| Knee osteoarthritis is one of the common diseases of middle and old people in China and even in the world,and its occurrence and development process is closely related to age,weight,trauma,exercise habits,geographical environment and many other factors[1].The pathological process includes wear of articular cartilage,degeneration of ligaments,inflammation of surrounding synovium and other pathological changes[2].The occurrence of knee osteoarthritis has caused great pain and inconvenience to the majority of patients in their daily life.According to statistics,among the population over 60 years old in China,the incidence rate of the population over 65 years old is more than 50%,and the incidence rate of the population over 75 years old is higher than 80%[3].When treating such diseases,professor huang guicheng from jiangsu hospital of traditional Chinese medicine believes that the pathogenesis includes three pathogenic factors,namely wind,cold and humidity,which invade the skin texture and cause local pain,swelling and motor disorders.In the treatment process,the method of dispelling wind and cold,warming the meridians,promoting blood circulation and relieving pain can often achieve the effect of effective medicine.In the study of the mechanism of osteoarthritis,it was found that during the development of knee osteoarthritis,with the proliferation and invasion of the blood vessels in the subchondral bone,the blood vessels invaded the cartilage,resulting in many pathological changes such as the rapture,defect and deformation of the cartilage.Under continuous stimulation,cartilage tissues secrete vascular endothelial growth factor(VEGF),which induces endothelial cells to proliferate and move and invade the cartilage in a directional manner.At the same time,cartilage tissue is continuously stimulated to secrete matrix metalloproteinase-13(MMP13)and other proteases,thereby opening up new tissue Spaces for vascular invasion.Animal model tests showed that in the early stages of KOA lesions,angiogenesis was more frequent in subchondral bone than in healthy tissue,and vessels invaded the bone-cartilage junction.Angiogenesis is closely related to the growth of sensory nerves.With the increase in the number of vessels,sympathetic and sensory nerves extend from the subchondral bone into the bone-cartilage channel,which is the basis of early pain in osteoarthritis and increases the expression of nerve growth factor in the vascular pathway.During the development of KOA disease,abnormal subchondral bone remodeling,increased number of neovascularization,and the formation of microcracks promote the transposition and interaction of molecular signals between bone and cartilage.The activation of these signaling pathways and the release of secretory factors play a key regulatory role in the interaction between cells and molecules.Accordingly,the study was carried out smoothly,providing an experimental basis for observing the effect of "wenjing tongluo soup" on VEGF.Based on the success of the first part of the experiment,we carried out the second part of the experiment,which was to observe whether"wenjing tongluo soup" could inhibit the expression of HIF1,VEGF and MMP13 in mouse knee cartilage.In addition,excess secretion of hypoxia-inducing factor-1,or HIF1,will lead to an increase in VEGF expression.As a regulator of angiogenesis,VEGF plays a key role in the progression of osteoarthritis,and determines the invasion of new blood vessels from the bone-cartilage junction to the cartilage layer.With the invasion of the blood vessels in the cartilage,a series of pathological manifestations such as fracture,separation and stripping of the diseased cartilage will be directly caused.Therefore,effectively inhibiting the expression of VEGF will help to slow down the process of destruction of articular surface cartilage in knee osteoarthritis.Since sensory nerves are often accompanied by new blood vessels,and overstimulation of sensory nerves is one of the internal causes of knee osteoarthritis pain,when"wenjing tongluo soup" effectively inhibits the process of vascular invasion of cartilage,can it further reduce the secretion of inflammatory factors related to knee osteoarthritis?The results showed that "wenjing tongluo soup" could effectively inhibit the expression of HIF1 PCR and VEGF in the knee joint of mice,so as to slow down the degeneration process of the knee joint cartilage and further reduce the expression of MMP13.To clear "warm by t2dm soup" is applied to the diseased tissue,through what way we conducted the third part of the experiment,the part of the experiment we enzyme linked immunosorbent experiment in the tested groups of mice serum HIF1 alpha,the expression of VEGF and MMP13 situation,found its expression in knee joint cartilage with groups of mice has certain relevance,and lays the foundation for subsequent in vitro experiment.The author believed that "wenjing tongluo decoction" could significantly inhibit the expression of HIF1 PCR,VEGF and MMP13 in the serum and knee cartilage of mice,thereby slowing down the angiogenesis at the bone-cartilage junction and its invasion into the knee cartilage,thereby maintaining the institutional integrity of articular cartilage and delaying the development of KOA.Modeling and Identification of knee osteoarthritis in mice:Objective:To improve the modeling method.After the completion of modeling,the success of modeling was identified by general observation,HE staining and other experimental methods,so as to