| Objective:Knee osteoarthritis?KOA?,with characters of high morbidity and disability rate,has limited treatment methods at present.The subject study the mechanism relation of Huangqi guizhi wuwu Tang's effect with VEGF network regulation in treating KOA,so that to provide novel evidence for KOA's clinical treatment and Huangqi guizhi wuwu Tang's application.Methods:?1?The critical target and pathway prediction of Huangqi guizhi wuwu Tang in intervening Knee osteoarthritis on the basis of network pharmacologyWith the help of Chinese and PubMed Database,filter and summarize the types of Huangqi guizhi wuwu Tang in treating disease clinically.Take advantage of TCMIP platform,predict drug targets of Huangqi guizhi wuwu Tang and disease targets of KOA,then set and draw the key targets of the drug and KOA targets network for the function and signal pathway analysis.?2?KOA rats'model Selection40 rats were randomly divided into control group?N?,plaster fixation group?M1?,cold stimulation and plaster fixation group?M2?,plaster fixation combined with multiple physical stimulation group?M3?and lower limbs upright in cold water group?M4?.Except control group,all groups'rats were modeled with respective methods for continuous 42 days?the 1st day as d1?,ie,plaster fixation in straight position for 12 hours?M1?,plaster fixation with lower limbs stand in cold water for separate 6 hours as well as cold environment?M2?,swim and starve or overeating stimulation combined with M2 group method,lower limbs upright stand in cold water for 12 hours.Detect rats'common state,and joint swelling during experiment.After experiment,detect rats'joint morphological and pathological change,as well as hemorheology,and EPO,TSH,ADR and VEGF,cAMP/cGMP expression in blood.?3?VEGF network regulating study in Huangqi guizhi wuwu Tang treating KOA rats model60 rats were randomly divided into control group?N,10?and modeling group?50?.After successfully modeling with cold stimulation and plaster fixation method,modeling groups'rats,were randomly divided into model group?M?,celecoxib group(Y:20.82 mg·kg-1),Huangqi guizhi wuwu Tang low(Hl:3.24 g·kg-1),median(Hm:6.48g·kg-1),and high dosage groups(Hh:12.96 g·kg-1).All rat were gavaged with 5mL·kg-1 volume and weight relation in each group for 28 days.Control group and model group were gavaged with same volume of distilled water.During experiment,rats'common state and joint swelling in every groups were observed.After experiment,joint pathological structure change,blood routine examination,joint NGF,SP,VEGF and ES expression and IL-17,NF-?B,TGF-?1,eNOS?PGE-2 which were VEGF network's relative core cytokines were tested.Results:?1?Respective common target of Huangqi,Guizhi,Baishao,Shengjiang and Dazao in Huangqi guizhi wuwu Tang occupied 74%,80%,70%,51%and 36%of their predicted target number.The common target number betwen Huangqi and Baishao,Guizhi and Baishao separately occupied more than 50%of their own common number,meanwhile,the total predicted common target of Huangqi,Guizhi and Baishao,accounted for 1.41,1.5 and 1.9 times when they shared common target with other herbs.The critical targets of Huangqi guizhi wuwu Tang in treating KOA were mainly GNB1,PRKCD,GCK,ADCYs and CROT in drug,and HTR2C,PRKAA2,MAPK3,CXCR1 and TP53 in disease,and their common targets were NF-?B2,NF-?B1A,TP53 and PPKAA2.The key targets'function enriched in mitochondrial's ATP energy metabolism,amino acid metabolism,cell signal transmission and transduction,and signal pathway enriched in nervous system,circulatory system,and chemokine,endocrine and VEGF signal pathway.?2?During modeling,all model groups rats showed poor hair and fur,and less joint activity compared with control group,and 3 plaster fixation model groups showed claudication and inflexible joint movement after 28th model day.Rats in