Objective To explore the mechanism of Danggui Sini Decoction in treating knee osteoarthritis(KOA)by using network pharmacology,combined with in vitro experimental verification.Methods The active components of Danggui Sini Decoction were retrieved and screened through TCMSP,CNKI and PubMed databases,and their corresponding targets were predicted using the SwissTarget Prediction platform.KOA related targets were retrieved from GeneCards and OMIM databases.Intersection of drug targets and KOA targets was obtained,and key components,core targets,and their related biological mechanisms were identified through network topology analysis,GO and KEGG pathway enrichment analysis.Molecular docking was used to verify the degree of binding between key components and core targets.Danggui Sini Decoction containing serum was prepared,and an in vitro KOA model was constructed by inducing rat articular chondrocytes with lipopolysaccharide.The CCK-8 method was used to screen for the optimal concentration of drug containing serum intervention,fluorescent probes were used to detect intracellular ROS content,immunofluorescence was used to detect NF-κB p65 nuclear translocation,RT-qPCR was used to detect the expressions of IL-6,IL-1β and TNF-α mRNA,Western blot was used to detect the expression of Toll-like receptor 4(TLR4),myeloid differentiation factor 88(MyD88),NF-κB p65 and p-NF-κB p65 proteins.Results The results of network pharmacology analysis indicated that the treatment of KOA with Danggui Sini Decoction may involve regulating Toll like receptors,MAPK,TNF,apoptosis and other inflammatory and aging signaling pathways,compounds such as quercetin,kaempferol and glycyrrhizin may play important roles.Core targets included IL6,IL1B,TNF,TLR4,NFKB1,JUN,MAPK1,RELA.In vitro experiments showed that compared with the blank group,the ROS content in chondrocytes of the model group significantly increased(P<0.01),NF-κB p65 protein was significantly nuclear translocation(P<0.01),and mRNA expressions of IL-6,IL-1β,TNF-α mRNA and protein expressions of TLR4,MyD88 and p-NF-κB p65 significantly increased(P<0.01);compared with the model group,the intervention of Danggui Sini Decoction containing serum could significantly reduce intracellular ROS content(P<0.05),inhibit NF-κB p65 nuclear translocation(P<0.01),and reduce the expression of inflammatory factor mRNA and related proteins(P<0.05,P<0.01).Conclusion Danggui Sini Decoction can significantly reduce the inflammatory response of rat articular chondrocytes induced by lipopolysaccharide and exert therapeutic effects on KOA.Its mechanism may be related to the inhibition of TLR4/MyD88/NF-κB pathway.

Objective To explore the mechanism of Danggui Sini Decoction in treating knee osteoarthritis(KOA)by using network pharmacology,combined with in vitro experimental verification.Methods The active components of Danggui Sini Decoction were retrieved and screened through TCMSP,CNKI and PubMed databases,and their corresponding targets were predicted using the SwissTarget Prediction platform.KOA related targets were retrieved from GeneCards and OMIM databases.Intersection of drug targets and KOA targets was obtained,and key components,core targets,and their related biological mechanisms were identified through network topology analysis,GO and KEGG pathway enrichment analysis.Molecular docking was used to verify the degree of binding between key components and core targets.Danggui Sini Decoction containing serum was prepared,and an in vitro KOA model was constructed by inducing rat articular chondrocytes with lipopolysaccharide.The CCK-8 method was used to screen for the optimal concentration of drug containing serum intervention,fluorescent probes were used to detect intracellular ROS content,immunofluorescence was used to detect NF-κB p65 nuclear translocation,RT-qPCR was used to detect the expressions of IL-6,IL-1β and TNF-α mRNA,Western blot was used to detect the expression of Toll-like receptor 4(TLR4),myeloid differentiation factor 88(MyD88),NF-κB p65 and p-NF-κB p65 proteins.Results The results of network pharmacology analysis indicated that the treatment of KOA with Danggui Sini Decoction may involve regulating Toll like receptors,MAPK,TNF,apoptosis and other inflammatory and aging signaling pathways,compounds such as quercetin,kaempferol and glycyrrhizin may play important roles.Core targets included IL6,IL1B,TNF,TLR4,NFKB1,JUN,MAPK1,RELA.In vitro experiments showed that compared with the blank group,the ROS content in chondrocytes of the model group significantly increased(P<0.01),NF-κB p65 protein was significantly nuclear translocation(P<0.01),and mRNA expressions of IL-6,IL-1β,TNF-α mRNA and protein expressions of TLR4,MyD88 and p-NF-κB p65 significantly increased(P<0.01);compared with the model group,the intervention of Danggui Sini Decoction containing serum could significantly reduce intracellular ROS content(P<0.05),inhibit NF-κB p65 nuclear translocation(P<0.01),and reduce the expression of inflammatory factor mRNA and related proteins(P<0.05,P<0.01).Conclusion Danggui Sini Decoction can significantly reduce the inflammatory response of rat articular chondrocytes induced by lipopolysaccharide and exert therapeutic effects on KOA.Its mechanism may be related to the inhibition of TLR4/MyD88/NF-κB pathway.

