| ObjectiveKnee osteoarthritis is a common chronic disease of the elderly,if the patient can not be treated effectively in the early stage,there may be a higher rate of disability in the later stage.Du Huo Ji Sheng Tang is a classic knee osteoarthritis prescription,has been widely used in clinical with good clinical results.Many scholars have confirmed that NLRP3/NF-?B signaling pathway is associated with rheumatoid arthritis and gouty arthritis,but whether this pathway is associated with knee osteoarthritis has not been confirmed.At the same time,Whether Du Huo Ji Sheng Tang can regulate NLRP3/NF-?B signaling pathway also needs to be studied.This experiment focuses on the NLRP3/NF-?B signaling pathway,from four aspects including clinical trial,in vivo experiment,in vitro experiment and analysis of active component,to research the correlation between knee osteoarthritis and NLRP3/NF-?B signaling pathway and if Du Huo Ji Sheng Tang can treat knee osteoarthritis by repressing inflammation via NLRP3/NF-?B signaling pathway.Methods1.Clinical trial:Serum of 15 healthy adults were collected.A total of 60 knee osteoarthritis patients with liver and kidney deficiency type were randomly divided into Du Huo Ji Sheng Tang group and Diclofenac Sodium Enteric-coated Tablets group,with 30 cases in each group.The patients were treated with drugs for 3 weeks.Before the treatment and after 3 weeks of the treatment,serum and synovial fluid of patients with knee osteoarthritis were collected.IL-1?,IL-6,IL-10,TNF-a,NLRP3,ASC,Caspase-1,p-NF-KB-P65 and p-I?Ba level were tested by ELISA kit in serum of healthy adults and serum and synovial fluid of patients with knee osteoarthritis.2.In vivo experiment:Fifty healthy SD rats were randomly divided into normal group,model group,diclofenac sodium group(5mg/kg),Du Huo Ji Sheng Tang low dose group(0.5g/kg)and Du Huo Ji Sheng Tang high dose group(lg/kg).Except the normal group,right knee joints of the rest group were modeled.Drainage method was used to determine the swollen volume of the rats' feet in each group on the 7,14,21 day after treatment.On the 21 day after treatment,blood was taken from the eyelid and synovial fluid was taken from the knee joint in each group,the percentage of white blood cells was counted and stated.The knee synovium was taken from the knee joint in each group,HE staining was used to evaluate the pathological changes.Blood was taken from the carotid arteries and the synovial fluid was taken from the knee joint,the levels of IL-6,IL-1? and TNF-? were tested by ELISA kit in serum and synovial fluid.The knee synovium was taken from the knee joint,the protein expressions of NLRP3?ASC?Caspase-1?IL-1??p-NF-?B-P65?NF-?B-P65?p-I?Ba?I?Ba were detected by Western blotting.3.In vitro experiment:Eighty healthy SD rats were randomly divided into 4 groups:normal group,diclofenac sodium group(5mg/kg),Du Huo Ji Sheng Tang low dose group(0.5g/kg)and Du Huo Ji Sheng Tang high dose group(1g/kg),in order to prepare serum containing drugs.Ten 24h neonatal female SD rats were used for primary chondrocyte culture.The chondrocytes were randomly divided into normal group(10%rat serum from normal group),model group(8?g/mL lipopolysaccharide+ 10%rat serum from normal group),serum containing group 1(8?g/mL lipopolysaccharide + 10%Diclofenac sodium-containing serum),serum containing group 2(8?g/mL lipopolysaccharide + 10%Du Huo Ji Sheng Tang 0.5g/kg-containing serum),serum containing group 3(8?g/mL lipopolysaccharide +10%Du Huo Ji Sheng Tang 1 g/kg-containing serum).The chondrocytes were stimulated for 24 hours.Cell viability was measured by MTT assay.SOD activity and MDA content in the supernatant of the cells were detected by assay kits.IL-6,IL-1?and TNF-a in the cell supernatant were determined by ELISA kits.The protein expressions of NLRP3?ASC?Caspase-1?IL-1??p-NF-?B-P65?NF-?B-P65?p-I?Ba?I?Ba in