| Objectives:1.To conduct a cross-sectional study of the clinical characteristics of patients with kidney deficiency and blood stasis evidence and damp-heat stasis evidence and transcription sequencing and DIA proteomics analysis,to comprehensively resolve the biological basis of kidney deficiency and blood stasis in AS.2.We conducted a prospective self-control study to observe the effect of Bushen Qiangji Decoction in the treatment of AS,and comprehensively assess the clinical efficacy of TCM in the treatment of AS in terms of disease activity,physical and chemical indicators,quality of life,and psychological status,etc.Among the screened out merit population,we initially explored the pharmacological efficacy biomarkers of Bushen Qiangji Decoction by quantitative proteomics technology to provide reference data for the precision medicine of AS.Methods:1.Relying on the established ankylosing spondylitis patient registry of the Chinese Traditional Chinese Medicine Rheumatology Registry Information Platform Patients with AS treated at the outpatient and inpatient departments of Guang’anmen Hospital of the Chinese Academy of Traditional Chinese Medicine were consecutively collected from October 2019 to October 2022,while 94 healthy volunteers were recruited at our physical examination center as a control group.The dimensions of kidney deficiency and blood stasis evidence and damp-heat stasis evidence in terms of ①patient disease activity and joint function:Bath Ankylosing Spondylitis Disease Activity Index(BASDAI),Bath Ankylosing Spondylitis Function Index(BASFI),Bath Ankylosing Spondylitis Measurement Index(BASMI),Patient Global Assessment(PGA),Visual Analogue Score(VAS)of Pain,Ankylosing Spondylitis Disease Activity Score(ASDAS);② laboratory-related indicators:complete blood count(CBC),C-reactive protein(CRP),erythrocyte sedimentation rate(ESR);③Patient Reported Outcome scales:ASAS Health Index(ASAS-HI),Functional Assessment of Chronic Illness Therapy-Fatigue(FACIT-F),Work Productivity Activity Impairment(WPAI),Depression Anxiety Stress Scales-21(DASS-21).On this basis,100 AS patients and 10 healthy volunteers were screened for transcriptomic sequencing,while 100 AS patients and 52 healthy volunteers were sequenced for quantitative plasma proteomics,integrating multi-omics to characterize the biological basis of AS patients with kidney deficiency and blood stasis.2.A prospective self-control study was conducted,in which patients with kidney deficiency and blood stasis were treated with the Bushen Qiangji Decoction(Epimedium Herba Epimedium,Semen Cuscutae,Rhizoma Dioscorea,Radix Angelicae Sinensis,Radix Paeoniae Alba,Curcuma Longa,etc.).The effect of the formula was observed at 0,4 and 12 w as the observation time points,and the ASDAS-CRP attainment rate was used as the main efficacy index to observe the effect of the formula in the treatment of AS attainment,and the disease activity and joint function(BASDAI,BASFI,BASMI,PGA,pain VAS score,ASDAS score),laboratory-related indicators(blood count,CRP,ESR),and patient-reported outcome scales(ASAS-HI,FACIT-F,WPAI,DASS-21)based on the dimensions of comprehensive assessment of the clinical efficacy of TCM for AS.On the basis of this,proteomic analysis of plasma samples from 12 patients who achieved clinically significant improvement(≥1.1 improvement in ASDAS-CRP compared to baseline period)after treatment to characterize the biomarkers before and after treatment with Bushen Qiangji Decoction,and to initially explore the pharmacodynamic biomarkers of Bushen Qiangji Decoction to provide reference data for precision medicine of AS.Results:1.biological basis study of patients with AS with kidney deficiency and blood stasis evidence:Study 1:(1)Comparison of basic patient data:A total of 199(60.67%)patients with kidney deficiency and blood stasis evidence and 129(39.33%)patients with damp-heat stasis evidence were included,while 94 healthy volunteers were recruited.The mean BMI of the patients with kidney deficiency and blood stasis was 25.36±6.32 and the HLA-B27 positivity rate was 66.8%;the mean BMI of the patients with damp-heat stasis was 24.79±8.12 and the HLA-B27 positivity rate was 40.3%,with statistically significant differences between the two groups(BMI:P=0.036,HLA-B27:P=0.001).The remaining demographic characteristics were not statistically different between the two groups.(2)Analysis of disease activity characteristics:the mean value of ASDAS-CRP score was 2.62±1.13 and the mean value of BASDAI score was 4.63± 1.85 for patients in the kidney deficiency and blood stasis group;the mean value of ASDAS-CRP score was 2.53± 1.21 and the mean value of BASDAI score was 4.60± 1.89 for patients in the damp-heat and stasis group.