An Analysis On The Relationship Between TCM Syndromes And MMPs And TIMP-1Indicators In Synovial Fluid Of Knee Osteoarthritis

ObjectiveAfter comparing matrix metalloproteinases (MMP-1,-3,-8,-13) and tissue inhibitor of metalloproteinase1(TIMP-1) contents in synovial fluid of patients with knee osteoarthritis(KOA) and normal people, this research aims to analyze the relationship between MMP-1,-3,-8,-13and TIMP-1contents in synovial fluid and indexes of severity for knee osteoarthritis and X-ray grade. It hopes to seek biological indicators of early diagnosis of KOA and condition assessment.Through investigations of TCM syndrome characteristics of KOA patients, physical and chemical properties of synovial fluid, X-ray grade and Lequesne indexes of severity for KOA, this research tries to explore the relationship between TCM syndrome characteristics and such indicators as physical and chemical properties of synovial fluid, X-ray grade, severity indexes, MMP-1,-3,-8,-13and TIMP-1. By doing so, this research aims to provide objective evidence of TCM syndrome characteristics of KOA and provide new ideas and methods for future research on TCM syndrome.By observing MMP-1, MMP-3, MMP-8, MMP-13and TIMP-1contents in synovial washing fluid in rats and severity of their articular cartilage degeneration, this research aims to explore correlation between these indices and degeneration of articular cartilage and thus further validate relevant clinical research conclusions.Method1.literature research Domestic and foreign studies on KOA were first reviewed, including names of TCM diseases, TCM syndrome differentiation and epidemiological study of knee osteoarthritis. After that, a summary was made of pathogenesis of KOA and research progress on matrix metalloproteinases and tissue inhibitor of metalloproteinase in KOA. Finally, relevant domestic KOA TCM syndrome research was analyzed.2. Clinical research A clinical questionnaire on KOA was developed according to diagnostic criteria for KOA and TCM syndrome diagnosis standard.78patients diagnosed with Knee Osteoarthritis were selected from Orthopedic clinic and ward department of Guangdong Provincial Second TCM Hospital affiliated to Guangzhou University of Traditional Chinese Medicine. Three of the patients were not included in the study since their synovial fluid could not be extracted. Survey contents included the general situation of patients, characteristics of TCM syndrome, physical and chemical properties of synovial fluid, X-ray features and Lequesne indexes of severity for KOA. At the same time, synovial fluid was collected from75cases of KOA patients and10normal people. MMP-1,-3,-8,-13and TIMP-1contents in synovial fluid determined analyzed following enzyme-linked immunosorbent assay (ELISA). Correlation of these objective indicators and TCM syndrome was analyzed by adopting modern statistical methods.3. Experimental study32SD rats were divided into four groups:sham operation group, operation group4W, operation group6W and operation group8W. Each group consisted of8rats, half males and half females. Modified Hulth modeling method was adopted for operation groups. Rats were killed at4W,6W and8W respectively to observe the knee X-ray characteristics of each rat. In addition, visual observation of the articular cartilage was made, Mankin scores were determined according to microscopy of articular cartilage degeneration, immunohistochemical detection of articular cartilage was made to check MMP-1,-3,-8and-13protein contents, and MMP-1,-3,-8,-13, TIMP-1levels of joint flushing fluid were checked following ELISA. Correlation between MMPs and TIMP-1contents in joint flushing fluid and cartilage degeneration was explored.Result1. Literature researchPathogenesis of knee osteoarthritis is complex and matrix metalloproteinases and tissue inhibitors of metalloproteinase play an important role in the process of articular cartilage matrix degradation of KOA. According to traditional Chinese medicine, knee osteoarthritis belongs to'arthralgia', with insufficiency of the liver, kidney, spleen as its internal causes and wind, cold, wet, heat, phlegm and blood stasis being its external causes; in addition, deficient root and excessive superficial is the