| Purposes:To explore the core symptoms of kidney-yang deficiency syndrome by Delphi method and screen diagnostic items for the establishment of "Kidney-Yang Deficiency Syndrome Scale".To evaluate the reliability,validity and applicability of "Kidney-Yang Deficiency Syndrome Scale" for clinical kidney-yang deficiency syndrome differentiation diagnosis.To explore the risk factors and clinical characteristics of primary osteoporosis with kidney-yang deficiency,and to predict the possible causative genes of primary osteoporosis with kidney-yang deficiency.Material and methods:1 Construction of the “Kidney-Yang Deficiency Syndrome Scale” based on the Delphi methodA total of 31 clinical experts of traditional Chinese medicine from 20 tertiary first-class Chinese medicine hospitals in 10 provinces and cities across the country were selected,covering 15 professional fields.Two rounds of structured network consultation questionnaires were issued to analyze the positive coefficient of experts,the authority coefficient of experts and the coordination coefficient of expert opinions.According to the three indicators of arithmetic mean,coefficient of variation and full score rate of each item,the items were screened by boundary value method.2 Reliability and validity evaluation of the “Kidney-Yang Deficiency Syndrome Scale”Using the convenience sampling method,a total of 1100 patients who attended outpatient clinics and medical checkups at the Liaoning University of Chinese Medicine Affiliated Hospital between February 2020 and June 2021 were selected as respondents.The results were compared with the results of independent and consistent diagnosis by two senior chief Chinese medicine practitioners to test the reliability of the Kidney Yang Deficiency Evidence Diagnostic Scale,and the structural validity was tested by exploratory factor analysis.3 Study of risk factors for primary osteoporosis of the kidney-yang deficiency typePatients aged ≥20 years were recruited between November 2019 and April 2021,and those diagnosed with POP by T-value and low bone mass(LBM)by Z-value were included in the study based on the BMD diagnostic criteria for POP,and patients with POP and LBM were recorded together as abnormal bone mass(ABM).Finally,all study subjects were divided into a kidney-yang deficiency ABM group,a non-kidney-yang deficiency ABM group,and a normal control group.According to the different diagnostic criteria,the respondents with T diagnosis were divided into the kidney-yang deficiency POP group,the non-kidney-yang deficiency POP group,and the T normal control group.A 30-item questionnaire related to personal history and lifestyle was conducted for all enrolled patients,including:gender,age,monthly income,dietary preferences,age at menarche,number of pregnancies,history of miscarriage,alcohol,tea,and milk consumption.The results were analyzed for between-group differences,and one-way and multi-way logistic regression analyses were performed for statistically significant difference analysis results.4 Study of clinical characteristics of primary osteoporosis with kidney-yang deficiency typeSixty investigative subjects recruited at the orthopedic outpatient clinic of the Affiliated Hospital of Liaoning University of Chinese Medicine between November 2019 and April2021 were selected and naturally divided into POP kidney-yang deficiency group,POP non-kidney-yang deficiency group,non-POP kidney-yang deficiency group and normal control group according to their diagnosis.Fasting peripheral venous blood was drawn from the investigated subjects,and physicochemical examinations of P1 NP,BALP,β-CTX,Irisin,OCN,and MSTN were performed.Analysis of variance,correlation test,and construction of unordered multicategorical logistic model were used to study the distribution characteristics of each index in the kidney-yang deficiency type POP.5 Whole-exome sequencing study of primary osteoporosis with kidney-yang deficiency typeIn this study,8 female patients with kidney-yang deficiency type POP were selected as the case group and 8 healthy females of the same age group were selected as the healthy control group,and DNA was extracted from peripheral venous blood,and the DNA samples were quality-checked and subjected to whole-exon capture and high-throughput sequencing,and the sequencing results were evaluated for quality and variation analysis,and then the genetic variants associated with the disease were screened by comparison between groups,and finally bioinformatics analysis and NCBI literature review were performed on the variant genes to further identify the possible causative genes.Results:1.Two rounds of Delphi method issued 31 and 31 questionnaires,respectively.The positive coefficients of experts were 100 % and 90.32 %.The authority coefficients of experts were0.87 and 0.88,respectively.The coordination coefficients of expert opinions were 0.185 and0.342.Nine items were deleted and three items were added according to the boundary value method.2.A total of 1100 natural population samples were collected in this survey,including 913 valid samples.The age range of the respondents was 16-97 years old,with an average age of(57.55±15.991)years old.There were 373 males(38.3%),with an average age of(54.84±16.419)years old,and 600 females(61.7%),with an average age of(59.23±15.496)years old.The total Cronbach “s Alpha of the 10 items used by 913 patients was 0.937,and the Spearman-brown correlation coefficient R=0.943,suggesting that the scale had good internal consistency.The results of adaptability test showed that the KMO test value was0.952,the first factor analysis characteristic value was 6.937,and the cumulative variance contribution rate was 69.37%.Kappa consistency test,test coefficient=0.650,P<0.05,statistically significant,suggesting that the patient”s self-evaluation and doctor dialectical two dialectical methods are in good