Clinical Analysis Of Gubi Zhitong Formula In The Treatment Of Knee Osteoarthritis And Its Mechanism Of Regulating Inflammatory Microenvironment

Objective:1 To retrospectively analyze the clinical efficacy of Gubi Zhitong Formula in treating patients with knee osteoarthritis;2 To investigate the relationship between the chemical composition of Gubi Zhitong Formula and knee osteoarthritis based on LC-MS;3 To evaluate the efficacy of Gubi Zhitong Formula in the treatment of osteoarthritis in a rat knee osteoarthritis model;4 To investigate the effect of Gubi Zhitong Formula on macrophage polarization and cartilage repair based on OA inflammatory environment;5 To investigate the molecular biological mechanism of M2 macrophage vesicles in promoting the repair of OA chondrocyte injury.Methods:1 To retrospectively analyze the clinical efficacy of Gubi Zhitong Formula in treating patients with knee osteoarthritisTo investigate the clinical efficacy of Gubi Zhitong Formula before and after treatment in patients with knee osteoarthritis,and to evaluate the clinical efficacy of Gubi Zhitong Formula through the knee osteoarthritis index score（WOMAC）,knee function score（Lysholm score）,etc.,and to provide a clinical basis for the subsequent study of the mechanism of action of Gubi Zhitong Formula.2 To investigate the relationship between the chemical composition of Gubi Zhitong Formula and knee osteoarthritis based on LC-MSTaking Gubi Zhitong Formula as the research object,the chemical composition of Gubi Zhitong Formula was analyzed by liquid chromatography-mass spectrometry to investigate the correlation between the chemical composition of Gubi Zhitong Formula and osteoarthritis.3 To evaluate the efficacy of Gubi Zhitong Formula in the treatment of osteoarthritis using a rat knee osteoarthritis modelIn vivo,the rat knee osteoarthritis model was established,and different doses of Gubi Zhitong Formula and positive drug Celecoxib were administered for intervention for 8weeks.After the end of administration,gait,quadrupedal weight bearing,and grip strength analysis were performed in each group to compare the behavioral status changes of rats in each group.All rats were sacrificed and sampled,and the histomorphological changes and the degree of articular cartilage lesions were compared between the groups of samples by H&E staining,safranin-fast green staining,Mankin score,and Micro-CT examination.Elisa,RT-q PCR and Western blot were used to detect the changes of pro-inflammatory factors（i NOS,IL-6,IL-1β）and cartilage anabolic factors（SOX9,COL2A1,ACAN）in serum and cartilage tissue to determine the effectiveness of Gubi Zhitong Formula in the treatment of osteoarthritis.4 To investigate the effect of Gubi Zhitong Formula on macrophage polarization and cartilage repair based on OA inflammatory environmentRat knee joint fluid was labeled with CD68-Alexa 555（macrophage surface marker）,CD86-Alexa 647（M1 macrophage surface marker）,and CD206-Alexa 488（M2macrophage surface marker）and analyzed by flow cytometry;The ratio of M1 and M2macrophages in each group was detected by flow cytometry;CCK-8 and cell cycle experiment were used to detect the effect of Gubi Zhitong Formula on macrophage proliferation activity;Flow cytometry was used to detect the regulatory effect of Gubi Zhitong Formula on M1 and M2 polarization direction of macrophages;The co culture model of M2 macrophages and OA chondrocytes was constructed by Transwell cell culture plate.The effects of M2 macrophages on the synthesis and catabolism of OA chondrocytes after Gubi Zhitong Formula intervention were determined by morphological observation of OA chondrocytes,Alcian staining,and protein detection.5 To investigate the molecular biological mechanism of M2 macrophage vesicles promoting the repair of OA chondrocyte injuryThe supernatant of M2 macrophages from rats and mice was used to intervene the OA chondrocytes of their respective species,and the protective effect of M2 macrophage supernatant on OA chondrocytes was clarified;The supernatant of M2 macrophages treated with proteinase K and RNase A was used to interfere with OA chondrocytes.The types of vesicles in the supernatant of M2 macrophages that promote the anabolism of OA chondrocytes were screened,and the vesicles were characterized and analyzed（transmission electron microscopy,nanoflow cytometry,Western blot）.Vesicles were extracted by ultracentrifugation and labeled with PKH67 and incubated with OA chondrocytes to observe the uptake of OA chondrocytes.The effective dosage gradient of vesicles was screened by CCK-8 experiment,and the m RNA levels of factors related to synthesis and catabolism of OA chondrocytes were detected by QRT PCR;The contents of M2 macrophage vesicles were subjected to mi RNA sequencing,and mi RNA minics were constructed to transfect