Clinical And Mechanism Study Of Wen San Ding Transdermal Therapy In The Treatment Of Pulmonary Alveolar Proteinosis Based On Multi-Omics

BackgroundPulmonary alveolar proteinosis(PAP)is a diffuse lung disease characterized by the accumulation of alveolar surfactant and dysfunction of alveolar macrophages,which results in progressive dyspnea of insidious onset.PAP is associated with abnormal production or clearance of alveolar surfactant.Autoimmune alveolar proteinosis(aPAP)accounts for>90%of all cases,which is mediated by autoantibodies targeting granulocyte-macrophage colony-stimulating factor(GMCSF).As a rare disease,PAP has a prevalence of at least 7 cases per million individuals in large population studies and affects men,women and children of all ages,ethnicities and geographical locations irrespective of socioeconomic status.In 2018,PAP is included in the first edition of China’s rare diseases list.Therapeutic management of PAP is limited.The current standard of care in PAP is whole-lung lavage(WLL).However,the median duration of benefit,or time to next WLL,was 15 months according to a report.Further research is needed to find emerging therapies and management for PAP.Traditional Chinese medicine(TCM)has a long history of treating diseases and promoting health in China and other Asian countries,which may provide ideas for the treatment of PAP.Although there is no record of this disease in the classical books,according to the literature search and clinical experience,PAP is most likely identified as phlegm and stasis blocking the ligaments.Taking phlegm-releasing herbs can relieve the condition.However,PAP has a long disease duration.Long-term oral administration of such herbal medicines may cause adverse events.For this reason,we innovatively proposed transdermal therapy in the treatment of PAP based on TCM theory.We developed Wen San Ding(WSD),a phlegm-releasing herbal compound.In addition,we adopted Transdermal Therapeutic Systems(TTS),a transdermal drug delivery device which can automatically adjust pressure and temperature.TTS can develop effective drug concentration rapidly at the lesion site,overcoming the problem of low efficiency in TCM transdermal administration.In the preliminary stage,we have treated 5 patients with aPAP using this therapy.After 3 months,their oxygenation function,activity endurance,and quality of life(QOL)have improved.However,the underlying mechanism of the transdermal therapy is still unclear.Since Thomas Roderick created the concept of the genome in 1986,other postgenomics technologies such as epigenomics,transcriptomics and metabolomics have undergone significant development in recent years.These technologies have revolutionized the field of life sciences by enabling the study of biological systems at a molecular level.With the advent of high-throughput sequencing technologies,it has become possible to generate large amounts of data in a relatively short period.The integration of these different omics,also known as multi-omics,allows for a more holistic and integrated analysis of biological systems.By combining information from different omics levels,researchers can obtain a more accurate and comprehensive understanding of the underlying mechanisms of diseases and other biological processes.Therefore,the use of multi-omics has become an increasingly important tools for understanding the complex mechanisms underlying TCM.TCM emphasizes a holistic view,which is in line with the concept of multi-omics.By using multi-omics technologies,researchers can gain a comprehensive understanding of the biological mechanisms of TCM,which can lead to the elucidation of synergistic effects of TCM formulas and optimize their therapeutic efficacy.Based on the previous study,we expanded the sample size of clinical observation.Some important outcomes we compared for the first time,such as pulmonary function tests(PFT),arterial blood gas(ABG),and related serum markers before and after treatment.Meanwhile,mRNA sequencing and DNA methylation microarray were also integrated to analyze the differences in gene expression levels and methylation levels before and after treatment,and to explore the targets and pathways of WSD via TTS in the treatment of pulmonary alveolar proteinosis.ObjectiveThe purpose of this study:1)To observe the clinical efficacy of WSD transdermal therapy in the treatment of autoimmune pulmonary alveolar proteinosis.2)To explore the molecular basis of WSD transdermal therapy using mRNA sequencing and DNA methylation microarray analysis.Methods1.Clinical observation of the WSD transdermal therapy in the treatment of autoimmune pulmonary alveolar proteinosis:We included patients with a clear previous diagnosis of aPAP from January 2018 to December 2022.After they signed an informed consent,we performed a single-arm study to compare the clinical efficacy before and after the WSD transdermal therapy.The clinical observation was divided into 2 parts:clinical manifestations,QOL and exercise capacity.The observation of clinical outcomes included:the TCM efficacy index,the visual analog score(VAS)of cough,mMRC dyspnea score,PFT,ABG,and disease-related serum markers.The observation of QOL and exercise capacity included:St.George’s respiratory questionnaire(SGRQ)and a 6-minute walk test(6WMT).Baseline data were collected from patients before the treatment.Afterward,WSD transdermal therapy was given via TTS.Bilateral Feishu points(BL13)and Geshu points(BL17)were chosen for treatment,administered 5 times per week for 60 minutes each time.After 6 months of treatment,the outcome indicators were evaluated again.During the treatment period,liver and kidney function as well as skin irritation reactions of patients were also monitored.SPSS 25.0 was used for statistical analysis of observational outcomes before