus pulposus cells and inhibit necroptosis,thereby slowing the progression of intervertebral disc degeneration.Its mechanism may be related to the RIPK1/RIPK3/MLKL pathway mediated necroptosis.

Objective:To investigate the key targets and mechanisms of compound Huangbai liquid in promoting wound healing of perianal abscess using network pharmacology and machine learning.Methods:Active components of compound Huangbai liquid and their target genes were screened and corrected using the TCMSP and HERB databases.Target genes related to wound healing were collected from the GeneCard and GEO databases.Common targets were identified using SangerBox online tool,followed by KEGG and GO enrichment analyses to explore potential biological functions.A PPI network was constructed to analyze core gene interactions,and immune cell infiltration was evaluated using the CIBERSORT algorithm.Key genes were screened using machine learning methods such as Boruta,random forest,XGBoost,and SVM-RFE.Finally,the binding affinity between active components and target genes was validated using AutoDock Vina.Results:Four key target genes(CYP19A1,IL10RA,ALOXE3,EGFR)were significantly correlated with components such as quercetin and berberine.These genes were involved in PI3K-Akt signaling pathway,and closely related to immune response and cell proliferation.The PPI network showed that these genes played important roles in angiogenesis and cell adhesion.Immune infiltration analysis showed that key genes were strongly correlated with immune cells such as macrophages.Conclusion:Compound Huangbai liquid may promote wound healing in perianal abscess by regulating multiple biological pathways and immune responses.

Objective:To investigate the key targets and mechanisms of compound Huangbai liquid in promoting wound healing of perianal abscess using network pharmacology and machine learning.Methods:Active components of compound Huangbai liquid and their target genes were screened and corrected using the TCMSP and HERB databases.Target genes related to wound healing were collected from the GeneCard and GEO databases.Common targets were identified using SangerBox online tool,followed by KEGG and GO enrichment analyses to explore potential biological functions.A PPI network was constructed to analyze core gene interactions,and immune cell infiltration was evaluated using the CIBERSORT algorithm.Key genes were screened using machine learning methods such as Boruta,random forest,XGBoost,and SVM-RFE.Finally,the binding affinity between active components and target genes was validated using AutoDock Vina.Results:Four key target genes(CYP19A1,IL10RA,ALOXE3,EGFR)were significantly correlated with components such as quercetin and berberine.These genes were involved in PI3K-Akt signaling pathway,and closely related to immune response and cell proliferation.The PPI network showed that these genes played important roles in angiogenesis and cell adhesion.Immune infiltration analysis showed that key genes were strongly correlated with immune cells such as macrophages.Conclusion:Compound Huangbai liquid may promote wound healing in perianal abscess by regulating multiple biological pathways and immune responses.

Objective To observe the effects of Bushen Huoxue Prescription on the pathway related to necroptosis of nucleus pulposus cells in a model rabbit of intervertebral disc degeneration;To explore its mechanisms in delaying intervertebral disc degeneration.Methods A intervertebral disc degeneration rabbit model was established using the spinal instability method.Totally 40 model rabbits were randomly divided into model group,ibuprofen group and Bushen Huoxue Prescription low-,medium-and high-dosage groups.Additionally,a normal control group and a sham-operation group were set up,with 8 rabbits in each group.Each treatment groups received the corresponding drugs via gavage for two consecutive weeks.HE staining was used to observe morphology of nucleus pulposus tissue,transmission electron microscopy was used to observe ultrastructure in nucleus pulposus cells,immunohistochemical staining was used to assess the expressions of Aggrecan and Collagen Ⅱ in nucleus pulposus tissue,Western blot was used to detect the expressions of p-RIPK1,p-RIPK3 and p-MLKL protein in nucleus pulposus tissue.Results Compared with the sham-operation group,the model group showed a significant decrease in nucleus pulposus cells,disordered cell arrangement,reduced extracellular matrix,interrupted cell membrane continuity under transmission electron microscopy,organelle swelling,nuclear membrane disruption,partial chromatin loss,and positive expression of Aggrecan and Collagen Ⅱ in nucleus pulposus tissue decreased(P<0.01),while the expressions of p-RIPK1,p-RIPK3 and p-MLKL protein significantly increased(P<0.01).Compared with the model group,the treatment groups showed an increased number of nucleus pulposus cells with orderly arrangement and more extracellular matrix,the ultrastructural damage of the cell membrane,organelle and nucleus in nucleus pulposus cells was partially restored under transmission electron microscopy,the positive expressions of Aggrecan and Collagen Ⅱ significantly increased in Bushen Huoxue Prescription medium-and high-dosage groups and the ibuprofen group(P<0.05,P<0.01),while the expressions of p-RIPK1,p-RIPK3 and p-MLKL protein significantly decreased(P<0.05,P<0.01).Conclusion Bushen Huoxue Prescription may delay intervertebral disc degeneration of the model rabbit by inhibiting the expressions of p-RIPK1,p-RIPK3 and p-MLKL protein in nucleus pulposus cells,and promoting the generation of extracellular matrix components Aggrecan and Collagen Ⅱ.

