Molecular Mechanism of Prescription for the Syndrome of Cold-Dampness Obstructing the Lung in the Treatment of COVID-19 Based on Network Pharmacology

Objective:To explore the molecular mechanism of the prescription for the syndrome of cold-damp-ness obstructing the lung in the treatment of corona virus disease 2019(COVID-19).Methods:The medicinals for the treatment of the syndrome of cold-dampness obstructing the lung,such as Cangzhu(Rhizoma Atractylo-dis),Chenpi(Pericarpium Gitri Reticulatae),Houpo(Cortex Magnoliae Officinalis),Huoxiang(Herba Agastachis),Caoguo(Fructus Tsaoko),Mahuang(Herba Ephedrae),Qianghuo(Rhizoma et Radix Notoptery-gi),Shengjiang(Rhizoma Zingiberis Recens),Binlang(Semen Arecae)in the Diagnosis and Treatment Pro-gram of COVID-19(Trial Version 6)were taken as research subjects,and the combination of these nine me-dicinals can be called Hanshi Zufei Fang(寒湿阻肺方,HSZFF).The active components and targets of each single Chinese materia medica was screened and obtained through the Traditional Chinese Medicine Systems Pharmacology(TCMSP)database.The target information related to COVID-19 was retrieved through the Gene-Cards disease-related target database.The medicinal prediction targets were mapped to the disease target to ob-tain the intersection targets.The DAVID database was applied to perform gene ontology(GO)enrichment anal-ysis and kyoto encyclopedia of genes and genomes(KEGG)pathway analysis on the targets;GraphPad Prism 5.0 software was applied to plot the biological process(BP)of GO enrichment analysis,cellular component(CC),molecular function(MF)histograms;OmicShare online software was applied to make KEGG advanced bubble chart;Cytoscape software was applied to visualize the interaction with the targets and Chinese materia medica-components-targets results.Results:Totally 56 key active components of 9 Chinese materia medica for cold-dampness obstructing lung syndrome were screened,and 55 targets were obtained.The results of GO and KEGG enrichment analysis showed that the compound prescription mainly regulated the body's immune re-sponse and reduced inflammation by regulating such signaling pathways of inflammatory response and immune regulation as TNF signaling pathway,HIF-1 signaling pathway,Toll-like receptor signaling pathway,infuenza A signaling pathway,T cell receptor signaling pathway.Conclusion:HSZFF can eliminate infl ammation and inhibit virus by regulating immune inflammatory factors closely related to the occurrence and development of diseases through multi-component and multi-target.

Efficacy of TCM Fumigation Using Dampness-and-cold-dispelling Formula Combined with Methotrexate and Leflunomide Treating Rheumatoid Arthritis with Cold-dampness Bi Syndrome

Background:Rheumatoid arthritis(RA),a chronic autoimmune disease,has a high incidence and disability rate,causing patients significant discomfort.Although several medicines can be effective,they are also associated with significant adverse effects.In contrast,fumigation as one of the most often used traditional Chinese medicine(TCM)external therapy has shown both efficacy and safety with less side effect.In light of this,we comple-ment western medicine treatment with TCM fumigation therapy to improve patients’clinical efficacy,alleviate symptoms,and improve prognosis.Objective:To investigate the therapeutic effect of TCM fumigation and modern medicine combined therapy in treating RA patients with cold-dampness Bi syndrome.Methods:A single-center,randomized,controlled study was designed.From January 2022 to December 2022,a total of 60 RA patients with cold-dampness Bi syndrome were enrolled in the study.The control group(30 cases)received conventional modern medicine treatment with methotrexate for 4 weeks,while the observation group(30 cases)for 4 weeks received a combination of TCM fumigation treatment and conventional modern medicine.The effects of the two groups were comprehensively compared,including the changes in TCM symptom scores and laboratory indicators,as well as the use of visual analogue scale(VAS)and health status rating scale(HAQ)before and after treatment.Results:The total effective rate of the observation group was 93.3%,which was significantly higher than that of the control group,which is 70%(P<0.05).Before treatment,there were no significant differences in joint functional activity,pain visual analogue scale(VAS),health assessment questionnaire(HAQ-DI)and laboratory indexes between the two groups(P>0.05).After treatment,each group compared to their initial condition,respectively,showed major improvement of joint functional activity and significantly decreased VAS,HAQ-DI,and RA-related biomarkers(P<0.05);inter-group comparison of these indicators showed significanty further enhanced effect of TCM fumigation combined therapy on the RA-related biomarkers and joint functional activity of patients(P<0.05),yet the control group receiving only modern medicine showed better results of the VAS and HAQ-DI.Conclusion:The TCM fumigation combined therapy is effective to treat RA patients with cold-dampness Bi syn-drome,and to enhance their life quality,improving the joint function,and reducing inflammation.

