| Objective:In this study,we investigated the distribution characteristics of TCM syndrome elements in patients with moderate to severe NPDR and their correlation with fundus changes and miRNAs.Further to provide objective and standardized diagnostic basis for the study of syndrome in patients with moderate to severe NPDR by exploring the indexes of fundus changes that correlated with TCM syndrome elements and syndrome patterns from macroscopic and microscopic aspects.Method:1.Selection of syndrome element scale:An expert questionnaire on syndrome element assessment of moderate to severe non-proliferative diabetic retinopathy was developed by consulting the literature,and the Delphi method was used to determine the TCM syndrome element assessment form of moderate to severe non-proliferative diabetic retinopathy.2.Case source:A total of 223 patients with moderate to severe NPDR who visited the Eye Hospital of China Academy of Chinese Medical Sciences were included.The diagnostic quantitative criteria of TCM syndrome elements were used to form a score table of related factors of each syndrome element,and the maximum likelihood method was used to determine the type of syndrome elements in patients,and the distribution rule of syndrome elements and markers was further analyzed.3.Correlation analysis of TCM syndrome elements and fundus changes in patients with moderate to severe NPDR:fundus color photography and OCTA examination of the posterior pole of patients were collected,respectively.Fundus color photography was performed to analyze the area of hemorrhage,exudation and cotton wool spots in the posterior pole of the fundus using AI;OCTA examination was performed to collect the foveal thickness,macular vessel density,peripapillary vessel density,nerve fiber layer thickness,ganglion cell complex,and foveal avascular area of the patient.The correlation between each syndrome and fundus image acquisition index was analyzed by sperman correlation coefficient;Independent variables were screened by establishing a random forest model of sputum dampness syndrome and LASSO regression model,and independent variables were included in the logistic regression model for risk factor analysis.4.The correlation between TCM syndrome and miRNA in patients with moderate to severe NPDR:134 enrolled patients were randomly selected,4 ml of fasting venous blood was collected,serum was separated by centrifugation,and frozen in a-80℃refrigerator.Sixteen healthy individuals were also selected as the normal control group.The expression levels of serum miR-146a and miR-145-5p in patients with moderate to severe NPDR and their expression differences between syndromes were detected by real-time PCR,and their correlation with syndromes and markers was analyzed using sperman correlation coefficient.Independent variables were screened by establishing a random forest model of sputum dampness syndrome and LASSO regression model,and independent variables were included in the logistic regression model for risk factor analysis.Results:1.Syndrome element score scale:223 patients with moderate to severe NPDR completed the TCM syndrome element rating scale for patients with moderate to severe NPDR and the questionnaire.2.TCM syndrome elements:Of the 223 patients with moderate to severe NPDR,there were a total of 13 TCM syndrome elements,including 8 pathological syndrome elements,which were phlegm-dampness(62.3%),fire-heat(58.3%),qi stagnation(53.3%),qi deficiency(46.7%),blood stasis(37.7%),yin deficiency(34.1%),blood deficiency(22.9%),and yang deficiency(20.2%)according to the proportion;there were 5 pathological syndrome elements,which were liver(59.2%),kidney(45.3%),stomach(27.8%),spleen(22.9%),and lung(15.2%)according to the proportion.Syndrome element combination:Results analysis showed that 223 patients with moderate to severe NPDR had 139 inclusion of deficiency and deficiency(62.3%),which was the most frequent of all syndrome element combinations,followed by 69 cases(30.9%)of pure no deficiency syndrome and 5 cases(2.2%)of pure deficiency syndrome.Common distribution:Cluster analysis and correlation analysis were used to combine the disease location syndrome elements and disease syndrome elements,and a total of 21 common compound factors were obtained,the top three of which were liver qi stagnation syndrome,kidney qi deficiency syndrome,and yin deficiency and fire excess syndrome.3.Correlation between syndrome elements and fundus images:The correlation between TCM syndrome elements and fundus color photography related indicators in patients with moderate to severe NPDR was analyzed using sperman correlation coefficient,and the results suggested that fire-heat syndrome