Objective To observe the effects of Bushen Huoxue Prescription on pressure-induced necroptosis in human nucleus pulposus cells and the expressions of RIPK1/RIPK3/MLKL pathway;To explore its potential mechanism in delaying intervertebral disc degeneration.Methods Human primary nucleus pulposus cells were cultured in vitro,and a model of nucleus pulposus cell degeneration was established using continuous load pressure method.After modeling,the nucleus pulposus cells were divided into model group,Bushen Huoxue Prescription group and inhibitor group,blank serum,Bushen Huoxue Prescription containing serum and necroptotic apoptosis inhibitor(Nec-1)intervention were administered,respectively.Normal group nucleus pulposus cells were cultured routinely.AO/EB fluorescence dual staining method was used for detecting cell apoptosis,flow cytometry was used to detect the necroptosis rate of nucleus pulposus cells,Western blot was used to detect the protein expressions of p-receptor interacting protein kinase(RIPK)1,p-RIPK3 and p-mixed lineage kinase domain like protein(MLKL),RT-qPCR was used to detect the mRNA expressions of RIPK1,RIPK3 and MLKL.Results Compared with the normal group,the model group showed more red fluorescence under AO/EB staining of nucleus pulposus cells,which were round and condensed,the necroptosis rate of nucleus pulposus cells increased(P<0.05),the protein expressions of p-RIPK1,p-RIPK3 and p-MLKL increased(P<0.05,P<0.01),while there was no significant difference in RIPK1 mRNA expression(P>0.05),and RIPK3 and MLKL mRNA expression increased(P<0.01).Compared with the model group,Bushen Huoxue Prescription group and the inhibitor group had less red condensed chromatin in the nucleus pulposus cells,Bushen Huoxue Prescription group had a lower rate of necroptosis(P<0.05),while the inhibitor group showed a decreasing trend in necroptosis rate(P>0.05),the protein expressions of p-RIPK1,p-RIPK3 and p-MLKL decreased in Bushen Huoxue Prescription group and the inhibitor group(P<0.05,P<0.01),while there was no significant difference in RIPK1 mRNA expression(P>0.05),and RIPK3 and MLKL mRNA expressions decreased(P<0.01).Conclusion Bushen Huoxue Prescription can alleviate pressure-induced damage to nucleus pulposus cells and inhibit necroptosis,thereby slowing the progression of intervertebral disc degeneration.Its mechanism may be related to the RIPK1/RIPK3/MLKL pathway mediated necroptosis.

