Efficacy Of Platelet-rich Plasma Combined With Xianlinggubao Capsule In The Treatment Of Koa And Its Network Pharmacology

ObjectiveTo explore the effect of autologous platelet-rich plasma(PRP)combined with oral Xianlinggubao Capsule on knee osteoarthritis(KOA)in the early and middle stages and to evaluate its effect on serum levels of related inflammatory factors in KOA patients.We also carried out network pharmacological analysis of Xianlinggubao capsule,so as to provide new ideas and basis for clinical treatment of KOA.MethodsKOA patients who were treated in the First Affiliated Hospital of Guangzhou University of TCM from 01/09/2017 to 30/04/2018 were included.According to the inclusion and exclusion criterias.Patients were randomly divided into PRP combined with Xian Ling Gu Bao capsule treatment group(PRP group)and sodium hyaluronate group(HA group)according to the random number table.Basic data about patients(gender,height,age,weight,course of the disease,K-L grade)and VAS,IKDC,WOMAC score were collected before and after treatment.The expression levels of serous relevant inflammatory factors(IL-1 and TNF-a)were measured before and 6w after treatment in all patients.What's more,related complications and TCM disease diagnostic outcomes were recorded.Using SPSS22.0 statistical software to analyze the mentioned above data.The mechanism of Xianling Gubao Capsule and Xianling Gubao Capsule in the treatment of KOA were analyzed based on network pharmacology.The active ingredients of Xianlinggubao Capsule were retrieved from BATMAN-TCM platform,a Bioinformatics tool developed by the Beijing Proteomics Research Center.With Knee Osteoarthritis as the key word,the reported genes related to KOA were retrieved in Coo LGe N and Gene Cards databases.The biological process and signal pathway of the predicted therapeutic target of Xianlinggubao Capsule were enriched.After taking the intersection of the therapeutic target and the KOA gene,the enrichment analysis of biological processes and signal pathways is also performed.Screening of potential targets for Xianling Gubao capsules in the treatment of KOA.The medicated serum of Xianling Gubao Capsule interferes with chondrocytes to verify the significance of potential targets.ResultsA total of 71 KOA patients(71 knees)was included in this study.Among them,there were 36 patients(36 knees)in PRP group,16 males and 20 females.The mean age is 58.11±4.97y,BMI is 23.13±2.58 Kg/m2,and the time of disease is 3.48±2.02y.In the HA group,there were 35 patients(35 knees),16 males and 19 females,with an average age of 56.89±6.08y,BMI is 22.69±1.88 Kg/m~2,and the time of disease is 3.84±1.93y.There were no statistically significant differences in the basic data of patients between two groups,the knee scores before treatment and the levels of relevant serous inflammatory factors(P>0.05).The VAS score of the PRP group decreased from 4.93±1.04 points to2.72±0.81 and 1.33±0.99 points before and 6m after treatment respectively.While,the VAS score of the HA group decreased from 4.51±0.85 to 3.25±0.93points and 2.68±0.81 points respectively,and the VAS score of the PRP group was lower than that of the HA group,there were statistical differences(P<0.05).The IKDC score in the PRP group increased from 40.17±6.44 points before treatment to 68.61±7.02 and 77.47±2.29 points at 3m and 6m after treatment.The IKDC score in the HA group increased from 39.64±7.07 points to 63.52±5.75 and 67.91±6.00 points at 3m and 6m after treatment respectively,showing statistical difference(P<0.05).The WOMAC score of the PRP group decreased from 53.19±7.3 points to 27.25±4.59 and 22.08±1.95 points at 3m and 6m after treatment,and that in the HA group decreased from 52.75±6.18points before treatment to 30.94±5.58 and 27.26±4.39 points at 3m and 6m after treatment respectively.After 6w,the mean value of IL-1 in PRP group decreased from 16.05±0.99pg/ml before treatment to 5.79±0.75pg/ml,while the HA group decreased from 15.77±0.72pg/ml to 6.34±0.98pg/ml.In PRP group,the value of TNF-?decreased from 37.55±1.28 pg/ml to 15.38±1.15pg/ml,while HA group decreased from 36.95±1.94pg/ml to 18.18±1.65pg/ml.The mean values of IL-1,TNF-?in PRP group were significantly decreased compared with the HA group(P<0.05).The effective rate of PRP group and HA group was 91.7%and 82.9%,respectively,showing statistical