The Diagnostic Criteria of Blood-Stasis Syndrome:Considerations for Standardization of Pattern Identification

Pattern identification （PI）, also called Bian Zheng （辨证）, syndrome differentiation, pattem diagnosis, or pattem classification, is the basic principle and the key concept of Chinese medicine （CM）. The core of PI is CM syndrome, on which CM theory, therapeutic method, prescribing formula and the use of Chinese herbal medicine are basically based. PI, in fact, is another classification method anticipated to improve the clinical efficacy. How to make an exact PI seems to be very important for taking full advantage of PI in clinical practice. Therefore, the establishment of diagnostic criterion of pattern has been the prerequisite for the standardization of PI. In recent years, a lot of diagnostic criteria of different CM patterns have been formulated. Taking the diagnostic criteria for blood-stasis syndrome as a model, the methodologies and considerations in establishing a pattern diagnostic criterion were discussed in this paper, which might be of great reference value in future PI standardization research.

Expression of T-Helper 17 Cells and Signal Transducers in Patients with Psoriasis Vulgaris of Blood-Heat Syndrome and Blood-Stasis Syndrome

Objective： To investigate the levels of cytokines related to T-helper （Th） 17 cells in serum and signal transducers in the psoriatic lesions of patients with psoriasis vulgaris of blood-heat syndrome （BHS） and blood-stasis syndrome （BSS）. Methods： Sixty patients with psoriasis vulgaris were divided into the BHS and BSS groups according to the syndrome differentiation of Chinese medicine （CM）. Ten healthy subjects were considered as the control group. Cytokine levels of interleukin （IL）-17, IL-23 and IL-6 in serum were determined by enzyme-linked immunosorbent assay. Expression levels of signal transducer and activator of transcription 3 （STAT3）, p38-mitogen-activated protein kinase （MAPK） and STAT6 in the psoriatic lesions were determined using immunohistochemistry （IHC）, Western blot, and real-time quantitative reverse transcription polymerase chain reaction, respectively. Results： Production of IL-17, IL-23 and IL-6 in the BHS group and BSS group were significantly increased compared with those in the control group （P〈0.05）. Levels of IL-17 and IL-23 in the BHS group were higher than those in the BSS group （P〈0.05）. Compared with the control group, IHC positive expressions and protein expressions of STAT3 and p38-MAPK, and the STAT3 mRNA expressions in the BHS and BSS groups were significantly higher （P〈0.05 or P〈0.01）. The protein expression of STAT3 in the BHS group was significantly higher than that in the BSS group （P〈0.05）. Conclusions： Cytokines in serum and signal transducers in the psoriatic lesions alter with various CM syndromes of psoriasis. The results provide scientific basis for the treatment based on syndrome differentiation of CM in treating psoriasis vulgaris.

Relationship between Platelet Activation Related Factors and Polymorphism of Related Genes in Patients with Coronary Heart Disease of Blood-stasis Syndrome

Objective： To comparatively study the expressive conditions of platelet activation related factors （GPⅠb, GPⅡb-Ⅲa and GMP-140） in healthy subjects and patients with coronary heart disease （CHD） of blood-stasis （BS） or non-blood-stasis （non-BS） syndrome, and to analyze the relationship between the activities of various glycoproteins and the polymorphism of genes. Methods： With case control design adopted, patients with the CHD （40 of BS, 37 of non-BS） and 39 healthy subjects for control, all fitting to the inclusion criteria, were selected in this study. The number of affected coronary branches was recorded by the contrast examination. The mean fluorescence intensity （MFI） of GPⅠb, GPⅡb-Ⅲa, and GMP-140 （CD42b, CD61, CD62p） in patients and healthy persons was measured with flow cytometry, the polymorphism of HPA-3 gene was detected by Taqman probe technique and that of HPA-2 gene was determined by gene sequencing. Results： MFI of CD61 and CD62p was higher in the CHD patients than in the healthy control, which was also higher in patients of BS syndrome than in patients of non-BS syndrome （P〈0.05）; MFI of CD42b was lower in the CHD patients than in the healthy control （P〈0.05）, but showing insignificant difference between BS and non-BS syndrome （P〉0.05）; at the same time, no significant difference of all the above-mentioned three MFI could be found in patients with various numbers of affected coronary branches, neither in patients with different genotypes at GPⅡb HPA-3 and GPⅠb HPA-2 polymorphism loci （P〉0.05）. Conclusion： （1） The activities of GP Ⅱ b-Ⅲa and GMP-140 were obviously increased in the genesis and developing process of CHD and CHD of BS syndrome, and so they could be taken as one of the objective indexes for microscopic diagnosis of BS syndrome. （2） The level of GPⅠb was lower in CHD patients than in healthy persons, but it was not a sensitive indicator for BS syndrome of CHD. （3） Levels of GP Ⅱb-Ⅲa, GPⅠb and GMP-140 were not related with the number of affected coronary branches in CHD patients. （4） The changes in amino-acids expression induced by the two loci brought no significant influence on GPⅠb and GP Ⅱb-Ⅲa activities.