chondrocytes were detected by Western blotting.4.Analysis of active component:Combined with the relevant reference materials,we have screened out the four main components of Du Huo Ji Sheng Tang,namely glycyrrhizic acid,pachyrin acid,paeoniflorin and gentianine.MTT was used to detect the viability of chondrocytes after different active components were treated.In vivo experiments were performed to replicate the model of knee osteoarthritis.The rats were divided into normal group,model group,diclofenac sodium(5mg/kg)group,gentianine low dose group(20mg/kg)and high dose group(40mg/kg).Drainage method was used to determine the swollen volume of the rats' feet in each group on the 7,14,21 day after treatment.On the 21 day after treatment,the knee synovium was taken from the knee joint in each group,HE staining was used to evaluate the pathological changes.Blood was taken from the carotid arteries and the synovial fluid was taken from the knee joint,the levels of IL-6,IL-1 ? and TNF-a were tested by ELISA kit in serum and synovial fluid.Results1.Clinical trial:Compared with healthy adults,serum levels of IL-1?,IL-6,TNF-a,NLRP3,ASC,Caspase-1,p-NF-KB-P65,p-I?Ba were significantly increased(P<0.01),the level of IL-10was significantly decreased(P<0.01)in knee osteoarthritis patients with liver and kidney deficiency type.After treated,the levels of IL-1 ?,IL-6,TNF-a,NLRP3,ASC,Caspase-1,p-NF-KB-P65 and p-I?Ba in serum and synovial fluid of patients with knee osteoarthritis were significantly decreased(P<0.O1),while IL-10 was increased(P<0.01).2.In vivo experiment:Compared with the normal group,the swelling volume of the feet in the model group increased significantly(P<0.01).The degree of swelling of the feet in the Du Huo Ji Sheng Tang group decreased gradually,and after 2 weeks and 3 weeks of the treatment,compared with the model group,it decreased significantly(P<0.01).Compared with the normal group,the percentages of lymphocytes,neutrophils and eosinophils were significantly increased in the blood of knee osteoarthritis model rats(P<0.01).After the treatment of Du Huo Ji Sheng Tang or diclofenac sodium,the percentages of lymphocytes,neutrophils and eosinophils decreased significantly(P<0.01).Compared with the normal group,the percentage of leukocyte(P<0.01),lymphocyte(P<0.01),neutrophil(P<0.01),eosinophil(P<0.01)in synovial fluid increased significantly.The percentages of leukocyte,lymphocytes,neutrophils and eosinophils in the synovial fluid were significantly decreased after treatment with Du Huo Ji Sheng Tang or diclofenac sodium(P<0.01).Compared with the normal group,the model group rats' knee joint synovial tissue has hyperplasia in surface,showing significant inflammatory cell infiltration,synovial vascular congestion,edema,cell proliferation.Compared with the model group,the synovial swelling and hyperemia and the proliferation of synovial cells were relieved in treatment group,the same as the inflammatory cell infiltration and vascular proliferation.The diclofenac sodium(5mg/kg)group was more effective.Compared with the normal group,the levels of IL-6,IL-1? and TNF-a in serum and synovial fluid of knee osteoarthritis rats were significantly increased(P<0.01).The levels of IL-6,IL-1? and TNF-a in serum and synovial fluid were significantly decreased(P<0.01)after treated with Du Huo Ji Sheng Tang or diclofenac sodium.The protein expressions of NLRP3,ASC,Caspase-1,IL-1?,p-NF-?B-P65 and p-I?Ba in synovial tissue of knee osteoarthritis rats were significantly increased.The values of p-NF-?B-P65/NF-?B-P65 and p-I?Ba/I?Ba were significantly increased(P<0.01).The expressions of NLRP3/NF-?B signaling pathway related proteins were significantly decreased(P<0.01)after treated with Du Huo Ji Sheng Tang or diclofenac sodium.3.In vitro experiment:The survival rate of chondrocytes in model group decreased significantly after LPS