and after treatment,to comprehensively evaluate the clinical efficacy and safety of the therapy.2.Multi-omics study of the WSD transdermal therapy in the treatment of autoimmune pulmonary alveolar proteinosis:The peripheral blood samples were collected from patients with aPAP in the clinical study before and after transdermal treatment.The pre-treatment samples were marked as the"pre-treatment group",the post-treatment samples were marked as the "post-treatment group",and healthy blood samples matched strictly by gender and age with the patients were marked as the "volunteer group".Transcriptome sequencing(RNA-seq)and DNA methylation microarray(850K)technologies were integrated to study these samples.The differentially expressed genes and differentially methylated genes before and after the transdermal treatment were screened by biomedical statistics.Using Gene Ontology(GO)and the Kyoto Encyclopedia of Genes and Genomes(KEGG)database to screen the target genes and pathways at the transcriptome and methylation levels,revealing the mechanism of the therapy based on multi-omics approaches.Results1.20 patients with aPAP enrolled in the study underwent 6 months of WSD transdermal therapy.The TCM comprehensive efficacy:Total efficiency rate based on the TCM syndrome score(TCMSS)reached 55%。TCMSS decreased significantly compared with the baseline(P<0.01).Specific syndrome scores such as chest tightness,shortness of breath,cough,sputum,wheezing,cyanosis,and chills improved significantly after treatment(P<0.05).As for clinical manifestations,cough VAS score and mMRC score were significantly reduced after treatment(P<0.05).PFT showed that the percentage of predicted DLco and FVC both improved after treatment(P<0.05).ABG including PaO2,P(A-a)O2,PaCO2,and SaO2 did not show significant statistical differences after treatment(P>0.05),but 72%of patients(8/11)showed an improvement in P(A-a)O2 after the intervention.Serum markers such as total cholesterol and low-density lipoprotein cholesterol also improved significantly after treatment(P<0.05).Regarding QOL and exercise capacity,the SGRQ total score showed a trend of a decrease after treatment,but the difference was not statistically significant(P>0.05).The 6-minute walk distance and resting oxygen saturation did not show significant statistical differences after treatment(P>0.05),but the post-exercise oxygen saturation increased compared to baseline(P<0.05).All patients did not experience abnormal liver or kidney function or significant skin irritation during WSD transdermal therapy.2.Transcriptome analysis revealed that aPAP patients had 2171 differentially expressed genes(394 up-regulated and 1777 down-regulated)compared to healthy volunteers.These genes were enriched in 1051 GO terms and 323 KEGG pathways,related to functions and pathways such as infection,immunity,antigen presentation,and inflammation,and lipid metabolism.After transdermal therapy,420 differentially expressed genes(413 upregulated and 7 downregulated)were identified,which were enriched in 353 GO terms and 256 KEGG pathways related to immunity,antigen presentation,inflammation,and lipid metabolism.A total of 1903 effective genes were identified for WSD transdermal therapy,and the Venn diagram showed that this therapy significantly reversed the expression of 314 differentially expressed genes in aPAP patients,including HLA-DRA,HLA-DRB1,APOA2,CCL1,GZMA,involving functions and pathways such as antigen processing and presentation,Th1 and Th2 cell differentiation,Th17 cell differentiation,cholesterol metabolism,PPAR signaling pathway,chemokine signaling pathway,and cytokine-cytokine receptor interaction.3.Methylation analysis revealed that there were 284 abnormal methylation sites(all hypomethylated)in the peripheral blood of aPAP patients compared to healthy individuals,including 163 hypomethylated genes.After transdermal therapy with WSD,a total of 74 differentially methylated sites were identified(49 hypermethylated and 25 hypomethylated),including 59 differentially methylated genes(39 hypermethylated genes and 20 hypomethylated genes).Combined analysis of DNA methylation and transcriptome revealed that the differential genes in aPAP patients after the transdermal treatment mainly included HLA-DMA and SKAP2,which were related to functions and pathways such as peptide antigen assembly with MHC Ⅱproteins,lymphocyte activation,antigen processing and presentation,Thl and Th2 cell differentiation,and Th17 cell differentiation.Conclusion1.WSD Transdermal therapy via TTS can significantly improve the clinical manifestations of patients with aPAP,such as coughing and chest tightness.It can also restore the patients’ pulmonary diffusion function,increase alveolar effective ventilation,and reduce the patient’s blood lipid levels.It shows great potential in improving the QOL and exercise capacity of aPAP patients.In summary,WSD transdermal therapy via TTS has a high degree of safety and shows a promising clinical application prospect.It is likely to become a novel type of TCM transdermal treatment for aPAP patients.2.The mechanism of WSD transdermal therapy for autoimmune pulmonary alveolar proteinosis may be related to HLA class Ⅱ genes such as HLA-DRA,HLA-DRB1,HLA-DMA,as well as APOA2,CCL1,GZMA,and SKAP2,mainly involving pathways such as antigen processing and presentation,Th1 and Th2 cell differentiation,Th17 cell differentiation,chemokine signaling pathways,cytokinecytokine receptor interactions,lymphocyte activation,cholesterol metabolism,and PPAR signaling pathways.WSD transdermal therapy via TTS may improve the pulmonary autoimmune deficiency and the accumulation of pulmonary surfactant by regulating the mRNA expression levels,DNA methylation levels,and related pathways of these genes,and thus exerting a therapeutic effect.