OBJECTIVE To investigate the mechanism of Xihuang pill （XHP） against diffuse large B-cell lymphoma （DLBCL）. METHODS The active ingredients of XHP and potential therapeutic targets for DLBCL were identified using TCMSP， GeneCards and DisGeNET databases. Protein-protein interaction networks were constructed using the String database and Cytoscape software to screen core components and core targets. Gene ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses were then performed. The clinical relevance of core targets was analyzed using the GEPIA and PanCanSurvPlot databases. Molecular docking and molecular dynamics （MD） simulation were conducted to verify the interactions between core components and core targets， and the binding free energy was calculated using the molecular mechanics Poisson-Boltzmann surface area （MM-PBSA） method. The effects of XHP on DLBCL and the related molecular mechanisms were validated using CCK-8 assay， flow cytometry and Western blot. RESULTS Network pharmacology analysis identified 108 active ingredients of XHP and 410 potential therapeutic targets for DLBCL. Six core components （e.g.， 17 beta-estradiol， quercetin） and ten core targets [e.g.， tumor protein 53 （TP53）， proto-oncogene tyrosine-protein kinase Src （SRC）] were obtained. Enrichment analysis indicated that the anti-DLBCL effects of XHP were primarily associated with the apoptotic signaling pathway， the phosphoinositide 3-kinase （PI3K）/protein kinase B （Akt） signaling pathway and so on. Clinical correlation analysis revealed that TP53 and SRC expression were significantly up-regulated in DLBCL tissues and associated with poor patient prognosis （P＜0.05）. Molecular docking， MD simulations and MM-PBSA calculations confirmed that the SRC-quercetin complex had a mail：stronger and more stable binding affinity. In vitro experiments demonstrated that XHP concentration-dependently inhibited the proliferation of DLBCL cells； compared with control group， XHP medium- and high-dose groups could significantly induce the apoptosis of SU-DHL2 and SU-DHL4 cells， and significantly down- regulated the expressions of SRC protein， phosphorylated （p）-PI3K/PI3K and p-Akt/Akt in SU-DHL4 cells （P＜0.05）. CONCLUSIONS XHP may inhibit the proliferation and induce the apoptosis of DLBCL cells by regulating the SRC/PI3K/Akt signaling pathway.

Objective To observe the effect of pirfenidone on myocardial fibrosis in rats and inves-tigate its underlying mechanism.Methods Twenty-four SD rats were randomly divided into sham-operation group,model group,low-and high-dose pirfenidone groups,with 6 rats in each group.Rat model of myocardial fibrosis was established by injecting isoprenaline into the tail vein,while normal saline was given to the sham operation group.Pirfenidone of 150 and 300 mg/(kg·d)were infused gastrically to the rats of low-and high-dose pirfenidone groups after modeling.Mas-son staining was used to observe the severity of myocardial fibrosis,immunohistochemical assay was employed to detect the expression of Collagen-1,NOD-like receptor thermal protein domain associated protein 3(NLRP3)inflammasome,cysteinyl aspartate-specific protease-1(Caspase-1),and Western blotting was performed to detect the protein levels of Collogen-1 and atrial desmo-plakin D(gasdermin D,GSDMD).Results The model group showed obvious myocardial fibrosis,and elevated expression of Collogen-1,NLRP3,Caspase-1 and GSDMD when compared with the sham operation group(P＜0.05).Low-and high-dose pirfenidone treatment resulted in signifi-cantly reduced myocardial fibrosis and reduced expression of Collogen-1,NLRP3,Caspase-1 and GSDMD[(8.14±1.40)％,(6.56±0.75)％vs(22.15±2.57)％,P＜0.05;0.14±0.03 vs 0.33±0.05,0.42±0.13,P＜0.05;(10.34±1.40)％,(10.33±3.40)％vs(23.22±1.99)％,P＜0.05;(15.67±0.56)％,(17.33±0.78)％vs(22.87±1.92)％,P＜0.05;0.43±0.06,0.46±0.11 vs 0.65±0.03,P＜0.05].Conclusion Pirfenidone inhibits cardiomyocyte pyroptosis and attenuates myocar-dial fibrosis through the NLRP3/Caspase-1/GSDMD signaling axis.