Differential Expression of MicroRNA in Endothelial Cells Incubated with Serum of Hypertension Patients with Blood-Stasis Syndrome

Objective： To screen out blood-stasis syndrome （BSS）-associated microRNA and therefore determine the possible target for treating hypertension. Methods： A high-energy sequencing method and digital gene expression sequencing theory were adopted to sequence microRNA （miRNA） and messenger RNA （mRNA）, and to determine differential expression in human umbilical vein endothelial cells incubated with serum samples from hypertension patients with or without BSS, and healthy controls. The results were confirmed using gene prediction software. Results： A total of 13 miRNAs and 11 mRNAs showed statistical difference both in the BSS/normal groups and BSS/non-BSS groups, respectively. Four pairs of target mRNNmiRNA were identified： FRMD4Nhsa-miR-34a, MAP3K14/hsa-miR-34a, PER1/hsa-miR-34a, and FGF2/hsa-miR-132. Conclusion： Four mRNNmiRNA pairs mentioned above seem to be involved in pathogenesis and maintenance of hypertension with BSS.

Circulating Levels of Inflammatory Cytokines in Patients with Psoriasis Vulgaris of Different Chinese Medicine Syndromes

Objective： To investigate whether the serum levels of inflammation-related cytokines might be different between the healthy individuals and the psoriatic patients diagnosed of three varied Chinese medicine （CM） syndromes [blood-stasis syndrome （BSS）, blood-dryness syndrome （BDS） and wind-heat syndrome （WHS）]. Methods： A total of 62 psoriatic patients were recruited and assigned to 3 groups according to their CM syndromes, including 27 patients of BSS, 21 of BDS and 14 of WHS. Another 20 sex- and age- matched healthy subjects were enrolled into the control group. Serum concentrations of multiple cytokines, including monocyte chemotactic protein-1 （MCP-1）, macrophage inflammatory protein-1α （MIP-1α）, soluble CD40 ligand （SCD40L）, tumor necrosis factor-α （TNF-α）, epidermal growth factor （EGF）, interleukin-8 （IL-8）, interleukin-17 （IL-17）, interferon γ inducible protein-10 （IP-10） and vascular endothelial growth factor （VEGF）, were measured by a multiplexed flow cytometric assay. Results： The circulating levels of MIP-1α, TNF-α, IL-8, and IP-10 were significantly increased in the psoriatic patients compared with the healthy controls （P〈0.01）. Male and female patients tended to have higher serum levels of MCP-1 and IP-10, respectively （P〈0.05）. Interestingly, compared with the control group, 6 out of the 9 cytokines （MCP-1, MIP-1α, TNF-α, EGF, IL-8 and IP-10） were substantially increased in the BSS group （P〈0.05 or P〈0.01）, whereas only MIP-1α and IL-8 levels were elevated in the BDS group （P〈0.05 or P〈0.01） concurrent with lowered concentrations of SCD40L and IL-17 （P〈0.05）. In the WHS group, MIP-1α was the only cytokine whose level was evidently increased （P〈0.01）, in contrast to IL-17 which was decreased as compared with the control （P〈0.05）. The psoriatic patients overall owned higher levels of MIP-1 a and IL-8 in the circulation which were comparable among the 3 groups of CM syndromes （P〈0.01）. In contrast, TNF-α level of the BSS group was the highest among the three （P〈0.01）, followed by the BDS and the WHS groups. Conclusions： The expression profiles of cytokines in the circulation might not be necessarily identical for psoriatic patients with different CM syndromes. Accordingly, the serum concentrations of certain cytokines could potentially be used as the ancillary indices for the clinical classification of psoriatic CM syndromes.

