Aim To construct and analyze the genotype of G protein-coupled receptor kinase 2(GRK2)conditional knockout mice in synoviocytes,and to provide an animal model for stud-ying the function of GRK2 in synoviocytes.Methods Grk2flox/+mice were bred to generate Grk2flox/flox mice,Grk2flox/flox mice were bred to Col1a1-iCre+mice,Grk2flox/+Col1a1-iCre+mice were bred to Grk2flox/flox mice.Grk2flox/flox Col1a1-iCre+mice were ob-tained as target mice.DNA was extracted and amplified by PCR to identify the genotype.Western blot was used to verify the effect of Grk2 knockout in synovium,liver and kidney tissues.HE staining was used to detect the effects of Grk2 conditional knockout in synovial cells on ankle synovium,liver and kidney tissues.Multiple immunofluorescence was used to detect GRK2 expression in synovial cells.Results The results of gene iden-tification showed that Grk2flox/flox Col1a1-iCre+mice had both Flox and Col1a1-iCre genotypes.Western blot results showed that GRK2 expression decreased in synovial tissues of Grk2flox/flox Col1a1-iCre+mice,but there was no significant change in the expression of GRK2 in liver and kidney tissues.HE staining showed that Grk2flox/flox Col1a1-iCre+mice had no significant pathological changes in the ankle synovium,liver and kidney.The results of multiple immunofluorescence showed that GRK2 expression in synovial cells of Grk2flox/flox Col1a1-iCre+mice de-creased.Conclusion Grk2 conditional knockout mice in syno-viocytes are successfully constructed and identified,which pro-vides an animal model for further study of the role of GRK2 in synovial-related diseases.

Objective:To compare the relative proportion of direct action (ray particles directly destroy biological molecules such as DNA and indirect action (radical-mediated oxidative damage) in the damage caused by X-ray and proton irradiation of lung cancer cells.Methods:Unirradiated human lung adenocarcinoma A549 cells and human large cell lung cancer NCI-H460 cells were cultured in media containing 0, 0.125, 0.25, 0.5, 0.75 mol/L dimethyl sulfoxide (DMSO) for 1 h to obtain plating efficiency (PE) values, thereby determining whether DMSO affected cell survival. Following pretreatment with each DMSO concentration, cells were exposed to X-ray irradiation at physical doses of 2, 4, 6, 8 Gy and proton irradiation at equivalent doses of 2, 4, 6, 8 GyE, respectively. Survival fractions (SF) and maximum protection (MP) values were calculated to evaluate the effects of varying DMSO concentrations on post-irradiation cell survival and to quantify the contribution of indirect radiation damage mechanisms (higher MP indicates greater indirect effect contribution). PE, SF, and MP values were determined using clonogenic assays. Comparisons among multiple groups were performed using one-way ANOVA followed by Tukey's multiple comparison, and comparisons between irradiation groups were analyzed using independent samples t-tests. Results:The PE of unirradiated cells treated with varying DMSO concentrations showed no statistically significant differences. Following pretreatment at different DMSO concentrations and subsequent irradiation with X-rays or protons, the protective effect of DMSO reached saturation at 0.5 mol/L. At this concentration, comparison of the average MP values across 4 radiation doses revealed: In A549 cells, the MP value was 54.21%±1.73% for X-ray irradiation group and 39.69%±0.72% for proton irradiation group ( t=16.82, P<0.001); in NCI-H460 cells, the MP value was 52.04%±1.00% for X-ray irradiation group and 41.31%±0.70% for proton irradiation group ( t=10.19, P<0.001). Conclusions:Under biologically equivalent doses, proton irradiation demonstrates greater reliance on direct effects in lung cancer cells killing compared with X-ray irradiation.