induction(P<0.01).The survival rate of chondrocytes was significantly increased in each drug-containing serum group(P<0.01).After LPS induction,the activity of SOD in the chondrocyte supernatant was significantly decreased(P<0.01)and MDA content was significantly increased(P<0.01).The drug-containing serum can decrease the SOD activity and reduce the content of MDA(P<0.01).The levels of IL-6,IL-1? and TNF-? in the supernatant of chondrocytes in model group were significantly increased after LPS induction(P<0.01),and all the drug-containing serum groups significantly reduced the levels of IL-6,IL-1? and TNF-a in the chondrocyte supernatant(P<0.01).The expressions of NLRP3,ASC,Caspase-1,IL-1?,p-NF-?B-P65 and p-I?Ba were significantly increased in LPS-treated chondrocytes compared with normal cells,the values of p-NF-?B-P65/NF-?B-P65 and p-I?Ba/I?Ba were significantly increased(P<0.01).The drug-containing serums significantly depressed the expressions of NLRP3/NF-?B signaling pathway related proteins(P<0.01).4.Analysis of active component:Compared with the normal group,LPS-induced model group chondrocyte cell survival rate significantly reduced.After given four active components of Du Huo Ji Sheng Tang,including glycyrrhizic acid,pachyrin acid,paeoniflorin and gentianine,the cell activity of each group was significantly increased(P<0.01),especially the gentianine.Compared with the normal group,the swelling volume of the feet in the model group increased significantly(P<0.01).The degree of swelling of the feet in the gentianine group decreased gradually,and after 2 weeks and 3 weeks of the treatment,compared with the model group,it decreased significantly(P<0.01).Compared with the normal group,the model group rats' knee joint synovial tissue has hyperplasia in surface,showing significant inflammatory cell infiltration,synovial vascular congestion,edema,cell proliferation.Compared with the model group,the synovial swelling and hyperemia and the proliferation of synovial cells were relieved in treatment group.The diclofenac sodium(5mg/kg)group was more effective.Compared with the normal group,the levels of IL-6,IL-1?and TNF-a in serum and synovial fluid of knee osteoarthritis rats were significantly increased(P<0.01).The levels of IL-6,IL-1? and TNF-? in serum and synovial fluid were significantly decreased(P<0.01)after treated with gentianine or diclofenac sodium.Conclusion1.The NLRP3/NF-?B signaling pathway was significantly activated in knee osteoarthritis patients with liver and kidney deficiency type.The levels of NLRP3/NF-?B signaling pathway related factors such as IL-1??IL-6?TNF-??NLRP3?ASC?Caspase-1?p-NF-?B-P65?p-I ? Ba in serum and synovial fluid of the patients with knee osteoarthritis were significantly decreased by Du Huo Ji Sheng Tang,while IL-10 increased.NLRP3/NF-?B signaling pathway was significantly suppressed by Du Huo Ji Sheng Tang.2.Du Huo Ji Sheng Tang has a good therapeutic effect for knee osteoarthritis rats,it can relieve feet swelling of knee osteoarthritis in rats and reduce the percentages of inflammatory cells and the levels of IL-6,IL-1? and TNF-?,and attenuated the synovial inflammation of the joints.Du Huo Ji Sheng Tang maybe can inhibite the protein expressions of NLRP3/NF-?B signaling pathway-related factors and reduced the inflammation of the knee osteoarthritis rats.3.Du Huo Ji Sheng Tang has a remarkable protective effect on LPS-induced chondrocytes by inhibiting the inflammation.4.The four main active ingredients of Du Huo Ji Sheng Tang,including glycyrrhizic acid,pachyrin acid,paeoniflorin and gentianine,have a protective effect on LPS-induced chondrocytes.In vivo experiment,gentianine has an inflammatory inhibition effect on knee osteoarthritis rats.The inhibiting effect on the inflammation of gentianine was similar to Du Huo Ji Sheng Tang. |
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