model group's cold syndrome score,weight,joint swelling and Paw temperature were significantly lower than group N separately on d7,d21,d28,and d35?P<0.05?,and the difference lasted until the end of the modelling.After modeling,all model rats showed morphological and pathological lesion in different degrees.VEGF and type?collagen in model groups were significantly higher than group N?P<0.05?,and cartilage and synovial's VEGF in M2 and M3 were significantly higher than M1?P<0.05?,while cartilage's VEGF in M4 was significantly lower than 3 plaster fixation groups?P<0.05?.Blood viscosity,TSH,and ADR in M1 and M2 were significantly lower than group N?P<0.05?,and the 2 groups'cAMP/cGMP ratio and group M2's cGMP were significantly higher than group N?P<0.05?.TSH and ADR in M2 and M3 were significantly lower than group M1?P<0.05?,whole blood viscosity?1/s?and TSH,cGMP in M3 were significantly higher than M2?P<0.05?,and cAMP were significantly higher than N and M1?P<0.05?.Blood viscosity in M4 were significantly higher than M2?P<0.05?,EPO,TSH and ADR were significantly higher than other model groups?P<0.05?,and cGMP was significantly higher than group M2?P<0.05?,while cAMP/cGMP was significantly lower than group M2 and M3?P<0.05?,as well as significantly lower expressed cAMP than M3 group?P<0.05?.?3?After administration of Huangqi guizhi wuwu Tang,Rats showed improved spirit,increased appetite,and alleviated joint swelling compared with control group.The weight,HGB and RBC count?except Hl?,Na+-K+-ATP activity?except Y and Hl?,IL-4?except Hl?,IL-4/IFN-?ratio,IL-10,IL-6 levels in blood,and ES expression were significantly increased than group M?P<0.05?.Paw temperature,joint swelling,cold syndrome score,pathological change score,along with PLT?except Hh?and WBC,IFN-?,TNF-?,eNOS,PGE-2,IL-17,VEGF and TGF-?1 levels in blood,and NGF,SP,IL-17,NF-?B and VEGF expression in joint were significantly decreased than M group?P<0.05?.Among drug groups,HGB,PLT,TNF-?,IL-10,IL-17 levels in blood and SP,NF-?B and VEGF expression in Hl group had weaker effect than group Y?P<0.05?.Group Hm and Hh's Na+-K+-ATP,IL-4/IFN-?ratio in blood,and NF-?B and VEGF expression in joint were significantly higher than group Y?P<0.05?.Conclusion:?1?Huangqi guizhi wuwu Tang take GNB1,PRKCD,GCK,ADCYs and CROT as drug target,to intervene NF-?B family and PPKAA2 which were the common target with KOA,in order to play a role in regulating the function of mitochondrial ATPase metabolism,amino acid metabolism,cell signal transmission and transduction,and mediating VEGF network with VEGF signal pathway as the core.Thus,it may promote energy metabolism,suppress joint inflammatory reaction and angiogenesis,to relieve pain sensation of patients.?2?Joint structure can certainly be damaged,through joint fixation in straight position.Cold stimulation and plaster fixation method can make rats'statement more serious,and TSH,ADR and VEGF levels can significantly different compared to purely plaster fixation method.While,there is no significantly difference between multiple stimulating method and simply cold stimulation and plaster fixation combination method,thus cold stimulation and plaster fixation combination method were chosen as KOA model preparation method.?3?Huangqi guizhi wuwu Tang can alleviate KOA rats'pathological lesion,decrease NGF and SP expression,hence Huangqi guizhi wuwu Tang had analgesic and joint's lesion prosthetic effect.Meanwhile,it can increase IL-4/IFN-?ratio,Na+-K+-ATPase activity,ES expression,and suppress VEGF network's core cytokines IL-17,PGE-2,NF-?B and VEGF expression,to play a role in regulating immunological function and control inflammation reaction and angiogenesis process,so as to relieve joint stasis,improve cartilage repair,relieve and treat KOA. |
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