Objective:To construct a mouse asthma model induced by mugwort pollen and to explore endotyping,providing methods for subsequent precision treatment.Methods:BALB/c mice were intraperitoneally injected with mugwort pollen extract(MPE)to sensitize,following MPE intranasal challenge to construct MPE allergic asthma murine model.Mice were randomly divided into PBS sensitization and PBS challenge(P-P),MPE sensitization and PBS challenge(M-P),MPE sensitization and MPE challenge model(M-M)groups.24 h after final challenge,mice were performed to examine airway responsiveness;bronchoalveolar lavage fluid(BALF)was harvested for cell counting and statistical classification of inflammatory cells through flow cytometry analysis.Pulmonary slides were collected for pathological examination,including HE,PAS,Masson and α-SMA immunohistochemical staining.ELISA was used to detect levels of IFN-γ,IL-4,IL-5,IL-13,IL-17A in lung tissue and serum,as well as serum total IgE and MPE-specific IgE,IgG1,IgG2a levels.Results:Pathological examination showed higher airway reactivity,more inflammatory cells infiltration around airway,obvious goblet metaplasia,thickening of airway smooth muscles and dramatical fibrin deposition around airway in model group.Total cell numbers of BALF were increased from<1×105 cells/ml in P-P group to>5×105 cells/ml in model group,in which eosinophils were predominant cellular type,levels of IL-4,IL-13,IL-17A in lung and IL-5,IL-13 levels in serum were significantly increased,as well as significant increasing levels of total IgE and MPE-specific IgE,IgG1,IgG2a.Conclusion:MPE-sensitized and challenged mice induces typical eosinophilic asthma featured with elevated eosinophils,as well as secretion of inflammatory factors of type 2 and type 17,IgE,IgG1 and IgG2a subtypes soars to high levels.

Epidemiological and animal studies indicate that pre-existing diabetes increases the risk of Parkinson's disease(PD).However,the mechanisms underlying this association remain unclear.In the present study,we found that high glucose(HG)levels in the cerebrospinal fluid(CSF)of diabetic rats might enhance the effect of a subthreshold dose of the neurotoxin 6-hydroxydopamine(6-OHDA)on the development of motor disorders,and the damage to the nigrostriatal dopaminergic neuronal pathway.In vitro,HG promoted the 6-OHDA-induced apoptosis in PC12 cells differentiated to neurons with nerve growth factor(NGF)(NGF-PC12).Metabolomics showed that HG promoted hyperglycolysis in neurons and impaired tricarboxylic acid cycle(TCA cycle)activity,which was closely related to abnormal mito-chondrial fusion,thus resulting in mitochondrial loss.Interestingly,HG-induced upregulation of pyruvate kinase M2(PKM2)combined with 6-OHDA exposure not only mediated glycolysis but also promoted abnormal mitochondrial fusion by upregulating the expression of MFN2 in NGF-PC12 cells.In addition,we found that PKM2 knockdown rescued the abnormal mitochondrial fusion and cell apoptosis induced by HG+6-OHDA.Furthermore,we found that shikonin(SK),an inhibitor of PKM2,restored the mito-chondrial number,promoted TCA cycle activity,reversed hyperglycolysis,enhanced the tolerance of cultured neurons to 6-OHDA,and reduced the risk of PD in diabetic rats.Overall,our results indicate that diabetes promotes hyperglycolysis and abnormal mitochondrial fusion in neurons through the upre-gulation of PKM2,leading to an increase in the vulnerability of dopaminergic neurons to 6-OHDA.Thus,the inhibition of PKM2 and restoration of mitochondrial metabolic homeostasis/pathways may prevent the occurrence and development of diabetic PD.