Differential Gene Expression Profiles in Coronary Heart Disease Patients of Blood Stasis Syndrome in Traditional Chinese Medicine and Clinical Role of Target Gene

Objective:To investigate the differential gene expression profiles in coronary heart disease(CHD) patients of blood-stasis syndrome(BSS) by oligonucleotide microarray technique,and the clinical significance of target gene.Methods:Subjects were assigned to CHD patients with BSS(n=8),CHD patients without BSS (n=8),and BSS patients without CHD(n=8) based on coronary angiography and the diagnostic criteria of BSS. The sex- and age-matched healthy volunteers(n=8) were enrolled as the control group.Venous blood s...

Logistic Regression Analysis of Damp-Heat and Cold Damp Impeding Syndrome of Rheumatoid Arthritis:A Perspective in Chinese Medicine

Objective： To investigate a method for quantitative differential diagnosis of damp-heat and cold-damp impeding syndrome of rheumatoid arthritis （RA） in Chinese medicine （CM）. Methods： Laboratory parameters were collected from 306 patients with RA. The clinical symptoms and laboratory parameters were compared between patients with these two syndromes （158 with RA of damp-heat impeding syndrome, and 148 with RA of cold-damp impeding syndrome）, and a regression equation was established to facilitate discrimination of the two RA syndromes. Results： There were significant differences in disease activity score in 28 joints [DAS28 （4）], erythrocyte sedimentation rate （ESR）, white blood cell count （WBC）, C-reactive protein （CRP）, platelet count （PLT）, albumin （ALB） and globulin （GLB） between the two syndrome of RA （P〈0.05）. Logistic regression analysis showed that the parameters ESR, WBC, CRP, joint pyrexia, joint cold, thirst, sweating, aversion to wind and cold, and cold extremities were statistically useful to discriminate damp-heat from cold-damp impeding syndrome. The regression equation was as follows： P=1/{1＋exp[-（3.0-0.021X1-0.196X2-0.163X3- 1.559X4＋1.504X5-0.927X6-1.039X7＋1.070X8＋1.330X9）]}. The independent variables X1-X9 were ESR, WBC, CRP, hot joint, cold joint, thirst, sweating, aversion to wind and cold, and cold limbs. A P value 〉 0.5 signified cold-damp impeding syndrome, and a P value 〈 0.5 signified damp-heat impeding syndrome. The accuracy was 90.2%. Conclusion： The regression equation may be useful for discriminating damp-heat from cold-damp impeding syndrome of RA.

Study on the Relationship between Blood Stasis Syndrome and Clinical Pathology in 227 Patients with Primary Glomerular Disease

To investigate the relationship between the severity of Chinese medicine(CM) bloodstasis syndrome(BSS) with clinical features and renal lesion indexes of the primary glomerular disease. Methods:An epidemiological survey was conducted to collect the data of 227 patients diagnosed as chronic primary glomerular diseases,and their severity of BSS were scored three days before renal biopsies were performed.The following clinical indexes were analyzed:age,course of glomerular diseases,24-h urine protein ration(Up...

Constructing protein-protein interaction network of hypertension with blood stasis syndrome via digital gene expression sequencing and database mining

OBJECTIVE： To construct a protein-protein interaction（PPI） network in hypertension patients with blood-stasis syndrome（BSS） by using digital gene expression（DGE） sequencing and database mining techniques.METHODS： DGE analysis based on the Solexa Genome Analyzer platform was performed on vascular endothelial cells incubated with serum of hypertension patients with BSS. The differentially expressed genes were f iltered by comparing the expression levels between the different experimental groups. Then functional categories and e nriched pathways of the unique genes for BSS were analyzed using Database for Annotation, Visualization and Integrated Discovery（DAVID） to select those in the enrichment pathways. I nterologous Interaction Database（I2D） was used to construct PPI networks with the selected genes for hypertension patients with BSS. The potential candidate genes related to BSS were identif ied by comparing the number of relationships among genes. Confi rmed by quantitative reverse transcription-polymerase chain reaction（q RTPCR）, gene ontology（GO） analysis was used to infer the functional annotations of the potential candidate genes for BSS.RESULTS： With gene enrichment analysis using DAVID, a list of 58 genes was chosen from the unique genes. The selected 58 genes were analyzed using I2 D, and a PPI network was constructed. Based on the network analysis results, candidate genes for BSS were identifi ed：DDIT3, JUN, HSPA8, NFIL3, HSPA5, HIST2H2 BE, H3F3 B, CEBPB, SAT1 and GADD45 A. Verif ied through qRT-PCR and analyzed by GO, the functional annotations of the potential candidate genes were explored.CONCLUSION： Compared with previous methodologies reported in the literature, the present DGE analysis and data mining method have shown a great improvement in analyzing BSS.