(P>0.05).The VAS score of spinal pain in the kidney deficiency and blood stasis group was 6.42±2.17,higher than that of the damp-heat stasis group at 4.53±2.30,with a statistically significant difference(P<0.05);(3)Analysis of the joint function characteristics:there was no statistical difference between the two symptoms.There was no statistical difference.(4)Analysis of the characteristics of inflammatory indexes:AS patients had higher CRP,ESR,Systemic Immunoinflammatory Index(SII)and Systemic Inflammatory Response Index(SIRI)than healthy controls(P<0.05),and there was no statistically significant difference between the two evidence groups(P>0.05).(5)Characterization of platelet-related parameters:platelet count and platelet specific volume(PCT)were higher in AS patients than in healthy controls(P<0.05),and PLT levels were higher in the kidney deficiency and blood stasis group than in the damp-heat stasis group(P<0.05).There was no statistical difference in platelet distribution width(PDW),mean platelet volume(MPV)and large platelet ratio(P-LCR)between the three groups(P>0.05).(6)Analysis of the characteristics of CBC parameters:Statistically significant differences were found between AS patients and healthy controls in PLR,NLR and LMR(P<0.05),but no significant differences were observed between the kidney deficiency and blood stasis group and the damp-heat stasis group(P>0.05).The differences in the remaining indices PMR,PNR and MNR were not statistically significant among the three groups(P>0.05).(7)Analysis of reported outcome characteristics:kidney deficiency and blood stasis evidence was higher than the damp-heat stasis group in terms of ASAS-HI and FACIT-F,with statistically significant differences(ASAS-HI:P=0.038,Facit-F:P=0.00).There was no statistically significant difference between the two groups in terms of emotional state and work ability(P>0.05).(8)Correlation analysis of clinical indicators in patients with AS kidney deficiency and blood stasis:SIRI was positively correlated with CRP,ESR and SII(r 0.7,0.7 and 0.9,respectively,P<0.05).ASAS-HI was positively correlated with PGA and BASDAI,(r both 0.5,P<0.05).Study 2:(1)The transcriptome sequencing results showed that there were 2298 differentially expressed mRNAs between AS and healthy controls,mainly involved in functional pathways such as T-cell signalling pathways,autophagy and Salmonella infection.After normalization of the assay data by TPM,31 significantly differentially expressed genes were identified,with good discrimination between different genders,evidence and disease activity.The main enriched pathways included TNF signaling pathway,IL-17 signaling pathway,NF-κB signaling pathway,osteoclast differentiation and various viral infection pathways,among which CXCL8,NFKB1A,JUN,ICAM1,The genes such as CXCL8,NFKB1A,JUN,ICAM1 and IL1B were involved in several pathways related to inflammation.(2)Quantitative proteomics results showed that there were 723 differential proteins associated with the disease,mainly enriched in functional pathways such as regulation of the actin cytoskeleton,bacterial invasion of epithelial cells,pathogenic E.coli infection and platelet activation,suggesting that the pathogenesis of ankylosing spondylitis may be associated with infection,certain inflammatory conditions or dysbiosis of the intestinal flora.Protein interaction network analysis revealed that RPL6,RPL7,RPL4,RPS5 and other Hub genes have more interactions with each other;there are 788 differential proteins associated with evidence,and the main enrichment pathways include prion disease,regulation of the actin cytoskeleton,local adhesion,platelet activation and other functional pathways,and protein interaction network analysis revealed more interactions between Hub genes such as FN1,GSK3B,POSTN,FYN and SERPINE1.(3)Integrative transcriptomic and proteomic association analysis showed that 78 genes were statistically significant at both mRNA level and protein level,of which 45 genes showed the same trend in expression changes at mRNA level and corresponding protein level,mainly involved in platelet activation,pathogenic intestinal flora infection,actin cytoskeleton regulation and phagocytosis and other related functional The key pathways are mainly enriched in the immune system,endocrine system,digestive system and nervous system,and are associated with a variety of bacterial or viral infectious diseases,cardiovascular diseases,tumours and neurodegenerative diseases,with differential molecules such as ACTB,ACTG1,RIIOA and SYK involved in most of the enriched pathways.2.Exploring the pharmacological targets of Bushen Qiangji Decoction in the treatment of AS with kidney deficiency and blood stasis:(1)General data of patients:106 patients with AS completed clinical observation,of which 77 were male,accounting for 72.6%,the average age was 43.57± 12.31 years,the average age of onset was 32.06± 11.85 years,and 65 were HLA-B27 positive,accounting for 61.3%.