difference(P<0.05).No obvious adverse reactions were found in both groups.The results showed that there were 58 active ingredients and 109 potential gene targets in Xianlinggubao Capsule,among which the top 5 components and targets were Bakuchiol,Cis-Hinokiresinol,Nicotine,Tingenone and Magnoflorine;ESR1,DRD2,AR,PGR and WNT4,which indicated that Xianlinggubao Capsule might play a therapeutic role mainly through these ingredients and targets.Fifty-three potential targets were identified by intersecting 109potential targets with KOA-related disease targets in Cool Ge N and Gene Cards databases.After gene enrichment analysis,it was found that biological processes were mainly concentrated in cytokine-mediated signaling pathway,regulation of inflammatory response,response to lipopolysaccharide,regulation of cytokine production,regulation of secretion;molecular functions were mainly concentrated on receptor ligand activity,cytokine activity,cytokine receptor binding,hormone activity.Signaling pathways are mainly concentrated in Inflammatory bowel disease(IBD),cytokine-cytokine receptor interaction,Chagas disease(American trypanosomiasis),Leishmaniasis,TNF signaling pathway.Ginkgolic acid,Nicotine,Spongesterol,Stigmasterol,Cis-Hinokiresinol and PTGS2,IGF1,IL1B,IL6 and TNFA in target sites all occupy the core of the network,indicating that they may play an important role in the treatment of KOA with Xianlinggubao Capsule.Molecular docking scoring results showed that Ginkgolic Acid and Spongesterol had a good binding ability,to each potential target,while PTGS2 and IL1B targets played an important role in the treatment process.Cell experiments show that low concentration of medicated serum can promote the proliferation of chondrocytes.The PCR results showed that compared with the normal chondrocyte group,the IL-1?group chondrocyte PTGS2,IGF1,IL-6,TNF-?and LEP m RNA expression levels were significantly increased,while the SOX9 m RNA expression level was significantly reduced(P<0.05).Compared with the IL-1?group,the chondrocyte PTGS2,IGF1,IL-6,TNF-?and LEP m RNA expression levels in the low-concentration Xianling Gubao group were significantly reduced,while the SOX9 m RNA expression levels were significantly increased(P<0.05).The expression levels of PTGS2,IGF1,IL-6and TNF-?m RNA in the high-concentration Xianling Gubao group were lower than those in the IL-1?group,and the difference was statistically significant(P<0.05).There was no significant difference between LEP and SOX9 between the two groups(P>0.05).Compared with the low-concentration Xianling Gubao group,the high-concentration drug-containing serum group had higher levels of PTGS2,IGF1,and IL-6 m RNA expression(P<0.05).ELISA results showed that compared with the normal chondrocyte group,the IL-1?group chondrocyte PTGS2,IGF1,IL-6,TNF-?and LEP protein expression levels were significantly increased,while the SOX9 protein expression level was significantly reduced(P<0.05).Compared with the IL-1?group,the chondrocyte PTGS2,IGF1,IL-6and TNF-?protein expression levels in the low-concentration Xianling Gubao group were significantly reduced,while the SOX9 protein expression levels were significantly increased(P<0.05).The expression levels of PTGS2,IGF1,IL-6,TNF-?and LEP protein in the high-concentration Xianling Gubao group were lower than those in the IL-1?group,and the difference was statistically significant(P<0.05).There was no significant difference in SOX9 protein between the two groups(P>0.05).Compared with the low-concentration Xianling Gubao group,the high-concentration drug-containing serum group had higher expression levels of PTGS2,IGF1,IL-6,TNF-?and LEP protein(P<0.05).ConclusionPRP combined with Xian Ling Gu Bao capsule could significantly release the pain and knee function scores for early and middle KOA patients.And the overall effect is better than that of the HA treatment group.In terms of relevant serous inflammatory factors,PRP combined with Xian Ling Gu Bao capsule can reduce the expression levels of IL-1 and TNF-?in serum and regulate the inflammatory response.It can effectively improve knee pain and function and delay the course of KOA.Xianling Gubao Capsule may down-regulate the expression of factors such as PTGS2,IGF1,IL-6,TNF-?and LEP by regulating inflammation-related pathways such as TNF,thereby achieving therapeutic effects.