| Objective1.Using the research method of network pharmacology,we constructed the network map of Zelan-active ingredient-action target-disease,so as to find out the potential target of Zelan in preventing and treating knee osteoarthritis,and further explain the mechanism of Zelan’s intervention on knee osteoarthritis.2.By studying the expression of AQP1,AQP3,AQP9 and TNF-alpha,MMP3 and IL-17 in experimental SD rats with knee osteoarthritis combined with synovitis,the mechanism of early prevention and treatment of OA in Zelan was preliminarily explored,so as to find an effective target for early treatment of OA,providing data support for "blood disadvantage is water" and early prevention and treatment of OA with traditional Chinese medicine.On the basis of geographical theory.MethodsExperiment 1:Screening and prediction of possible bioactive components and targets of Zeeland by querying TCMSP database;obtaining relevant targets of disease by searching Genecards and OMIM;searching proteins through Uniprot database,constructing protein interactions by using String The PPI network uses Cytosacpe software to construct the Zeeland-Component-Target-Disease interaction network,using the Funrich tool for GO enrichment analysis and the R language for KEGG enrichment analysis.Experiment 2:1.Eighteen SPF male SD rats of 8 weeks old,weighing an average of 220-250 g,were randomly divided into sham operation group(group A),control group(group B),and experimental group Zelan low.The dose group(C),the experimental group Zeeland middle dose group(D),the experimental group Zeeland high dose group(E),18 in each group.3.Modeling method:(1)Sham operation group—after anesthesia,only the incision skin and knee joint capsule are exposed to the knee joint cavity,and then sutured layer by layer.(2)Control group—lateral knee incision,cross before cutting The ligament and medial collateral ligament,partial excision of the medial meniscus anterior horn,pre-draw drawer test,knee joint extension and valgus stress test confirmed that the ligament has been cut off,suture the knee joint cavity layer by layer;drive movement for 2 hours per day;(3)experimental group-After successful surgical modeling,different doses of Zeeland were administered to the stomach from the first day after modeling,and once a day,the stomach was administered to the end of the test,and the animals were sacrificed for examination.4.The gastric lavage was performed for 6 weeks from the first day of molding.The gastric lavage was as follows:A group was given daily saline of the same volume as the drug group.Group B was given daily saline of the same volume as group B.Group C was given Zelenia daily at 1/2 times the dose of clinical adults.Group D was given a daily dose of Zelenan equivalent to that of a clinical adult.Group E was given a daily dose of Zelenan equal to 2 times the dose for clinical adults.5.Six rats were randomly selected from each group at 2 weeks,4 weeks,and 6 weeks,and executed by means of spinal dislocation method.AQP1,AQP3,AQP9,MMF3,TNF-α,and IL-17 in synovial tissue of the left knee were determined.(1)Histomorphological observation:The intra-articular condition was observed by gross macroscopic examination.The Mankin method was used to evaluate the medial cartilage of the left femur,and the degree of lesion of knee osteoarthritis was determined according to the score.(2)ELISA was used to detect the expression of AQP1,AQP3,AQP9,MMF3,TNF-α,IL-17 in synovial tissue,and operated according to the kit instructions;(3)The joint edema index was measured by the foot volume method according to the degree of lesion between the control group and the control group,0 points:swelling degree≤5%;1 point:swelling degree ≤15%;2 points:15%<Swelling degree<30%;3 points:30%<severe swelling degree<60%;4 points:swelling degree>60%.(4)Statistical analysis6.Statistical analysis:Statistical analysis was performed using the statistical software SPSS 25.0.The measurement data in accordance with the normal distribution was expressed as mean ± standard deviation(x±s),and the median(quartile)was used in the normal distribution.)said.The Shapiro-Wilk test was used to test the normality of the data.For the normal distribution and the homogeneity of the variance,the one-way analysis of variance was used for comparison between groups.The LSD test was used for comparison between groups.The pair did not conform to the normal distribution.The Kruskal-Wallis test with nonparametric test was used.The Kruskal-Wallis one-way ANOVA was used to compare the two groups.The difference was statistically significant at P<0.05.Results1.The appearance of knee joint cartilage in SD rats of sham operation group was basically normal at each time point.The specific manifestations were:bright red color,smooth surface,no crack and softening and no signs of hyperplasia.The model group and Zeeland low,medium and high dose groups showed different degrees of joint degeneration,while the model group was more severe than the experimental group.2.There was no obvious inflammatory change in the synovial tissue of the knee joint in the sham operation group under the naked eye and light microscope.The synovial tissue of the knee joint in the model group had obvious inflammatory changes.The synovial membrane of the experimental group had inflammatory pathological changes compared with the blank group.,but significantly lighter than the model group.3.Gross cartilage observation results:There was no significant change in cartilage after sham operation group.The cartilage was observed significantly at 2 weeks,4 weeks,and 6 weeks,and the degree of cartilage degeneration was increased.Zeeland low,medium,and high dose groups Compared with the control group,the gross cartilage observation was changed and the degree of degeneration was reduced.4.Mankin score of cartilage:The cartilage morphology was significantly altered at weeks 2,4,and 6 in the model group,and the Mankin score increased over time.Mankin scores were lower in the Zelene-medium dose group at 2 weeks than in the model group(P<0.05)and in the Zelene-advanced dose group at 6 weeks than in the model group(P<0.05).5.The AQP1,AQP3 and AQP9 in the synovial tissue,the low,medium and high dose groups of Zeeland and the model group were lower than the model group at 2 weeks,4 weeks and 6 weeks(P<0.05).The difference was statistically significant.6.The content of MMP3 in synovial tissue was lower in the low,middle and high dose groups of Zeeland and the model group at 2 weeks,4 weeks and 6 weeks(P<0.05).The difference was statistically significant.7.The content of TNF-α in synovial tissue was lower in the low,middle and high dose groups of Zeeland and the model group at 2 weeks,4 weeks and 6 weeks(P<0.05).The difference was statistically significant.8.The content of IL-17 in synovial tissue was lower in the low,middle and high dose groups of Zeeland and the model group at 2 weeks,4 weeks and 6 weeks(P<0.05).The difference was statistically significant.9.Joint edema index:Compared with the sham operation group,the joint edema index of the model group was higher than that of the sham operation group at 2,4,and 6 weeks(P<0.05),2 weeks,6 weeks.The joint edema index was the lowest in the high-dose group of Zeeland and the model group(P<0.05).ConclusionZelan treatment of knee osteoarthritis has multi-component and multi-target characteristics;it may play a therapeutic role through P53 signaling pathway,IL-17 signaling pathway and TNF signaling pathway,which is consistent with the current mainstream therapeutic OA mechanism..Zelan can reduce the content of AQP1,AQP3,AQP9,IL-17,MMP3 and TNF-α in the synovial tissue of rats with KOA model,improve the inflammation of synovial tissue of knee osteoarthritis in rats,and delay the cartilage retreat.Change to improve joint function. |
PDF Full Text Request