elements,qi stagnation syndrome elements,blood stasis syndrome elements,yin deficiency syndrome elements,and qi deficiency syndrome elements in patients with moderate to severe NPDR were positively correlated with fundus hemorrhage area(rs>0,P<0.05),indicating that patients with fire-heat syndrome elements,qi stagnation syndrome elements,blood stasis syndrome elements,yin deficiency syndrome elements,and qi deficiency syndrome elements may be more likely to have fundus hemorrhage.There was a positive correlation between yang deficiency syndrome elements and fundus exudation area(rs>0,P<0.05),indicating that patients diagnosed with yang deficiency syndrome elements may be more likely to have fundus exudation;there was a positive correlation between qi stagnation syndrome elements and cotton wool spot area(rs>0,P<0.05),indicating that patients diagnosed with qi stagnation syndrome elements are more likely to have cotton wool spots in the fundus.The correlation between TCM syndrome elements and fundus OCTA related indicators in patients with moderate to severe NPDR was analyzed by sperman correlation coefficient.The results suggested that phlegm-dampness syndrome elements were compared with non-phlegm-dampness syndrome elements,blood stasis syndrome elements were positively correlated with foveal thickness compared with non-blood stasis syndrome elements(rs>0,P<0.05),indicating that patients diagnosed with phlegm-dampness syndrome elements and blood stasis syndrome elements were more likely to have increased foveal thickness;blood deficiency syndrome elements were negatively correlated with macular vascular density compared with non-blood deficiency syndrome elements(rs>0,P<0.05),indicating that patients with blood deficiency syndrome elements may be more likely to have decreased macular vascular density;patients with blood deficiency syndrome elements were positively correlated with FAZ area(rs>0,P<0.05),indicating that patients with blood deficiency syndrome elements were more likely to have increased FAZ area;patients with phlegm-dampness syndrome elements were negatively correlated with RPC density compared with non-blood deficiency syndrome elements(rs>0,P<0.05),The results showed that patients diagnosed with phlegm-dampness syndrome were more likely to have decreased RPC density;compared with non-blood deficiency syndrome,blood stasis syndrome and blood stasis syndrome were positively correlated with GCC thickness,that is,patients with blood deficiency syndrome and blood stasis syndrome were prone to thinning GCC thickness.Sperman correlation analysis was used to analyze the correlation between common TCM and fundus color photography related indicators in patients with moderate to severe NPDR.The results suggested that yin deficiency and fire excess syndrome was positively correlated with fundus hemorrhage area in patients with moderate to severe NPDR compared with non-yin deficiency and fire excess syndrome(rs>0,P<0.05),indicating that patients diagnosed with yin deficiency and fire excess may be more likely to have fundus hemorrhage;kidney qi deficiency was positively correlated with cotton wool spots compared with non-kidney qi deficiency syndrome(rs>0,P<0.05),indicating that patients with kidney qi deficiency may be more likely to have cotton wool spots.By establishing the random forest and Lasso regression model of sputum dampness syndrome,the independent variables gender,age,history of hypertension,CMT,RPC,and macular vascular density were selected,and the independent variables were included in the multivariate logistic regression model.After analysis and screening using the stepwise regression method,it was concluded that CMT and RPC density were important influencing factors of sputum dampness syndrome.4.Correlation between TCM syndrome elements and miRNAs in patients with moderate to severe NPDR:Correlation between TCM syndrome elements and miRNA-146a in patients with moderate to severe NPDR:Through real-time PCR detection of miRNA-146a in 134 patients with moderate to severe NPDR and 16 healthy controls,it was found that miR-146a expression was decreased in patients with moderate to severe NPDR compared with healthy controls,and the difference was statistically significant(P<0.05);compared with healthy controls,it showed the correlation between miRNA-146a expression and each syndrome element,and the results showed that serum miRNA-146a expression was negatively correlated with pulmonary syndrome elements.Compared with healthy controls,serum miRNA-146a expression decreased in phlegm-dampness syndrome element,fire-heat syndrome element,qi stagnation syndrome element,qi deficiency syndrome element,blood stasis syndrome element,yin