Objective To screen immune-related long non-coding RNAs(LncRNAs)in the TCGA database pancreatic cancer dataset and construct a prognostic risk assessment model with immune-related LncRNAs to explore prognosis-related potential molecular mechanisms.Methods RNA-seq data of 171 pancreatic cancer samples and corresponding clinical information were obtained by The Cancer Genome Atlas(TCGA)database,and two classical immune-related gene datasets(GO0006955/IMMUNE RESPONSE and GO0002376/IMMUNE SYSTERM PROCESS)and gene annotation information were used to identify immune-related LncRNAs.The immune-related LncRNAs associated with pancreatic cancer prognosis were used for univariate and multivariate Cox analyses to establish a model for the assessment of pancreatic cancer prognostic risk based on immune-associated LncRNAs.This risk model was used for survival analysis,clinical correlation analysis,immune cell infiltration analysis,pathway enrichment analysis,and prognostic column line plot modeling.Results We screened 119 immune-related LncRNAs in pancreatic cancer,and five immune-related LncRNAs(AC064836.3,LINC00941,ZNF236-DT,TMEM161B-AS1 and AC068580.2)were identified for the development of pancreatic cancer prognostic risk assessment model.According to the prognostic risk assessment model,pancreatic cancer patients were divided into low-risk group(n=86)and high-risk group(n=85).Compared with the low-risk group,the high-risk group showed a significant negative enrichment trend for immune-related signaling pathways,the 5-year overall survival of pancreatic cancer patients was significantly increased in the low-risk group compared with the high-risk group.The expression of low-risk immune-related LncRNAs(AC064836.3,ZNF236-DT and TMEM161B-AS1)gradually decreased with increasing clinical stage of pancreatic cancer patients.Patient age(P=0.031,risk ratio and 95％CI:1.025/1.002-1.048)and prognostic risk score(P＜0.001,risk ratio and 95％ confidence interval 1.801/1.465-2.215)could be used as independent prognostic risk factors for overall survival in pancreatic cancer.In addition,the prognostic risk assessment model had better predictive efficiency(area under the curve=0.695)compared with the disease predictive ability of common clinical characteristics.Steroid biosynthesis,pentose phosphate pathway,intercellular linkage,cytoskeletal rearrangement and other pathways related to energy metabolism and invasive migration of pancreatic cancer cells were significantly activated in the high-risk group.Meanwhile,pancreatic cancer patients in the high-risk group had lower levels of naive B cells,plasma cells and neutrophils with anti-tumor activity,but their macrophage infiltration levels were significantly higher than those in the low-risk group.Conclusion The prognostic risk assessment model constructed based on five immune-related LncRNAs can effectively predict the survival status,clinical characteristics,molecular pathways,and immune cell infiltration differences of pancreatic cancer patients.Meanwhile,relying on this model,the prognosis of pancreatic cancer patients can be prospectively predicted,which enhances the usefulness of this risk prediction model.

Objective To observe the effect of pirfenidone on myocardial fibrosis in rats and inves-tigate its underlying mechanism.Methods Twenty-four SD rats were randomly divided into sham-operation group,model group,low-and high-dose pirfenidone groups,with 6 rats in each group.Rat model of myocardial fibrosis was established by injecting isoprenaline into the tail vein,while normal saline was given to the sham operation group.Pirfenidone of 150 and 300 mg/(kg·d)were infused gastrically to the rats of low-and high-dose pirfenidone groups after modeling.Mas-son staining was used to observe the severity of myocardial fibrosis,immunohistochemical assay was employed to detect the expression of Collagen-1,NOD-like receptor thermal protein domain associated protein 3(NLRP3)inflammasome,cysteinyl aspartate-specific protease-1(Caspase-1),and Western blotting was performed to detect the protein levels of Collogen-1 and atrial desmo-plakin D(gasdermin D,GSDMD).Results The model group showed obvious myocardial fibrosis,and elevated expression of Collogen-1,NLRP3,Caspase-1 and GSDMD when compared with the sham operation group(P＜0.05).Low-and high-dose pirfenidone treatment resulted in signifi-cantly reduced myocardial fibrosis and reduced expression of Collogen-1,NLRP3,Caspase-1 and GSDMD[(8.14±1.40)％,(6.56±0.75)％vs(22.15±2.57)％,P＜0.05;0.14±0.03 vs 0.33±0.05,0.42±0.13,P＜0.05;(10.34±1.40)％,(10.33±3.40)％vs(23.22±1.99)％,P＜0.05;(15.67±0.56)％,(17.33±0.78)％vs(22.87±1.92)％,P＜0.05;0.43±0.06,0.46±0.11 vs 0.65±0.03,P＜0.05].Conclusion Pirfenidone inhibits cardiomyocyte pyroptosis and attenuates myocar-dial fibrosis through the NLRP3/Caspase-1/GSDMD signaling axis.