agreement,"Kidney-Yang Deficiency Syndrome Scale" has good reliability and validity.3.A total of 106 subjects were included in the study of risk factors.According to the different diagnosis of ABM/POP,kidney-yang deficiency syndrome/non-kidney-yang deficiency syndrome,the subjects were divided into 58 kidney-yang deficiency ABM group,24non-kidney-yang deficiency ABM group and 24 normal control group.There were 47 patients in kidney-yang deficiency POP group,14 in non-kidney-yang deficiency POP group and 6 in normal control group.The analysis of variance showed that the kidney-yang deficiency type ABM group had a higher risk for "age" "sex" "age at menarche" "physical activity level" "stool characteristics" "dietary preferences" "black tea consumption" "milk consumption" coffee " and " alcohol "(P<0.05),while the kidney-yang deficiency POP group differed in" gender "" age at menopause "" age at menopause " and " alcohol "(P<0.05)." age at menopause "" physical activity level "" stool characteristics "" black tea consumption " and" alcohol consumption."alcohol consumption"(P<0.05).Univariate logistic regression showed that "age"(OR=1.503;95%CI:1.025,1.083,P< 0.01),"sex as female"(OR=8.244;95%CI:2.800,24.272,P<0.01),"age at menarche"(OR=1.396;95%CI:1.073,1.815,P<0.05),"thin stools"(OR=8.936;95%CI:2.382,33.520,P<0.01),"sticky stools"(OR=7.096;95%CI:1.420,35.467,P<0.05)as risk factors.In the POP group,"female gender"(OR=18.409;95%CI:3.472,97.595,P<0.01)and "loose stools"(OR=8.00;95%CI:1.601,39.967,P <0.05)were risk factors and "increasing age at menopause"(OR=0.60;95%CI:0.423,0.851,P < 0.01)were protective factors.Multifactorial logistic regression analysis showed that "milk consumption 2-6 times per week"(OR= 0.09;95%CI:0.011,0.76,P<0.05),"meat and vegetarian" dietary preferences(OR=0.13;95%CI:0.018,0.945,P<0.05),and "high intensity physical activity level"(OR=0.096;95%CI:0.012,0.793,P<0.05)were protective factors for kidney-yang deficiency type ABM;"high intensity physical activity"(OR=0.093;95%CI:0.013,0.683,P < 0.05)was a protective factor for the kidney-yang deficiency type POP.4.Irisin(OR=0.92;95%CI:0.846,1.001,P<0.05),BMI(OR=0.765;95%CI:0.602,0.971,P<0.05)levels were associated with lower risk of kidney-yang deficiency POP as a protective factor for kidney-yang deficiency POP,and BALP(OR=1.105;95%CI:1.008,1.211,P<0.05),and age(OR=1.176;95%CI:1.039,1.332,P<0.05)were associated with a higher risk of kidney-yang deficiency-type POP.5.A total of 550,933 SNP variant loci and 36,262 In Del variant loci were detected in 16 samples by whole-exome sequencing and variant site analysis.After rare mutation screening,deleteriousness screening,inter-group comparison screening and ACMG classification,83 candidate genes were finally obtained.Enrichment analysis of the candidate genes revealed that they are involved in biological processes such as positive regulation of transcription and DNA templating,and are involved in regulating protein digestion and absorption,purine metabolism and ECM-receptor interactions.The candidate genes were ranked for gene-disease association,and the top 25 candidate genes for association were included to review the relevant literature in NCBI,and the possible pathogenic genes for Yang deficiency type POP were further identified as NDUFA8,CREB1,IAP,KAT2 B,Fox03.Conclusions:1.Experts are highly active and authoritative.Finally,a series of symptoms of "Deficiency Cold" were selected as important symptoms of kidney-yang deficiency syndrome.Ten syndrome items of “aversion to cold”,“cold limbs”,“soreness and lumbago”,“weakness and weakness of knees”,“pale complexion”,“mental malaise”,“clear and long urine”,“frequent urination at night”,“five more diarrhea” and “hyposexuality” were used as diagnostic items of kidney yang deficiency syndrome to construct a “Kidney-Yang Deficiency Syndrome Scale”.2.The "Kidney-Yang Deficiency Syndrome Scale" has good reliability and validity,with no special training,rapid assessment,high sensitivity.3.The Kidney-Yang Deficiency POP has obvious acquired risk factors,including "gender(female)","age","stool characteristics(thin stool)","eating Leftover food(eat frequently)" may be the main risk factor for kidney-yang deficiency type POP,and "milk consumption(drink frequently)" may be the protective factor for kidney-yang deficiency type POP.4.serum Irisin and serum OCN were highly consistent with the high bone turnover status of OP and maintained a significant positive correlation,and they were also significantly positively correlated in the group of elderly kidney yang deficiency evidence,suggesting a significant consistency in energy metabolism between the two,and their increased levels were associated with high metabolic status in vivo.Irisin and BMI were protective factors for kidney yang deficiency type POP,and BALP and age Irisin and BMI were protective factors for kidney-yang deficiency POP,and BALP and age were risk factors for kidney-yang deficiency POP.5.Whole exome sequencing revealed a large number of genetic variant loci in patients with renal yang deficiency type POP,and the variability of mutant loci compared with healthy individuals was obvious.The differential genes of renal yang deficiency type POP were significantly associated with energy metabolism,and NDUFA8,CREB1,IAP,KAT2 B,and Fox03 might be the causative genes in patients with renal yang deficiency type POP.The OCN→c AMP/PKA→CREB1→PGC-1α→FNDC5/Irisin pathway may be responsible for the expression of musculoskeletal traits,regulation of energy metabolism in vivo,and the development of "deficiency cold" symptoms in kidney-yang deficient POP patients.The pathway may be a target pathway for the expression of biological traits,regulation of energy metabolism and the development of "cold deficiency" symptoms in the kidney-yang deficiency POP. |
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