deficiency syndrome element,yang deficiency syndrome element,liver syndrome element,renin syndrome element,spleen syndrome element,lung syndrome element,liver qi stagnation syndrome,kidney qi deficiency syndrome,and yin deficiency and fire excess syndrome,and the differences were statistically significant(P<0.05),suggesting that phlegm-dampness syndrome element,fire-heat syndrome element,qi stagnation syndrome element,qi deficiency syndrome element,blood stasis syndrome element,yin deficiency syndrome element,yang deficiency syndrome element,liver syndrome element,renin,gastrin,spleen syndrome element,lung syndrome element,liver qi stagnation syndrome,kidney qi deficiency syndrome,and yin deficiency and fire excess syndrome may be associated with decreased serum miRNA-146a expression.In addition compared with liver syndrome,renin,and gastrin,the expression of serum miRNA-146a in lung syndrome decreased,and the difference was statistically significant(P<0.05),and the results of correlation analysis showed that miR-146a expression was negatively correlated with lung syndrome(rs<0,P<0.05).These results suggest that the decreased expression of lung syndrome miR-146a should be paid special attention to.Correlation between TCM syndrome elements and miRNA-145-5p in patients with moderate to severe NPDR:The results of this study showed that the expression of serum miRNA-145-5p increased in patients with moderate to severe NPDR compared with healthy controls,and the difference was statistically significant(P<0.05);the expression of serum miRNA-145-5p increased in fire-heat syndrome elements,qi stagnation syndrome elements,qi deficiency syndrome elements,blood stasis syndrome elements,yang deficiency syndrome elements,liver syndrome elements,renin,gastrin,liver qi stagnation syndrome,kidney qi deficiency syndrome,and yin deficiency and fire excess syndrome compared with healthy controls,and the difference was statistically significant(P<0.05);the expression of serum miRNA-145-5p increased in liver syndrome elements,renin,and gastrin compared with lung syndrome elements,and the difference was statistically significant(P<0.05).It is suggested that fire-heat syndrome element,qi stagnation syndrome element,qi deficiency syndrome element,blood stasis syndrome element,yang deficiency syndrome element,liver syndrome element,renin syndrome element,stomach syndrome element,liver qi stagnation syndrome,kidney qi deficiency group syndrome element,and yin deficiency and fire excess syndrome element may be related to the increase in the expression of serum miRNA-145-5p.Correlation analysis showed a negative correlation between serum miRNA-145-5p and pulmonary syndrome elements without clinical significance.Conclusion:1.Distribution of TCM syndrome elements:There are 13 main TCM syndrome elements in patients with moderate to severe NPDR.Among them,there were 8 disease syndrome elements,which were phlegm-dampness,fire-heat,qi stagnation,qi deficiency,blood stasis,yin deficiency,blood deficiency,and yang deficiency according to the proportion;there were 5 disease location syndrome elements,which were liver,kidney,stomach,spleen,and lung according to the proportion.2.The syndrome element combination of patients with moderate to severe NPDR is mainly based on deficiency and deficiency inclusion,of which the top three syndrome element combinations are liver qi stagnation syndrome,kidney qi deficiency syndrome and yin deficiency and fire excess syndrome.3.There is a correlation between TCM syndrome elements and fundus changes in patients with moderate to severe NPDR,which can provide the basis for local syndrome differentiation of TCM ophthalmology and expand the category of"TCM inspection".For example,patients with fire-heat,yin deficiency and qi deficiency syndrome are more likely to have fundus hemorrhage;patients with qi stagnation syndrome are more likely to have fundus hemorrhage and cotton wool spots;patients with blood stasis syndrome are more likely to have fundus hemorrhage,and are more likely to have increased CMT and thinning of GCC;patients with yang deficiency syndrome are more likely to have hard exudation of fundus;patients with phlegm-dampness syndrome are more likely to have increased CMT and decreased RPC density;patients with blood deficiency syndrome are more likely to have decreased vascular density in the macular area,increased FAZ area and thinning of GCC thickness.4.miR-146a expression decreased and miR-145-5P expression increased in the serum of patients with moderate to severe NPDR.miR-146a may be used as a syndrome biomarker for pulmonary syndrome in patients with moderate to severe NPDR. |
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