Objective To screen immune-related long non-coding RNAs(LncRNAs)in the TCGA database pancreatic cancer dataset and construct a prognostic risk assessment model with immune-related LncRNAs to explore prognosis-related potential molecular mechanisms.Methods RNA-seq data of 171 pancreatic cancer samples and corresponding clinical information were obtained by The Cancer Genome Atlas(TCGA)database,and two classical immune-related gene datasets(GO0006955/IMMUNE RESPONSE and GO0002376/IMMUNE SYSTERM PROCESS)and gene annotation information were used to identify immune-related LncRNAs.The immune-related LncRNAs associated with pancreatic cancer prognosis were used for univariate and multivariate Cox analyses to establish a model for the assessment of pancreatic cancer prognostic risk based on immune-associated LncRNAs.This risk model was used for survival analysis,clinical correlation analysis,immune cell infiltration analysis,pathway enrichment analysis,and prognostic column line plot modeling.Results We screened 119 immune-related LncRNAs in pancreatic cancer,and five immune-related LncRNAs(AC064836.3,LINC00941,ZNF236-DT,TMEM161B-AS1 and AC068580.2)were identified for the development of pancreatic cancer prognostic risk assessment model.According to the prognostic risk assessment model,pancreatic cancer patients were divided into low-risk group(n=86)and high-risk group(n=85).Compared with the low-risk group,the high-risk group showed a significant negative enrichment trend for immune-related signaling pathways,the 5-year overall survival of pancreatic cancer patients was significantly increased in the low-risk group compared with the high-risk group.The expression of low-risk immune-related LncRNAs(AC064836.3,ZNF236-DT and TMEM161B-AS1)gradually decreased with increasing clinical stage of pancreatic cancer patients.Patient age(P=0.031,risk ratio and 95％CI:1.025/1.002-1.048)and prognostic risk score(P＜0.001,risk ratio and 95％ confidence interval 1.801/1.465-2.215)could be used as independent prognostic risk factors for overall survival in pancreatic cancer.In addition,the prognostic risk assessment model had better predictive efficiency(area under the curve=0.695)compared with the disease predictive ability of common clinical characteristics.Steroid biosynthesis,pentose phosphate pathway,intercellular linkage,cytoskeletal rearrangement and other pathways related to energy metabolism and invasive migration of pancreatic cancer cells were significantly activated in the high-risk group.Meanwhile,pancreatic cancer patients in the high-risk group had lower levels of naive B cells,plasma cells and neutrophils with anti-tumor activity,but their macrophage infiltration levels were significantly higher than those in the low-risk group.Conclusion The prognostic risk assessment model constructed based on five immune-related LncRNAs can effectively predict the survival status,clinical characteristics,molecular pathways,and immune cell infiltration differences of pancreatic cancer patients.Meanwhile,relying on this model,the prognosis of pancreatic cancer patients can be prospectively predicted,which enhances the usefulness of this risk prediction model.

Objective:To investigate the effect of circular RNA (circRNA)_005987 on contrast-associated acute kidney injury (CA-AKI) and its mechanism, and provide new ideas for the prevention and treatment of CA-AKI.Methods:CA-AKI rat models and HK-2 cell injury models were established using iopromide, and CA-AKI-related circRNA_005987 was screened based on circRNA expression chip and real-time quantitative PCR (RT-qPCR). Knockdown and overexpression of circRNA_005987 were performed in HK-2 cell model, respectively. Cell counting kit-8 (CCK-8) and Edu staining assays were used to evaluate cell proliferation. Western blotting was used to detect the protein expression of autophagy-related protein microtubule-associated protein 1 light chain 3B (LC3B), P62, beclin-1 and autophagy-related gene 14 (ATG14). Immunofluorescence staining was used to detect protein expression of LC3B. Electron microscope was used to observe the autophagosome formation. Autophagy activator rapamycin and autophagy inhibitor 3-methyladenine were used for in vitro rescue experiments to observe the changes of the above indicators. Mechanistically, bioinformatics analysis was applied to analyze the binding site among circRNA_005987, miR-129-5p and ATG14, and dual luciferase reporter assay was used to verify their interactions. CircRNA_005987 was knocked down and overexpressed in HK-2 cell model, and RT-qPCR was used to detect the expression of miR-129-5p. HK-2 cells were treated with miR-129-5p inhibitor and mimic, Western blotting was used to detect the protein expression of ATG14, and CCK8 and Edu staining assays were used to evaluate cell proliferation. Results:CircRNA_005987 expression was up-regulated in vitro and vivo CA-AKI models (both P<0.05). Overexpression of circRNA_005987 inhibited cell proliferation and promoted cell autophagy, while knockdown of circRNA_005987 had opposite effects (all P<0.05). In vitro rescue experiments confirmed that circRNA_005987 inhibited cell proliferation by activating autophagy ( P<0.05). The dual luciferase reporter assay suggested that there was an interaction between circRNA_005987, miR-129-5p and ATG14. Knockdown of circRNA_005987 increased miR-129-5p expression, while overexpression of circRNA_005987 inhibited miR-129-5p expression (both P<0.05). Knockdown of miR-129-5p inhibited cell proliferation, while overexpression of miR-129-5p reversed the effect (both P<0.05). Conclusion:CircRNA_005987 promotes CA-AKI through activating autophagy via sponging miR-129-5p, suggesting that circRNA_005987 plays an important role in the pathological process of CA-AKI.