Objective:To compare the relative proportion of direct action (ray particles directly destroy biological molecules such as DNA and indirect action (radical-mediated oxidative damage) in the damage caused by X-ray and proton irradiation of lung cancer cells.Methods:Unirradiated human lung adenocarcinoma A549 cells and human large cell lung cancer NCI-H460 cells were cultured in media containing 0, 0.125, 0.25, 0.5, 0.75 mol/L dimethyl sulfoxide (DMSO) for 1 h to obtain plating efficiency (PE) values, thereby determining whether DMSO affected cell survival. Following pretreatment with each DMSO concentration, cells were exposed to X-ray irradiation at physical doses of 2, 4, 6, 8 Gy and proton irradiation at equivalent doses of 2, 4, 6, 8 GyE, respectively. Survival fractions (SF) and maximum protection (MP) values were calculated to evaluate the effects of varying DMSO concentrations on post-irradiation cell survival and to quantify the contribution of indirect radiation damage mechanisms (higher MP indicates greater indirect effect contribution). PE, SF, and MP values were determined using clonogenic assays. Comparisons among multiple groups were performed using one-way ANOVA followed by Tukey's multiple comparison, and comparisons between irradiation groups were analyzed using independent samples t-tests. Results:The PE of unirradiated cells treated with varying DMSO concentrations showed no statistically significant differences. Following pretreatment at different DMSO concentrations and subsequent irradiation with X-rays or protons, the protective effect of DMSO reached saturation at 0.5 mol/L. At this concentration, comparison of the average MP values across 4 radiation doses revealed: In A549 cells, the MP value was 54.21%±1.73% for X-ray irradiation group and 39.69%±0.72% for proton irradiation group ( t=16.82, P<0.001); in NCI-H460 cells, the MP value was 52.04%±1.00% for X-ray irradiation group and 41.31%±0.70% for proton irradiation group ( t=10.19, P<0.001). Conclusions:Under biologically equivalent doses, proton irradiation demonstrates greater reliance on direct effects in lung cancer cells killing compared with X-ray irradiation.

Aim To construct and analyze the genotype of G protein-coupled receptor kinase 2(GRK2)conditional knockout mice in synoviocytes,and to provide an animal model for stud-ying the function of GRK2 in synoviocytes.Methods Grk2flox/+mice were bred to generate Grk2flox/flox mice,Grk2flox/flox mice were bred to Col1a1-iCre+mice,Grk2flox/+Col1a1-iCre+mice were bred to Grk2flox/flox mice.Grk2flox/flox Col1a1-iCre+mice were ob-tained as target mice.DNA was extracted and amplified by PCR to identify the genotype.Western blot was used to verify the effect of Grk2 knockout in synovium,liver and kidney tissues.HE staining was used to detect the effects of Grk2 conditional knockout in synovial cells on ankle synovium,liver and kidney tissues.Multiple immunofluorescence was used to detect GRK2 expression in synovial cells.Results The results of gene iden-tification showed that Grk2flox/flox Col1a1-iCre+mice had both Flox and Col1a1-iCre genotypes.Western blot results showed that GRK2 expression decreased in synovial tissues of Grk2flox/flox Col1a1-iCre+mice,but there was no significant change in the expression of GRK2 in liver and kidney tissues.HE staining showed that Grk2flox/flox Col1a1-iCre+mice had no significant pathological changes in the ankle synovium,liver and kidney.The results of multiple immunofluorescence showed that GRK2 expression in synovial cells of Grk2flox/flox Col1a1-iCre+mice de-creased.Conclusion Grk2 conditional knockout mice in syno-viocytes are successfully constructed and identified,which pro-vides an animal model for further study of the role of GRK2 in synovial-related diseases.