prepare for subsequent experiments.Methods:Through the anterior cruciate ligament is broken(ACLT)building method,in order to achieve the building effect of knee osteoarthritis in mice,continue to raise 4 weeks after building,do not take other special intervention,during 4 weeks after materials,and general observations made the surface of the mice after knee joint structure change,through HE slice staining observation made the mold after the articular cartilage and subchondral bone histological morphological structures,etc.Results:After successful modeling,the gross observation showed that the knee joint surface structure of mice in the blank group and the sham group did not change significantly,while the knee joint surface of mice in the model group showed local pathological changes such as hyperplasia,crack and peeling.HE section staining showed that the surface cartilage structure of the knee joint of mice in the blank group and the sham group was complete,with a clear boundary between cartilage and subchondral bone and a clear tidal line.In the model group,the articular cartilage of knee joint was cracked and separated,and the bone-cartilage boundary was not obvious,and no obvious tidal line was observed.Conclusion:After the anterior cruciate ligament dissection(ACLT)modeling,the results were satisfactory and could be used in subsequent experiments.Inhibitory effect of WJTLT on HIF1 a?VEGF?MMP13 in mouse knee joint:Objective:To observe the effect of WJTLT on HIF1??VEGF?MMP13 in mouse knee joint.Methods:The decoction with high concentration and low concentration was prepared and concentrated.Meloxicam was used as the positive drug.The experimental animals were divided into five groups:sham operation group(group A),model group(group B),high-dose WJTLT group(group C),low-dose WJTLT group(group D),and positive drug group(group E).After successful modeling,group A and group B were given 10ml/kg·d-1 normal saline for gavage,group C and group D were given 10ml/kg·d-1 WJTLT for gavage,and group E were given 10ml/kg·d-1 meloxicam solution for gavage for 2 weeks and 4 weeks,respectively.After gavage,the expression of VEGF in the knee joint of mice was detected by immunohistochemistry and enzyme-linked immunoadsorption.Results:After 2 weeks of gavage,the immunohistochemical results showed that the VEGF expression in the knee cartilage of mice in the high-dose group,the low-dose group and the positive group decreased significantly compared with the model group,and the VEGF expression in the positive group was the lowest,close to the level of the sham group.The expression of VEGF in the high-dose group,the low-dose group and the positive group showed a decreasing trend,and the difference was statistically significant.Results of enzyme-linked immunosorbent assay showed that compared with the sham group,the expression of VEGF in the knee joint of mice in other groups was significantly increased,and the expression of VEGF in the knee joint of mice in the model group was the highest.Compared with the model group,the expression of VEGF in the high-dose group,the low-dose group and the positive group were all decreased,with statistically significant differences.Conclusion:WJTLT has a clear inhibitory effect on VEGF expression in knee joint during the course of knee osteoarthritis in mice.It can effectively intervene the development of knee osteoarthritis,inhibit the inflammatory response,and delay the degeneration of knee cartilage.Effect of WJTLT on HIF1,VEGF and MMP13 in mouse serum:Objective:To observe the effect of WJTLT on HIF1,VEGF and MMP13 in mouse serum.Methods:The high and low concentration of WJTLT was prepared and concentrated,and meloxicam was used as the positive drug for comparison.The experimental animals were divided into five groups:sham operation group(group A),model group(group B),high dose group(group C),low dose group(group D)and positive drug group(group E).After successful modeling,group A and group B were given 10ml/kg·d-1 normal saline,group C and group d were given 10ml/kg·d-1 WJTLT,and group E was given 10ml/kg·d-1 meloxicam aqueous solution,lasting for 2 weeks.After gavage,enzyme-linked immunosorbent assay were used to detect the expression of HIF1 PCR,VEGF and MMP13 in the serum of mice.Results:After 2 weeks and 4 weeks of gavage,the immunohistochemical results showed that the expression of MMP13 in the knee cartilage of mice in the high-dose group,the low-dose group and the positive group decreased significantly compared with the model group,and the expression of MMP13 in the positive group was the lowest,close to the level of the sham group.The expression of MMP13 in the high-dose group,the low-dose group and the positive group showed a decreasing trend,and the difference was statistically significant.The results of enzyme-linked immunosorbent assay showed that the expression of MMP13 in the knee joints of mice in other groups was significantly increased compared with that in the sham group,among which the expression of MMP13 in the knee joints of mice in the model group was the highest,and the expression of MMP13 in the high-dose group,the low-dose group and the positive drug group were all decreased compared with the model group,with statistically significant differences.Conclusion:WJTLT can regulate the expression of VEGF in the knee joint of mice,thereby delaying the development of osteoarthritis of the knee and inhibiting the expression of inflammatory factors in osteoarthritis of the knee. |
PDF Full Text Request