Objective:To investigate the effect of circular RNA (circRNA)_005987 on contrast-associated acute kidney injury (CA-AKI) and its mechanism, and provide new ideas for the prevention and treatment of CA-AKI.Methods:CA-AKI rat models and HK-2 cell injury models were established using iopromide, and CA-AKI-related circRNA_005987 was screened based on circRNA expression chip and real-time quantitative PCR (RT-qPCR). Knockdown and overexpression of circRNA_005987 were performed in HK-2 cell model, respectively. Cell counting kit-8 (CCK-8) and Edu staining assays were used to evaluate cell proliferation. Western blotting was used to detect the protein expression of autophagy-related protein microtubule-associated protein 1 light chain 3B (LC3B), P62, beclin-1 and autophagy-related gene 14 (ATG14). Immunofluorescence staining was used to detect protein expression of LC3B. Electron microscope was used to observe the autophagosome formation. Autophagy activator rapamycin and autophagy inhibitor 3-methyladenine were used for in vitro rescue experiments to observe the changes of the above indicators. Mechanistically, bioinformatics analysis was applied to analyze the binding site among circRNA_005987, miR-129-5p and ATG14, and dual luciferase reporter assay was used to verify their interactions. CircRNA_005987 was knocked down and overexpressed in HK-2 cell model, and RT-qPCR was used to detect the expression of miR-129-5p. HK-2 cells were treated with miR-129-5p inhibitor and mimic, Western blotting was used to detect the protein expression of ATG14, and CCK8 and Edu staining assays were used to evaluate cell proliferation. Results:CircRNA_005987 expression was up-regulated in vitro and vivo CA-AKI models (both P<0.05). Overexpression of circRNA_005987 inhibited cell proliferation and promoted cell autophagy, while knockdown of circRNA_005987 had opposite effects (all P<0.05). In vitro rescue experiments confirmed that circRNA_005987 inhibited cell proliferation by activating autophagy ( P<0.05). The dual luciferase reporter assay suggested that there was an interaction between circRNA_005987, miR-129-5p and ATG14. Knockdown of circRNA_005987 increased miR-129-5p expression, while overexpression of circRNA_005987 inhibited miR-129-5p expression (both P<0.05). Knockdown of miR-129-5p inhibited cell proliferation, while overexpression of miR-129-5p reversed the effect (both P<0.05). Conclusion:CircRNA_005987 promotes CA-AKI through activating autophagy via sponging miR-129-5p, suggesting that circRNA_005987 plays an important role in the pathological process of CA-AKI.

Objective To study the role of microRNA(miR)-483-3p in reducing myocardial fibrosis in rats,and explore the relationship between its mechanism and autophagy.Methods A total of 24 male SD rats were randomly divided into sham operation group,model group,blank transfec-tion group and high expression group,with 6 rats in each group.The blank transfection group and the high-expression group were pretreated with a single injection of adeno-associated virus(AAV)-blank transfection and AAV-miR-483-3p(5×1011 vg)in the tail vein,respectively.In 14 d later,the sham group was injected with 2.5 ml/(kg·d)normal saline for 14 d,and rat model of myocardial fibrosis was established by 2 mg/ml isoproterenol[2.5 ml/(kg·d)]injection through tail vein for 14 consecutive days.Myocardial pathological damage,severity of myocardial fibrosis,and expression levels of collagen-Ⅰ,microtubule-associated protein light chain 3(LC3),autoph-agy-related protein 5(Atg5)and autophagy degradation substrate(P62)in cardiomyocytes were evaluated and measured.Results Compared with the sham operation group,the model group had obviously larger myocardial fibrosis area,higher positive expression of Collagen-Ⅰ,and increased protein levels of Atg5 and LC3-Ⅱ/LC3-Ⅰ,and decreased expression level of P62 protein(P＜0.05).The myocardial fibrosis area,positive expression of Collagen-Ⅰ,the expression levels of Atg5 and LC3-Ⅱ/LC3-Ⅰ protein[(13.64±1.51)％vs(27.47±1.55)％,(13.48±3.07)％vs(30.91±2.45)％,0.98±0.17 vs 1.24±0.28,0.66±0.05 vs 1.26±0.09,P＜0.05]were significant-ly decreased,and the expression level of P62 was notably increased(0.91±0.11 vs 0.74±0.06,P＜0.05)in the high expression group than the model group.Conclusion MiR-483-3p attenuates myocardial fibrosis in rats,and the mechanism may be related to the inhibition of cardiomyocyte autophagy.