Objective:To explore the effects of hydroxychloroquine(HCQ)on immune mediators dysregulation in severe infection after chemotherapy in acute myeloid leukemia(AML)and its molecular mechanism.Methods:Bone marrow or peripheral blood samples of 36 AML patients with severe infection(AML-SI)and 29 AML patients without infection(AML-NI)after chemotherapy were collected from the First Affiliated Hospital of Gannan Medical University from August 2022 to June 2023.In addition,the peripheral blood of 21 healthy subjects from the same period in our hospital was selected as the control group.The mRNA expressions of CXCL12,CXCR4 and CXCR7 were detected by RT-qPCR technology,and the levels of IL-6,IL-8 and TNF-α were detected by ELISA.Leukemia-derived THP-1 cells were selected and constructed as AML disease model.At the same time,bone marrow mesenchymal stem cells(BM-MSCs)from AML-SI patients were co-cultured with THP-1 cells and divided into Mono group and Co-culture group.THP-1 cells were treated with different concentration gradients of HCQ.The cell proliferation activity was subsequently detected by CCK-8 method and apoptosis was detected by Annexin V/PI double staining flow cytometry.ELISA was used to detect the changes of IL-6,IL-8 and TNF-α levels in the supernatant of the cell co-culture system,RT-qPCR was used to detect the mRNA expression changes of the core members of the CXCL12-CXCR4/7 regulatory axis,and Western blot was used to detect the expressions of apoptosis regulatory molecules and related signaling pathway proteins.Results:CXCL12,CXCR4,CXCR7,as well as IL-6,IL-8,and TNF-α were all abnormally increased in AML patients,and the increases were more significant in AML-SI patients(P＜0.01).Furthermore,there were statistically significant differences between AML-NI patients and AML-SI patients(all P＜0.05).HCQ could inhibit the proliferation and induce the apoptosis of THP-1 cells,but the low concentration of HCQ had no significant effect on the killing of THP-1 cells.When THP-1 cells were co-cultured with BM-MSCs of AML patients,the levels of IL-6,IL-8 and TNF-α in the supernatance of Co-culture group were significantly higher than those of Mono group(all P＜0.01).After HCQ intervention,the levels of IL-6,IL-8 and TNF-α in cell culture supernatant of Mono group were significantly decreased compared with those before intervention(all P＜0.01).Similarly,those of Co-culture group were also significantly decreased(all P＜0.001).However,the expression of the core members of the CXCL12-CXCR4/7 regulatory axis was weakly affected by HCQ.HCQ could up-regulate the expression of pro-apoptotic protein Bax,down-regulate the expression of anti-apoptotic protein Bcl-2,as well as simultaneously promote the hydrolytic activation of Caspase-3 when inhibiting the activation level of TLR4/NF-κ B pathway,then induce the programmed death of THP-1 cells after intervention.Conclusion:The core members of CXCL12-CXCR4/7 axis and related cytokines may be important mediators of severe infectious immune disorders in AML patients.HCQ can inhibit cytokine levels to reverse immune mediators dysregulation and suppress malignant biological characteristics of leukemia cells.The mechanisms may be related to regulating the expression of Bcl-2 family proteins,hydrolytically activating Caspase-3 and inhibiting the activation of TLR4/NF-κ B signaling pathway.

Objective To analyze the literature related to diphenidol tablets poisoning,the characteristics of poisoning were summarized to provide reference for controlling the suicidal abuse risk of diphenidol tablets.Methods The global literature on suicide,overdose,poisoning,shock,and death related to difenidol published from January 1,2011 to December 31,2022 was analyzed,including gender,age,dosage,cardiac(blood)concentration,poisoning symptoms,etc.Results Young women were the majority of people with poisoning.The highest proportion of the age group is 11 to 30 years group.Patients who take medication doses greater than 3 000 mg may have a higher risk of death;patients with a heart(blood)concentration greater than 6 μg·mL-1 may have a higher risk of death.Malignant arrhythmia,consciousness disorders,coma,and apnea are common serious adverse events during poisoning.Conclusion It is recommended that the drug regulatory authorities should require the Listing permit holder of difenidol tablets to add the risk and symptoms of poisoning into the instructions.It is suggested that restricting individual consumers from purchasing large amounts of difenidol tablets in the short term.It is recommended that canceling the high-dose sales packaging of difenidol tablets.It is suggested that converting difenidol tablets into prescription drugs,even consider canceling the registration certificate of difenidol tablets.