OA chondrocytes.The anti-apoptosis ability of OA chondrocytes was observed and functional mi RNAs were screened.The targets of mi RNAs were predicted by targetscan database and verified by dual luciferase reporter experiment.Results:1 In this study,311 patients with knee osteoarthritis collected were statistically analyzed for age and gender,and the results showed that women aged 51-60 years and 61-70 years were the main affected groups.The results of WOMAC score showed that Gubi Zhitong Formula could effectively improve the symptoms of knee joint stiffness and pain,and effectively reduce the knee arthritis index（p<0.05）.The results of Lysholm score showed that Gubi Zhitong Formula significantly improved knee joint function（p<0.05）.The results of SF-36 score showed that the scores of patients in both groups before treatment reflected that the patients had significant quality of life decline and psychological disorders,and the scores of patients in each group increased after treatment,indicating that with the relief of pain and movement disorder function,the quality of life of patients could be improved and psychological disorders could be improved（p<0.05）.2 The chemical constituents of Gubi Zhitong Formula were identified by HPLC-MS.The results showed that the top five chemical components in Gubi Zhitong Formula were identified in positive ion mode:2-chlorophenylalanine,adenosine,pyroglutamic acid,l-choline,naringin;In the negative ion mode,the top five chemical components in Gubi Zhitong Formula were identified as naringin,androsterol-o-rutinoside,acanthoside B,d-quinic acid,citric acid,and maleic acid.3 The rat knee osteoarthritis model was established by treadmill exercise and divided into normal group,model group,low and high dose groups of Gubi Zhitong Formula and Celebrex group.Gait analysis showed that the contact area（cm²）of the right hind paw decreased in the model group compared with the normal group,while the contact area increased in the Gubi Zhitong Formula and Celebrex groups.In the model group,the step length（cm）was reduced,the swing phase time（s）and support time（s）were prolonged（p<0.01）,while there was no significant difference in the immobilization time（s）between the groups（p>0.05）;gross anatomy showed that the synovial hyperplasia was significant and the cartilage wear at the femoral trochlea was significant in the model group,and the synovial hyperplasia was reduced and the cartilage wear was reduced in the normal group,Gubi Zhitong Formula group,and Celecoxib group;Micro-CT lateral view showed some cartilage defects in the femoral trochlea,and anteroposterior view of the knee joint revealed that the subchondral bone porosity was reduced,while cartilage injury was reduced and the porosity was increased after intervention with Gubi Zhitong formula and Celecoxib;H&E results showed that the articular cartilage surface was uneven and rough,chondrocytes were hypertrophic,disorganized,the surface of the Gubi Zhitong formula group was smooth,and chondrocytes were arranged neatly;In the serum of the model group,the levels of pro-inflammatory cytokines nitric oxide synthase（i NOS）,interleukin-6（IL-6）,and interleukin-1β（IL-1β）were significantly increased（p<0.01）,and the levels of anti-inflammatory cytokines interleukin-10（IL-10）and transforming growth factor-β（TGF-β）were decreased（p<0.01）,and pro-inflammatory factors were reduced to varying degrees under the intervention of Gubi Zhitong formula and Celecoxib（p<0.05）,while the promoting effect of Celecoxib on anti-inflammatory factors was not significantly different from that of the model group（p>0.05）,and the total RNA and related protein results of cartilage tissues in each group showed that Gubi Zhitong formula up-regulated the expression of cartilage anabolic factors Acan,Col2a1,and Sox9 m RNA（p<0.01）and down-regulated the expression of catabolic factors Mmp3,Mmp13,and Adamts5 m RNA（p<0.01）.4 The detection results of the knee joint fluid of rats in each group showed that compared with the model group,the proportion of CD68+cells in the joint fluid of rats in the high-dose and low-dose groups of Gubi Zhitong Formula increased（31.14%to 56.65%）,indicating that Gubi Zhitong Formula can promote macrophage proliferation,and the proportion of CD68+and CD86+double positive cells has a downward trend（84.22%to80.72%）,but there was no statistical significance;The proportion of CD68+CD206+double positive cells was significantly increased（48.39%to 74.17%）,indicating that Gubi Zhitong formula can significantly increase the proportion of CD68+CD206+double positive cells in joint fluid.CCK-8 assay results showed that with the increase of the concentration of Gubi Zhitong Formula,the number of raw 264.7 cells increased in a dose-dependent manner（p<0.05）.Cell cycle detection found that Gubi Zhitong Formula