Objective To explore the therapeutic effect of bicyclol(BIC)on rat model of myocardial fibrosis and its possible mechanism.Methods Twenty-four SPF male SD rats were randomly di-vided into sham group,model group,low-and high-dose groups,with 6 rats in each group.Except for the sham group,all other groups were injected with 5 mg/(kg·d)isoproterenol by tail vein to establish myocardial fibrosis model,and the low-and high-dose groups were administered by ga-vage with 100 and 200 mg/(kg·d)BIC,respectively for 14 consecutive days.HE staining and Masson staining were used respectively to observe the severity of myocardial injury and fibrosis.Western blot assay was employed to detect the protein expression of Collagen Ⅰ,Collagen Ⅲ,al-pha smooth muscle actin(α-SMA),methyltransferase-like protein 3(METTL3),α-ketoglutarate-dependent dioxygenase AlkB homolog 5(ALKBH5)and YTH domain family protein 1(YTHDF1)in rat myocardium.Results Compared with the sham group,myocardial cell necrosis and myocardial fibrosis were significantly more serious in the model group.Low-and high-dose BIC treatment reduced myocardial cell rupture and necrosis and myocardial fibrosis when com-pared with the model group.The expression levels of Collagen Ⅰ,Collagen Ⅲ,α-SMA,METTL3 and YTHDF1(P＜0.05)were significantly higher,and that of ALKBH5(0.58±0.02 vs 0.88±0.07,P＜0.05)was notably lower in the myocardial tissues of the model group than the sham group.While,both doses of BIC treatment significantly reversed above changes in protein levels(P＜0.05).Conclusion BIC can effectively alleviate myocardial structural damage and interstitial collagen deposition in rats with isoproterenol-induced myocardial fibrosis,and its mechanism may be related to m6A methylation modification.

OBJECTIVE To reveal the role of the basal forebrain(BF)GABAergic neurons in the regulation of isoflurane anesthesia and to elucidate the underlying neural pathways.METHODS The activity of BF GABAer-gic neurons was monitored during isoflurane anesthesia using a genetically encoded calcium indicator in Vgat-Cre mice of both sexes.The activity of BF GABAer-gic neurons was manipulated by chemogenetic and opto-genetic approaches.Sensitivity,induction time and emer-gence time of isoflurane anesthesia were estimated by righting reflex.The electroencephalogram(EEG)power and burst-suppression were monitored by EEG recording.The effects of activation of GABAergic BF-thalamic reticu-lar nucleus(TRN)pathway on isoflurane anesthesia were investigated with optogenetics.RESULTS The activity of BF GABAergic neurons was generally inhibited during isoflurane anesthesia,obviously decreased during the induction of anesthesia and gradually restored during the emergence from anesthesia.Activation of BF GABAergic neurons with chemogenetics and optogenetics promoted behavioral emergence from isoflurane anesthesia,with decreased sensitivity to isoflurane,delayed induction and accelerated emergence from isoflurane anesthesia.Optogenetic activation of BF GABAergic neurons prom-oted cortical activity during isoflurane anesthesia,with decreased EEG delta power and burst suppression ratio during 0.8%and 1.4%isoflurane anesthesia,respectively.Similar to the effects of activating BF GABAergic cell bod-ies,photostimulation of BF GABAergic terminals in the TRN also strongly promoted cortical activation and behav-ioral emergence from isoflurane anesthesia.CONCLU-SION The GABAergic neurons in the BF is a key neural substrate for general anesthesia regulation that facilitates behavioral and cortical emergence from general anesthe-sia via the BF-TRN pathway.