Objective To investigate the role of Ras-related C3 botulinum toxin substrate 1/mitogen-activated protein kinase/extracellular signal-regulated kinase (Rac 1/MAPK/ERK) signal pathway in rats with ventilator induced lung injury (VILI) and its mechanism.Methods Thirty Sprague-Dawley (SD) rats were randomly divided into spontaneous respiration group,normal tidal volume (VT) group and high VT group with 10 rats in each group.The rats in spontaneous respiration group were kept their spontaneous breathing.The rats in normal VT group and high VT group were performed tracheal intubation after tracheostomy,and underwent mechanical ventilation on bilateral lungs with 6 mL/kg and 40 mL/kg VT respectively with maintenance anesthesia.After 4-hour ventilation,heart blood,bronchoalveolar lavage fluid (BALF) and lung tissues were harvested.The levels of interleukins (IL-1β,IL-6),tumor necrosis factor-α (TNF-α),myeloperoxidase (MPO) and macrophage inflammatory protein-2 (MIP-2) in serum and BALF were determined by enzyme linked immunosorbent assay (ELISA).Lung wet/dry radio (W/D) was determined.The lung tissues were stained with hematoxylin and eosin (HE),and pathological changes were observed,and pathological scores were evaluated.The ultra structure changes in type Ⅱ alveolar epithelial cells (AEC Ⅱ)were observed with transmission electron microscope.The positive expressions of phosphorylation of extracellular signal-regulated kinase (p-ERK) were determined by immunohistochemistry,and those of Racl and F-actin were determined by immunofluorescence.The mRNA expressions of ERK and Rac1 were determined by real-time fluorescent quantitation reverse transcription-polymerase chain reaction (RT-qPCR),and protein expressions of Rac-1,p-ERK and F-actin were determined by Western Blot.Results ① Compared with spontaneous breathing group,lung W/D in both mechanical ventilation groups was significantly increased,with more significant increase in the high VT group (6.64 ± 0.88 vs.1.79 ± 0.36,P ＜ 0.01).② There was no obvious pathological changes in the lung tissue and AEC Ⅱ of the spontaneously breathing group.In the normal VT group,there was slight edema and infiltration of inflammatory cells;AEC Ⅱ had less lamellar bodies and uniform distribution of the villi of the alveolar epithelium.In the high VT group,the edema of the lung tissue,the widening of the pulmonary septum,the alveolus congestion,the infiltration of inflammatory cells,and alveolar structure disorder were found;and AEC Ⅱ was irregular,the number of lamellar bodies in the plastids was decreased and was unevenly distributed.The pulmonary histopathological score in the high VT group was significantly higher than that in the spontaneous breathing group and the normal VT group (12.00 ± 2.00 vs.6.00 ± 1.51,8.50 ± 0.53,both P ＜ 0.01).③ Compared with spontaneous breathing group,IL-1β,IL-6,TNF-α,MPO,and MIP-2in serum and BALF in both mechanical ventilation groups were significantly increased,with more siguificant increase in the high VT group [serum IL-1 β (ng/L):104.2 ± 15.1 vs.20.3 ± 8.3,IL-6 (ng/L):46.6 ± 11.5 vs.22.7 ± 7.5,TNF-α (ng/L):39.4±6.5 vs.5.4± 1.9,MPO (ng/L):0.66±0.24 vs.0.06±0.03,MIP-2 (ng/L):109.2±25.8 vs.22.8±8.4;BALF IL-1 β (ng/L):121.5 ± 25.6 vs.24.0 ± 7.5,IL-6 (ng/L):136.7 ± 32.7 vs.31.4 ± 10.5,TNF-α (ng/L):98.0 ± 14.8vs.10.1 ±2.6,MPO (ng/L):0.80±0.31 vs.0.08±0.04,MIP-2 (ng/L):144.4±28.9 vs.41.2±20.7;all P ＜ 0.01].④ There were only a few p-ERK,Rac1 and F-actin positive expressions in the spontaneous breathing group.The positive expressions in normal VT group were increased.In high VT group,the positive expression of p-ERK was significantly increased;Rac1 and F-actin were mainly distributed in the cell membrane and cytoplasm respectively,the positive expressions were further enhanced.⑤ The gene expressions of ERK and Rac1,and protein expressions of p-ERK,Rac1 and F-actin in the high VT group were significantly higher than those in the spontaneous breathing group and normal VT group [ERK mRNA (2-△△Ct):8.23±2.83 vs.1,3.02± 1.38,p-ERK protein (gray value):1.15±0.36 vs.0.61 ±0.23,0.88±0.22;Rac1 mRNA (2-△△Ct):4.45 ±2.26 vs.1,1.22±0.39,Rac1 protein (gray value):0.91 ±0.16 vs.0.48±0.11,0.55 ± 0.10;F-actin protein (gray value):0.70± 0.09 vs.0.49 ± 0.08,0.55 ± 0.04;all P ＜ 0.01].Conclusion F-actin expression in lung tissue was up-regulated in rats with VILI,which resulted in reconstruction of AEC Ⅱ cyto-skeleton,and variation of cell membrane permeability through Rac 1 /MAPK/ERK sigualing pathway during VILI.