(2)Primary outcome index:The compliance rate for ASDAS-CRP<1.3 was 5.66%at week 4 and 10.38%at week 12;the compliance rate for ASDAS-CRP<2.1 was 28.40%at week 4 and 53.09%at week 12.(3)Secondary outcome indicator:①In terms of the efficacy of the TCM symptoms:The evidence points of kidney deficiency and blood stasis at weeks 4 and 12 were significantly lower than those at baseline(P<0.05),and the evidence points at week 12 were significantly lower than those at week 4(P<0.05).The total effective rate of TCM symptoms at week 12 was 51.9%,and the difference in the total effective rate of TCM symptoms at different time points was statistically significant(P<0.05).②In terms of ASAS20/40 response rate:At week 4,the ASAS20 response rate was 22.6%;the ASAS40 response rate was 11.3%;at week 12,the ASAS20 response rate was 56.6%;the ASAS40 response rate was 27.4%.③In terms of disease activity and joint function assessment:In terms of ASDAS-CRP scores,the ASDAS-CRP improved significantly at week 12 compared to the baseline period and week 4(P<0.05);in terms of BASDAI scores,there was a significant improvement at weeks 4 and 12 compared to baseline(P<0.05);in terms of nocturnal pain VAS,spinal pain VAS and PGA scores,there was a significant decrease at weeks 4 and 12 compared to baseline(P<0.05),and the differences in each score were statistically significant at weeks 4 and 12(P<0.05);in terms of BASFI scores,there was a statistically significant decrease at weeks 4 and 12 compared to baseline(P<0.05);and in terms of BASFI scores,there was a statistically significant decrease at weeks 4 and 12 compared to baseline(P<0.05);in terms of BASFI scores,significant improvements were observed at weeks 4 and 12 compared to baseline(P<0.05);no significant differences were observed in BASMI scores before and after treatment(P>0.05).④In terms of laboratory indicators:CRP levels decreased significantly at weeks 4 and 12 compared to baseline(P<0.05);ESR decreased significantly at weeks 4 and 12 compared to baseline(P<0.05),and ESR levels decreased significantly at week 12 compared to week 4(P<0.05);SII and SIRI indicators were significantly lower at week 12 compared to baseline and at week 4(P<0.05);PLT levels were significantly lower at weeks 4 and 12 compared to baseline(P<0.05);PCT and PLR levels were not statistically significantly different before and after treatment(P>0.05);⑤In terms of patient-reported outcome assessment:ASAS-HI and Facit-F were significantly improved at week 4 and 12 compared with the baseline period(P<0.05).Among the 17 dimensions included in the ASAS-HI,the most significant improvements in functional aspects such as pain,energy and power,and maintenance of posture were observed after 12 weeks of treatment with the TCM.(4)In terms of safety evaluation:No serious adverse events occurred in all patients and there was no statistical difference in the comparison of liver and kidney function before and after treatment.(5)Pharmacodynamic target analysis:Quantitative DIA proteomic analysis of pre-and post-treatment plasma from the euthymic population that achieved clinically important improvements screened a total of 434 differential proteins with functions reported to be associated with pathways such as metabolism and pathogenic intestinal flora infection,of which molecules such as PSMD1,PSMD3,RELA,MPO,RHEB,CXCR4 and TEC may be potential pharmacodynamic biomarkers,mainly closely related to functions such as inflammation,osteoblast differentiation and metabolism.Conclusion:1.Kidney deficiency and blood stasis evidence as the basic type of AS was significantly different from healthy controls and patients with damp-heat stasis evidence in terms of inflammation-related index,platelet parameters,spinal pain VAS,PLT count,ASAS-HI,and FACIT-F.Integrating transcriptomic and proteomic sequencing,we found a large number of differences in biological molecular characteristics among AS patients and different symptoms,which are important in the exploration of the pathogenesis and therapeutic targets of AS.2.Bushen Qiangji Decoction is clinically effective in treating patients with AS with kidney deficiency and blood stasis,and can improve patients’ clinical symptoms in terms of disease activity,joint function,laboratory indicators,and multi-dimensional patient-reported outcome scales based on clinical application with good safety.Proteomic analysis identified PSMD1,PSMD3,RELA,MPO,RHEB,CXCR4 and TEC as potential pharmacological biomarkers of Bushen Qiangji Decoction. |
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