basic characteristic of the disease. Objective indicators of immune cytokines and inflammatory mediators are related to KOA TCM syndrome. Therefore, objective analysis of TCM syndrome is feasible.2. Clinical research75patients with KOA patients were surveyed, including32cases (42.67%) of liver and kidney deficiency and stasis of the channels,21cases (28.00%) of spleen and kidney deficiency and damp flowing downward to condyle, and22cases (29.33%) of liver and kidney deficiency with phlegm and blood stasis accumulation.No statistically significant differences (P>0.05) were found in body mass index, obesity, occupational characteristics of three groups of TCM patients. Logistic regression analysis of course and TCM syndrome revealed no statistically significant difference (P=0.623>0.05) between the syndrome of liver and kidney deficiency and stasis of the channels and the syndrome of spleen and kidney deficiency and damp flowing downward to condyle. However, a statistically significant difference (P=0.036<0.05) was found between the syndrome of liver and kidney deficiency with phlegm and blood stasis accumulation and the syndrome of spleen and kidney deficiency and damp flowing downward to condyle.No statistically significant difference (P=0.966>0.05) was found in the colors of the three groups of TCM syndrome in patients with synovial fluid. Nevertheless, a statistically significant difference (P<0.05) was found in their transparency and viscosity.As a result of rank sum tests, a statistically significant difference (P <0.05) was found in the X-ray grade distribution of three groups of TCM patients. Besides, non-parametric statistics revealed a statistically significant difference (P<0.05) in KOA severity indices distribution of the three groups of patients.No MMP-8or MMP-13were detected in normal human synovial fluid. However, MMP-1,-3and TIMP-1were detected in the synovial fluid of75patients, including10cases with MMP-8and21cases with MMP-13. Statistically significant differences (P<0.05) were found in the MMP-1, MMP-3, TTMP-1, MMP-1/TIMP-1, MMP-3/TIMP-1of synovial fluid of three groups of TCM syndrome patients and those of normal people.Logistic regression analysis of MMP-1and MMP-3, TIMP-1, MMP-1/TIMP-1, MMP-3/TIMP-1of synovial fluid as well as X-ray grade revealed statistically significant differences (P<0.01) in the various indicators of four levels. A decreasing order of the four levels according to their MMP-1,-3, MMP-1/TIMP-1, MMP-3/TIMP-1contents is as follows:Level â…£> Level â…¢> Level â…¡> Level â… . A decreasing order of the different levels according to their MMP-1content is as follows:Level â…¢> Level â…¡> Level â… , with Level â…£ slightly lower than Level â…¢.Logistic regression analyses of the correlation between MMP-1, MMP-3, TIMP-1, MMP-1/TIMP-1and MMP-3/TIMP-1contents in synovial fluid and indexes of severity for KOA revealed a statistically significant difference (P<0.01) in the MMP-1and TIMP-1contents of four levels of knee osteoarthritis severity. A decreasing order of the different levels according to their contents is as follows:very or extremely serious) severe) moderate) mild. No significant differences (P>0.05) were found in the MMP-3, MMP-1/TIMP-1, MMP-3/TIMP-1contents of the four levels.Significant differences (P<0.05) were found among different groups of TCM syndrome patients in terms of MMP-1contents of synovial fluid; significant differences (P<0.01) were found between the syndrome of liver and kidney deficiency with phlegm and blood stasis accumulation and other two syndrome in terms of MMP-3, TIMP-1, MMP-1/TIMP-1and MMP-3/TIMP-1contents. No significant differences (P=0.176,0.069,0.106,0.208>0.05) were found between the syndrome of spleen and kidney deficiency and damp flowing downward to condyle and the syndrome of liver and kidney deficiency and stasis of the channels.3. Experimental researchMMP-1,-3and TIMP-1were detected in joint flushing fluid. No significant differences (p=0.057,0.053)0.05) were found between operation group4W and sham operation group in terms of TIMP-1, MMP-1/TIMP-1contents. Significant differences (P<0.05) were found between other groups and sham operation group. An increasing trend was observed in the MMP-1,-3, and