The Study Of Mechanism Of Polyphyllin Ⅶ In Preventing Hypoxic Pulmonary Hypertension In Rats Based On Transcriptomics

Objective:Hypoxic Pulmonary Hypertension(HPH),a progressive and destructive complication of various diseases such as chronic obstructive pulmonary disease and interstitial lung disease,is an important factor causing morbidity and death of various types of lung/heart disease patients.Pulmonary vascular remodeling is the main pathological feature,and the proliferation,migration,autophagy and inflammatory reaction of pulmonary artery smooth muscle are the main reasons to promote vascular remodeling.Polyphyllin Ⅶ(PPⅦ)has been proven to have anti-inflammatory effects.At the same time,it can inhibit the proliferation and migration of a variety of cells and promote autophagy.But PPⅦ has not been studied in HPH yet.Therefore,this study aims to study whether PPⅦ can inhibit pulmonary small blood vessel remodeling,prevent HPH,then based on complete transcriptome sequencing data,to explore the specific molecular mechanism of PPⅦ in the prevention of HPH from the transcriptome level,which providing a theoretical basis for in-depth understanding of the pathogenesis of HPH and new potential drugs for the effective prevention and treatment of HPH.Methods:1.HPH rat model construction and testing:(1)Experimental animal grouping and HPH rat model construction:18 SD rats were randomly divided into 3 groups(6 in each group):the Con group rats were fed under normoxia for 21 days;Rats in the HPH group were fed in a hypoxic environment(10%O2)for 8 h/day,16 h/day in a normoxic environment,and continuously fed for 21 days;rats in the PPⅦ group were fed under the same conditions as the HPH group,but from the first day of feeding,PPⅦ 1.0 mg/kg was injected intraperitoneally every day and fed continuously for 21 days.(2)HPH rat model test:After the intervention,testing the right ventricular systolic pressure(RVSP),right ventricular hypertrophy index(RVHI)and pulmonary small vessel remodeling in each group of rats to judge whether the HPH rat model is successfully constructed and whether PPⅦ can prevent the occurrence of HPH.2.Rat lung tissue RNA-sequencing and functional analysis:(1)Randomly select the lung tissues of 3 rats from the Con group,HPH group and PPⅦ group,after extracting total RNA and constructing a cDNA library,whole transcriptome sequencing was performed.Then,differential ncRNAs and mRNAs related to HPH or PPⅦpreventing HPH were screened;(2)Based on the results of differential expressed analysis,candidate ncRNAs and mRNAs were obtained;(3)Quantitative Reverse Transcription Polymerase Chain Reaction(qRT-PCR)technology was used to verify the accuracy of sequencing results;(3)Functional enrichment analysis of differentially expressed mRNAs and candidate mRNAs was performed using Metascape,including Gene ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway analysis;(4)Protein-Protein interaction analysis of the differentially expressed mRNAs and candidate mRNAs was performed;(5)Bioinformatics analysis was used to predict targets and the circRNAs-miRNAs-mRNAs ceRNA network,then lncRNAs-miRNAs-mRNAs ceRNA network related to PPⅦ preventing HPH were constructed respectively;(6)Metascape was used to perform functional enrichment analysis of the mRNAs in the ceRNA network.Results:1.Compared with the Con group,hypoxia induction significantly increased the RVSP(16.33±2.36 mmHg vs 28.70±2.93 mmHg,P<0.001),RVHI(0.12±0.01 vs 0.18±0.02,P<0.05)and pulmonary small blood vessel remodeling in the HPH group;and PPⅦ intervention significantly alleviated these changes caused by hypoxia(RVSP:23.73±1.27 mmHg vs 28.70±2.93 mmHg,P<0.05;RVHI:0.18±0.02 vs 0.13±0.03,P<0.05).The results showed that the HPH rat model was successfully constructed;and PPⅦ can reduce RVSP,RVHI and pulmonary small vessel remodeling in HPH rats,thereby PPⅦ could prevent the occurrence of HPH.2.According to the threshold of P<0.05 and |log2Fold Change|>1,in the differentially expressed analysis of HPH group vs Con group,71 differentially expressed circRNAs(45 up-regulated,26 down-regulated)and 155 differentially expressed were screened out lncRNAs(85 up-regulated,70 down-regulated),20 differentially expressed miRNAs(15 up-regulated,5 down-regulated),695 differentially expressed mRNAs(534 up-regulated,161 down-regulated)were screened.In the analysis of the