prolonged the S+G2M phase of RAW 264.7 cell cycle,indicating that Gubi Zhitong Formula can promote the proliferation of RAW 264.7 cells.In addition,flow cytometry revealed that LPS+INF-γAfter combined intervention with Gubi Zhitong Formula,the proportion of CD68+single positive RAW 264.7 cells did not change significantly（82.97%-87.55%）,while the proportion of CD68+CD86+double positive cells decreased（87.27%-76.36%）,and the proportion of CD68+CD206+double positive cells did not change significantly（0.6%-1.03%）;After the intervention of IL-4+IL-13 combined with Gubi Zhitong Formula,the proportion of CD68+single positive cells increased（66.14%-75.65%）,the proportion of C68+CD86+double positive cells decreased（32.34%-16.70%）,and the proportion of CD68+CD206+double positive cells increased（70.76%-83.07%）,indicating that Gubi Zhitong Formula promotes the proliferation of raw 264.7 cells and its polarization to M2 type.Through the co culture model of M2 macrophages and OA cells,it was found that the co-culture group with M2macrophages could maintain the intrinsic morphology of chondrocytes,increase the expression of COL2A1 and ACAN in OA chondrocytes,and reduce ADAMTS5,MMP13,TNF-αprotein levels.5 M2 polarization was induced in rat macrophages and mouse RAW 264.7 cell lines,respectively,and OA chondrocytes were added at different volume ratios after collecting the supernatants,and it was found that the supernatants of M2 macrophages from different species could up-regulate Col2a1 m RNA levels in OA chondrocytes,and OA chondrocytes were given after particle size screening,Proteinase K,and RNase A treatment of M2macrophage supernatants,respectively,and it was found that RNA in vesicles with a diameter between 0.1 and 0.22μm in M2 macrophage supernatants had a role in promoting OA chondrocyte injury repair,and M2 macrophage vesicles were characterized using transmission electron microscopy,nanoflow flow,and Western blot techniques,and co-culture of PKH-67-labeled vesicles with OA chondrocytes revealed that they could be taken up by OA chondrocytes,and the effective concentrations of M2 macrophage vesicles were selected as 4,8,and 16μg/ml by CCK-8 assay,and immunofluorescence and RT-q PCR.M2 macrophage vesicles can up-regulate Sox9,Col2a1,Acan m RNA and protein levels and down-regulate Mmp13,Mmp9,and Adamts5 m RNA levels in OA chondrocytes.miRNAs contained in M2 macrophage vesicles（mi R-709,mi R-5126,mi R-3960,mi R-2317）were enriched using RNA micro-array technology,mi RNAs minics were constructed and transferred into OA chondrocytes to find that mi R-3960 was superior to other mi RNAs in its ability to callback Col2a1 and Acan m RNA levels,while mi R-3960could reduce OA chondrocyte apoptosis,and RT-q PCR of apoptosis markers caspase3,caspase 12,caspase 8,and caspase 9 revealed that mi R-3960 could down-regulate its expression,of which caspase 12 could be specifically activated by Ca²⁺,and after labeling free Ca²⁺in OA chondrocytes,mi R-3960 was found to reduce free Ca²⁺concentrations in OA chondrocytes,and mi R-3960 targets were predicted by Target Scan database,and three involved Ca²+regulatory genes in OA chondrocytes were selected,and Cacng3,Camk2b,and Edh4 were detected by RT-q PCR.Direct binding of mi R-3960 to Cacng3 m RNA was confirmed by dual luciferase reporter assays.Conclusion:1 Gubi Zhitong Formula can improve the knee joint function score,reduce the arthritis index,relieve pain,stiffness,and other symptoms,and improve the SF-36 quality of life score.2 The principal components of Gubi Zhitong Formula include 2-chlorophenylalanine,adenosine,pyroglutamic acid,L-choline,naringin,androstenol-O-rutinoside,naringin,acanthoside B,dexquinic acid,citric acid,and maleic acid,and some of them have been shown to be useful in the prevention and treatment of knee osteoarthritis.3 Gubi Zhitong Formula can reduce the levels of inflammatory factors in serum and cartilage tissue of OA rats,promote the repair of knee cartilage injury,improve the weight bearing ability and grip strength level of quadrupedal feet of OA rats,improve the coordination ability of limbs of rats to regulate gait cycle,and improve the motor ability of knee osteoarthritis rats.4 Gubi Zhitong Formula can prolong the S+G2M phase of macrophage cell cycle and reduce macrophage polarization to M1 type and promote their polarization to M2 type,and up-regulate the anabolic level of OA chondrocytes by increasing the proportion of M2macrophages.M2 macrophage could up-regulate the expression of Col2a1 and Acan m RNA,down-regulate the expression of Mmps and Adamts m RNA,and protect OA chondrocytes.5 miR-3960 in M2 macrophage vesicles reduced free Ca²⁺concentration in OA chondrocytes by binding to Cacng3 m RNA and downregulating Cacng3 expression,thereby reducing OA chondrocyte apoptosis mediated by Caspase12.