Objective To explore the intervention effect and mechanism of Zhiwei Fuwei pills on precancerous lesions of gastric cancer(PLGC)model rats based on hypoxia-inducible factor-1α(HIF-1α)/vascular endothelial growth factor(VEGF)signaling pathway.Methods PLGC model rats were established by the combination of N-methyl-N'-nitro-N-nitrosoguanidine and ammonia drinking,hunger and satiation disorder,ethanol gavage and ranitidine feeding,and were randomly divided into model group,control group and experimental-H,-M,-L groups,with 8 rats in each group.Another 8 healthy rats were selected as blank group.Experimental-H,-M,-L groups were given 1.67,0.84 and 0.42 g·kg-1 Zhiwei Fuwei pills solution by intragastric administration,respectively;control group was given 2.00 × 10-3g·kg-1 folic acid solution by intragastric administration;blank group and model group were given 0.9％NaCl by intragastric administration,once a day for 4 weeks.The structural changes of gastric microvessels were detected by transmission electron microscopy,and the expression levels of HIF-1α,VEGF,vascular endothelial growth factor receptor-2(VEGFR-2)and Angiopoietin-2(Ang-2)were detected by western blot.Results Compared with the model group,the abnormality of gastric microvascular structure in experimental-H,-M,-L groups was improved to some extent,and the effect of high dose experimental group was the most obvious.The relative expression levels of HIF-1 α protein in experimental-H,-M groups and control group,model group and blank group were 0.43±0.03,0.66±0.04,0.77±0.01,0.80±0.02 and 0.37±0.02;the relative expression levels of VEGF protein were 0.97±0.06,1.21±0.06,1.51±0.07,1.54±0.05 and 0.88±0.02;the relative expression levels of VEGFR-2 protein were 1.03±0.06,1.18±0.04,1.37±0.05,1.45±0.02 and 0.70±0.02;the relative expression levels of Ang-2 protein were 0.51±0.03,0.61±0.02,0.71±0.01,0.78±0.03 and 0.34±0.01,respectively.Compared with the model group,the above indexes in the experimental-H,-M groups were statistically significant(all P＜0.01).Conclusion Zhiwei Fuwei pills may inhibit the abnormal activation of HIF-1α/VEGF signaling pathway,improve hypoxia microenvironment,reduce angiogenesis,and then effectively interfere with the progression of PLGC.

Objective To explore the intervention effect and mechanism of Zhiwei Fuwei pills on precancerous lesions of gastric cancer(PLGC)model rats based on hypoxia-inducible factor-1α(HIF-1α)/vascular endothelial growth factor(VEGF)signaling pathway.Methods PLGC model rats were established by the combination of N-methyl-N'-nitro-N-nitrosoguanidine and ammonia drinking,hunger and satiation disorder,ethanol gavage and ranitidine feeding,and were randomly divided into model group,control group and experimental-H,-M,-L groups,with 8 rats in each group.Another 8 healthy rats were selected as blank group.Experimental-H,-M,-L groups were given 1.67,0.84 and 0.42 g·kg-1 Zhiwei Fuwei pills solution by intragastric administration,respectively;control group was given 2.00 × 10-3g·kg-1 folic acid solution by intragastric administration;blank group and model group were given 0.9％NaCl by intragastric administration,once a day for 4 weeks.The structural changes of gastric microvessels were detected by transmission electron microscopy,and the expression levels of HIF-1α,VEGF,vascular endothelial growth factor receptor-2(VEGFR-2)and Angiopoietin-2(Ang-2)were detected by western blot.Results Compared with the model group,the abnormality of gastric microvascular structure in experimental-H,-M,-L groups was improved to some extent,and the effect of high dose experimental group was the most obvious.The relative expression levels of HIF-1 α protein in experimental-H,-M groups and control group,model group and blank group were 0.43±0.03,0.66±0.04,0.77±0.01,0.80±0.02 and 0.37±0.02;the relative expression levels of VEGF protein were 0.97±0.06,1.21±0.06,1.51±0.07,1.54±0.05 and 0.88±0.02;the relative expression levels of VEGFR-2 protein were 1.03±0.06,1.18±0.04,1.37±0.05,1.45±0.02 and 0.70±0.02;the relative expression levels of Ang-2 protein were 0.51±0.03,0.61±0.02,0.71±0.01,0.78±0.03 and 0.34±0.01,respectively.Compared with the model group,the above indexes in the experimental-H,-M groups were statistically significant(all P＜0.01).Conclusion Zhiwei Fuwei pills may inhibit the abnormal activation of HIF-1α/VEGF signaling pathway,improve hypoxia microenvironment,reduce angiogenesis,and then effectively interfere with the progression of PLGC.

Precancerous lesions of gastric cancer(PLGC)is a key pathological stage in the process of inflammation cancer transformation of the gastric mucosa.Early diagnosis and effective intervention in this stage have positive significance in preventing the occurrence of gastric cancer.Numerous studies have confirmed that traditional Chinese medicine can effectively intervene in PLGC by regulating cell proliferation and apoptosis,regulating inflammatory response,anti angiogenesis,inhibiting epithelial mesenchymal transition,inhibiting glycolysis,antioxidant stress,combating helicobacter pylori,regulating autophagy levels,and regulating gut microbiota through various pathways.The article systematically elaborates on the research status of the intervention mechanism of traditional Chinese medicine monomers or extracts,and traditional Chinese medicine formulas in PLGC,aiming to provide reference for the clinical treatment of this disease and related drug development.