differentially expressed RNA between the PPⅦ group and the HPH group,80 differentially expressed circRNAs(45 up-regulated,35 down-regulated),155 differentially expressed lncRNAs(76 up-regulated,79 down-regulated),and 12 differentially expressed miRNAs(7 up-regulated,5 down-regulated),447 differentially expressed mRNAs(201 up-regulated,246 down-regulated)were identified.Based on the differentially expressed genes of HPH group vs Con group and PPⅦgroup vs HPH group,we finally selected 21 differential circRNAs,53 differential lncRNAs,3 miRNAs and 199 differential mRNAs as candidate genes.The qPCR verification results were consistent with the sequencing results.The differential expression trend of candidate genes in the HPH group vs Con group was opposite to that PPⅦ group vs HPH group(PPⅦ reverses the change of candidate genes caused by hypoxia).Therefore,they are likely to be the targets of PPⅦ for preventing HPH.3.The functional enrichment of differential mRNA in HPH group vs Con group is mainly enriched in biological processes such as immune response,inflammatory response,cell migration and proliferation.GO analysis and KEGG functional enrichment analysis were performed on the differential mRNA of the PPⅦ group vs HPH group,then we found that the enrichment results mainly focused on:killing of cells of other organism,cytolysis,cell cycle,immune effect process and IL-17 signaling pathway,etc.GO and KEGG analysis of candidate mRNAs that are differentially expressed in the HPH group vs Con group and PPⅦ group vs HPH found these differential genes mainly affect peptidase regulator activity,cell cycle,proteolysis,apoptosis,ERBB2/3 signaling pathway,non-canonical Wnt signaling pathway,negative regulation of Rho protein signal transduction,extracellular matrix and humoral immune response,etc.4.There are 5204 edges and 400 nodes in the protein-protein interaction network of differential mRNAs between HPH group vs Con group,indicating that there are 5204 interactions between 400 proteins.At the same time,we also constructed a differential protein-protein interaction network of PPⅦ group vs HPH with 1212 edges and 193 nodes.By analyzing the protein-protein interaction of candidate mRNAs,we constructed a protein-protein interaction map with 794 edges and 80 nodes.5.In order to further understand the specific mechanism of PPⅦ inhibiting HPH,we constructed a circRNA-miRNA-mRNA ceRNA regulatory network in candidate RNAs using miRNA as a medium.The figure contains 5 circRNAs,2 miRNAs and 13 mRNAs.At the same time,the lncRNA-miRNA-mRNAceRNA regulatory network was also built,which contains 18 lncRNAs,2 miRNAs and 13 mRNAs.6.We performed GO analysis and KEGG functional enrichment analysis on mRNAs in the ceRNA regulatory network.The enrichment results were:peptidase inhibitor activity,peptidase modulator activity,positive regulation of cell cycle processes,negative regulation of proteolysis,Mitotic spindle,apoptosis-multiple,ERBB2 signaling pathway,ERBB3 signaling pathway,non-canonical Wnt signaling pathway through JNK cascade,non-canonical Wnt signaling pathway through MAPK cascade,negative regulation of Rho protein signal transduction,cell Extra matrix,non-standard Wnt signaling pathway,negative regulation of Ras protein signal transduction and humoral immune response.Conclusion:1.The HPH rat model was successfully constructed.At the same time,PPⅦ can reduce the RVSP,RVHI and pulmonary small blood vessel remodeling in HPH rats.Therefore,PPⅦ could prevent the occurrence of HPH.2.Full transcriptome genes(circRNAs,lncRNAs,miRNAs,mRNAs)were differential expressed in the lung tissues of rats in the Con group,HPH group and PPⅦgroup.These differentially expressed genes may play an important role in the occurrence and development of HPH and the process of PPⅦ preventing HPH.3.The candidate genes differentially expressed in the HPH group vs Con group and the PPⅦ group vs HPH group with opposite changing trends may be the target of PPⅦ in inhibiting the incidence of HPH.4.circRNAs and lncRNAs act as sponges of miRNAs to regulate mRNAs expression and play a role in the occurrence and development of HPH and the prevention of HPH by PPⅦ.Our research provides new ideas for the pathogenesis of HPH,and also provides a new potential drug for the prevention of HPH,but the experimental results still need a large number of clinical samples to verify.