Objective To investigate the value of quantitative analysis of the left ventricular longitudinal strain in patients with hypertrophic cardiomyopathy (HCM) and with normal left ventricular ejection fraction (LVEF) by using two-dimensional speckle tracking imaging.Methods Twenty-eight HCM patients with normal LVEF (all of the cases were non obstructive HCM),who were diagnosed by clinical and ultrasound echocardiography between January 1,2015 and January 1,2016 in the First Affiliated Hospital of Dalian Medical University,served as the experimental group.And twenty healthy volunteers served as the healthy control group.The peak longitudinal strain (LPS) of the left ventricle and the systolic peak of the left ventricle were calculated by the STE technique.The indexes such as the transmural gradient (△ LS=LPSEndo-LPSEpi)and the transmural gradient percentage (△ LS％=△ LS/LPSEndo) were calculated.The Peak systolic longitudinal strain of endocardium (LPSEndo),the peak systolic longitudinal strain of mid-cardium (LPSMid),the peak systolic longitudinal strain of epicardium (LPSEpi),the peak systolic longitudinal strain of basal segment (LPSb),the peak systolic longitudinal strain of middle segment (LPSm),the peak systolic longitudinal strain of apical segment (LPSa),the global peak systolic longitudinal strain (GLPS) and other left ventricular myocardial strain,such as △ LS,△ LS％,in both the HCM group and the healthy control group,were analyzed by using independent samples t test comparison.For each layer of the left ventricle and the overall myocardial longitudinal strain,two independent sample t test was used for comparison between groups,and LSD-t test was used for intra-group comparison.Results (1) There was a gradient of LPS among the three layers and the three segments in both of the two groups:LPSEndo and LPSMid [(18.36±4.97)％ vs (13.80±4.23)％,(26.41±2.93)％ vs (22.19±2.49)％],the difference was statistically significant (t=5.550,8.529,P ＜ 0.05);LPSEndo and LPSEpi [(18.36±4.97) ％ vs (11.91 ±3.63)％,(26.41±2.93)％ vs (19.43±2.20)％],the difference was statistically significant (t=5.550,8.529,P ＜ 0.05);There was significant difference between LPSMid and LPSEpi in the healthy control group [(22.19 ± 2.49)％ vs (19.43 ± 2.20)％,t=3.709,P ＜ 0.05)],that was,LPSEndo ＞ LPSMid ＞ LPSEpi.LPSa and LPSm,the difference was statistically significant (t=4.029,6.839,P ＜ 0.05);LPSa and LPSb,the difference was statistically significant (t=5.304,9.887,P ＜ 0.05);There was significant difference between LPSm and LPSb in the healthy control group (t=4.170,P ＜ 0.05);that was,LPSa ＞ LPSm ＞ LPSb.In the HCM group,LPS in the 3 layers,3 segments,and the whole left ventricular wall were lower than that of the the healthy control group,the differences were statistically significant [GLPS:(14.63± 3.75)％ vs (22.68±2.51)％,t=-8.347;LPSEndo to LPSEpi:t=-6.477,-7.909,-8.242;LPSa to LPSb:t=-6.647,-8.790,-7.267;all P ＜ 0.05).(2) Compared with the healthy control group,both the segmental gradient and global transmural gradient in the HCM group were found reduced,but the difference had no statistical significance (all P ＞ 0.05).(3) The transmural gradient percentage both in the healthy control group and the HCM group were reduced from the apical segment to the basal segment,the difference were statistically significant (HCM group:t=9.985,5.969;healthy control group:t=17.513,7.043;all P ＜ 0.05).Compared with the healthy control group,the △ LS％a and the △ LS％m of HCM group were significantly higher [(58.86± 11.32)％ vs (43.70±4.73)％,(28.43± 11.48)％ vs (20.30± 3.66)％],and the difference was statistically significant (t=5.634,3.049,all P ＜ 0.05).Conclusions (1) Using 2D-STI could accurately determine the regional or the global left ventricular systolic function in patients with HCM.(2) The transmural gradient percentage can be more sensitive to reflect the change of the transmural gradient,and more research needed to explore its value for clinical application.