TIMP-1, MMP-1/TIMP-1, MMP-3/T1MP-1of joint flushing fluid as postoperative time increased.Statistically significant differences (P<0.01) were found between operation groups4W,6W,8W and the sham operation group in terms of articular cartilage of MMP-1,-3,-8and-13protein expression. As postoperative time increased increased, MMP-1,-3,-8,-13protein expressions in articular cartilage increased.As regards relationship between two variables of MMP-1in joint flushing fluid and MMP-1in cartilage, Pearson-related coefficient test revealed a statistically significant difference (r=0.946, P<0.01). Regarding relationship between two variables of MMP-3in joint flushing fluid and MMP-3in cartilage, Pearson-related coefficient test revealed a statistically significant difference (r=0.843, P<0.01).No MMP-8or-13was detected in joint flushing fluid in sham operation group or operation group4W. Within operation group6w, MMP-8was detected in1case and MMP-13was detected in2cases. Within operation group8w, MMP-8was detected in3cases and MMP-13was detected in5cases.ConelusionLiver and kidney deficiency and stasis of the channels were more common in TCM syndromes of knee osteoarthritis. However, body mass index, body obesity, occupational characteristics and the color of the synovial fluid of knee osteoarthritis were not explicitly correlated with TCM syndromes.Duration, transparency and viscosity of synovial fluid, X-ray characteristics and severity indices were correlated with KOA TCM syndromes to some extent and could thus be used as points of reference of the TCM syndrome differentiation. With the extension of the duration, liver and kidney deficiency with phlegm and blood stasis accumulation increase. As regards the transparency and viscosity of the synovial fluid, transparency of liver and kidney deficiency and stasis of the channels syndrome were the highest, and their viscosity was also the highest. This was followed by spleen and kidney deficiency and damp flowing downward to condyle syndrome. Transparency of liver and kidney deficiency with phlegm and blood stasis accumulation syndrome was the lowest and their viscosity was also the lowest. As regards classifications of the X-ray, the syndrome of liver and kidney deficiency and stasis of the channels and the syndrome of spleen and kidney deficiency and damp flowing downward to condyle were more common in Classes â…  and â…¡, while the syndrome of liver and kidney deficiency with phlegm and blood stasis accumulation were more common in Classes â…¢ and â…£. Regarding severity indexes, the syndrome of liver and kidney deficiency and stasis of the channels was more common in patients with mild to moderate severity indexes, the syndrome of spleen and kidney deficiency and damp flowing downward to condyle was more common in patients of moderate severity, while the syndrome of liver and kidney deficiency with phlegm and blood stasis accumulation prevalent were more common with severe or extremely severe patients.MMP-1,-3, MMP-1/TIMP-1and MMP-3/TIMP-1were associated with X-ray level: as the level of X-ray increased, values of these indicators also increased. MMP-1and TIMP-1were associated with severity indices of KOA:the knee more severe osteoarthritis was, the higher MMP-1and TIMP-1. MMP-1and TIMP-1can be used for early diagnosis of knee osteoarthritis and as biological indicators of the severity and efficacy evaluation. MMP-1/TIMP-1and MMP-3/TIMP-1were higher in liver and kidney deficiency with phlegm and blood stasis accumulation; therefore, they can provide reference for knee osteoarthritis, liver and kidney deficiency with phlegm and blood stasis accumulation, and thus provide an objective and standardized basis for TCM.As time after surgery extended, articular cartilage degeneration gradually increased, the mean Mankin's score was elevated, and MMP-1,-3and TIMP-1, MMP-1/TIMP-1, MMP-1/TIMP-1in synovial fluid also increased. This indicated that these indicators were associated to some extent with cartilage degeneration. MMP-1and-3contents in joint flushing fluid can reflect the protein expression changes of cartilage MMP-1and-3. MMP-8and-13were detected to be low in joint flushing fluid, which was consistent with the results of clinical studies.