Knee osteoarthritis （KOA） is a common degenerative joint disease in the middle-aged and elderly. The incidence of KOA is rising as the population aging aggravates and the obese population grows. KOA seriously affects the health and daily life of the patients. The commonly used drugs for the symptomatic treatment of KOA include non-steroidal anti-inflammatory drugs， cartilage protective drugs， and opioid analgesics， which have limited therapeutic effects and induce obvious adverse drug reactions. Eucommiae Cortex is one of the commonly used Chinese herbal medicines for the treatment of KOA， while its pharmacological material basis and mechanism remain unclear， which limits its clinical application. The active ingredients of Eucommiae Cortex for treating KOA mainly include iridoids （geniposide， aucubin）， lignans （pinoresinol diglucoside）， flavonoids （quercetin， astragaloside， baicalein， hyperoside， and kaempferol）， phenylpropanoids （chlorogenic acid）， and polysaccharides. These compounds regulate the levels of inflammatory cytokines， inhibit oxidative stress， protect chondrocytes， balance the synthesis and degradation of extracellular matrix， and control the progression of KOA via the mitogen-activated protein kinase， nuclear factor-κB， phosphatidylinositol-3-kinase/protein kinase B， and Janus kinase 1/signal transducer and activator of transcription 3 signaling pathways. This paper introduces the mechanisms of Eucommiae Cortex and its active components in the treatment of KOA， aiming to provide a theoretical basis for the development of new drugs for KOA.

Objective:To select nucleic acid extraction reagents and amplification reagents by comparing the minimum detection limit, amplification efficiency, specificity, accuracy, and matching analysis with 6 nucleic acid extraction reagents of 6 novel coronavirus (2019-nCoV) nucleic acid detection reagents and provide scientific basis for clinical diagnosis and treatment.Methods:The national-standard material of 2019-nCoV nuclear acids was diluted as ths six fradient concentrations. Then, the diluted samples were analyzed by using the six kinds of agents/kits, Guangzhou Daan (DA), Beijing Jinhao (JH), Wuhan Mingde (MD), Sichuan Mike (MK), Jiangsu Shuoshi (SS) and Shanghai Zhijiang (ZJ). The minimum detection limit, amplification efficiency, accuracy, specificity and other properties of these kits were examined. The positive control levels 1, 2 (low and medium concentration) were extracted by using the six nucleic acid extraction reagents (numbered a-f) with six 2019-nCoV detective kits to detect target genes of 2019-nCoV genome, and compared between different nucleic acid extraction reagents and different nucleic acid detection reagents. Match between the different nucleic acid extraction reagents detection reagents, using two-way analysis of variance.Results:The minimum detection limit of Nucleocapsid (N) gene of DA, JH, MD, SS reagent was 7.0×10 2 copies/ml.The minimum detection limit of open reading frame 1ab (ORF1ab) gene of JH, MD, SS, MK reagent was 9.39 ×10 2 copies/ml.The minimum detection limit of envelope (E) gene of MK, ZJ reagent was 5.03×10 2 copies/ml. SS reagent N gene amplification efficiency was 89%, ORF1ab gene amplification efficiency was 90%, was the highest among six reagents.a, b, c, d extraction reagents and 6 nucleic acid detection reagents match well, But a extraction reagent was more suitable for use with SS,MK and ZJ reagents ( P<0.05), d extraction reagent was more suitable for MD,SS,DA,JH and MK nucleic acid detection reagents( P<0.05); e extraction reagent was more suitable for SS nucleic acid detection reagents( P<0.05), f extraction reagent was most suitable for DA, JH Nucleic acid detection reagents ( P<0.05); e and f reagents were not suitable for use with MK and ZJ. Conclusion:The 6 detection reagents have good performance, and the appropriate extraction reagents and detection reagents should be used in conjunction with the actual conditions of the laboratory.