| Purpose:This paper summarizes Professor Zuo Tao’s unique perspective of"eye kidney correlation"theory,discusses the relationship between eye and kidney in the pathogenesis evolution of consumptive thirst eye disease,and the clinical experience of using"Liuwei Dihuang Decoction"to treat consumptive thirst eye disease.To study the correlation between TCM syndrome types and renal function indexes of diabetic retinopathy,summarize the common TCM syndrome types and risk factors of DR,and provide objective basis for DR.This formula was then used to intervene in animal experiments to observe the effects of Liuwei Dihuang Decoction plus-flavoured on the symptoms of"three more one less",fundus changes and pathological changes of the eyes and kidneys in rats with DR.Meanwhile the TLR4/My D88/NF-κBp65 pathway was also used as the entry point for the study to analyze the mechanism of action of Liuwei Dihuang Decoction plus-flavoured in the treatment of DR from a microscopic perspective,which provides an experimental basis for the control of DR in Chinese medicine.Method:1.The case data of 120 DR patients were collected,the TCM syndrome type,clinical stage and renal function related indexes of DR patients were counted,and the correlation between TCM syndrome type and renal function indexes of diabetic retinopathy was investigated.2.80 SPF grade SD rats were acclimatized for 7 days.The body weights of all rats were measured.After measurement,20 rats were chosen randomly as normal control group and fed with normal diet,while the remaining 60 rats were fed on a high-carbohydrate,high-fat diet as the pre-modeled group.After 1 month of continuous feeding,the high-sugar obese rat model was replicated.The 60 rats in the pre-model group were fasted for 12 hours（without water）and given an intraperitoneal injection of STZ solution to selectively destroy pancreaticβ-cells and induce type II diabetes,while normal control rats were given an equal amount of sodium citrate-citrate buffer intraperitoneally,and after 72 hours,blood was collected from the caudal vein to measure blood glucose.Rats with FBG levels above 16.7 mmol/L for three consecutive days were considered successful.After successful modelling,20 rats in the model group,20 rats in the western medicine group and 20 rats in the Liuwei Dihuang Decoction plus-flavoured group were fed with high sugar and high fat diet to maintain the high glucose model status.After all the FBGs were above the modeling standard,the pharmacological intervention was started on the following day.Regular and sample control groups were instilled with the equivalent volumetric gavage of steam water for,while the Liuwei Dihuang Decoction plus-flavoured group was gavaged with 12.06g·kg-1·d-1 of raw medicine and the western medicine group was gavaged with 135mg·kg-1·d-1 of calcium hydroxybenzene sulfonate solution,both twice daily.The rats were gavaged for 16 weeks,water drinking,food intakes,urine volumes and body weight of rats were monitored on the first day of every 4weeks until the completion of the experiment.The effect of Liuwei Dihuang Decoction plus-flavoured on the vascularization of the fundus of DR rats was investigated by dynamic observation of fundus changes in each group of rats through fundus photography;the effect of Liuwei Dihuang Decoction plus-flavoured on the blood-retinal barrier was investigated by measuring the leakage of retinal EB in DR rats;the HE staining of retina and kidney demonstrated that hyperglycemia could cause ocular and renal pathological changes at the same time,and the effect of Liuwei Dihuang Decoction plus-flavoured on retinal and renal pathological changes in each group of rats was observed.The effects of the addition of Liuwei Dihuang Decoction plus-flavoured on the pathological changes of retina and kidney in each group of rats were observed.3.In the fourth part,the animals were kept,grouped and intervened in the same way as in the third part,and the expression of apoptotic proteins,inflammatory factors,related pathway proteins and m RNAs were analyzed from both retinal and renal perspectives with the premise of ocular-renal correlation.The expression of IL-1β,IL-6,IL-8 and TNF-αin serum and urine of each group of rats was measured by enzyme-linked immunosorbent assay（ELISA）;the expression of apoptotic proteins caspase-1,caspase-3 and related pathway proteins in retina and kidney of each group of rats was measured by immunohistochemistry（IHC）;the expression of apoptotic proteins caspase-1,caspase-3 and related pathway proteins in retina and kidney of each group of rats was measured by protein blotting（WB）.The expression of TLR4,My D88 and NF-κBp65 in the retina and kidney of rats was detected by WB;the expression of TLR4,My D88 and NF-κBp65 m RNA in the retina and kidney of rats was detected by q PCR;the apoptosis in the retina and kidney of rats was observed by TUNEL,and the mechanism of DR treatment by Liuwei Dihuang Decoction plus-flavoured was investigated.Results:1.The highest proportion of DR patients with different TCM syndrome type was syndrome of Yin deficiency and blood stasis,which was significantly higher than other syndrome type,and the proportion of other three syndrome types in descending order was:Qi-Yin two deficiency syndrome type>Spleen-Kidney two syndrome type>Phlegm-stasis obstruction syndrome type;the distribution of disease duration in different syndrome types in descending order was:Phlegm-stasis obstruction syndrome type>Spleen-Kidney two deficiency syndrome type>Yin deficiency with stasis syndrome type>Qi-Yin two deficiency syndrome type,statistically meaningful difference in disease length between different TCM syndrome types（P<0.05）;the differences in gender,age and BMI between the different TCM syndrome types were not statistically significant（P>0.05）.Statistically significant difference in systolic blood pressure between different TCM syndrome types,with the highest systolic blood pressure in Yin deficiency with stasis syndrome（P<0.05）.Yin deficiency with stasis evidence predominated in the NPDR stage and phlegm stasis obstruction evidence accounted for the highest percentage in the PDR stage.Comparison of FBG between different TCM syndrome types,with the lowest FBG level in Qi and Yin deficiency syndrome type（P<0.05）;Comparison of Hb Alc levels between different TCM syndrome types,with the highest Hb Alc levels in the Phlegm-Stasis Blockage syndrome type（P<0.05）.The differences in TC and TG between different TCM syndrome types were all statistically significant,with the highest level in the Phlegm-stasis block syndrome type（P<0.05）;there were no figures for HDL-C between the different TCM syndrome types（P>0.05）;comparison of LDL-C levels between different TCM syndrome types,with the highest levels in the two deficiencies of spleen and kidney.The differences in Scr and BUN between different TCM syndrome types were measured in terms of the maximum levels of Scr and BUN in Spleen and Kidney deficiency symptoms（P<0.05）.Comparison of BUA between different TCM syndrome types,with the highest level of BUA in phlegm-stasis blockage syndrome（P<0.05）.Comparison of Cys C and Hcy between different TCM syndrome types,with the highest level of phlegm and stasis blocking syndrome（P<0.05）.No risk factors for DR in Qi-Yin two deficiency syndrome were found by multiple logistic regression analysis,but risk factors for DR in Spleen-Kidney two deficiency syndrome were longer duration of diabetes,high FBG,high Hb Alc level,high LDL-C,high Scr,BUN,Cys C and BUA;risk factors for DR in Yin deficiency with stasis syndrome were longer duration of diabetes,high FBG,high Hb Alc The risk factors for DR with yin deficiency and stasis are long duration of diabetes,high FBG,high Hb Alc level,high SBP,high Hcy;the risk factors for DR with phlegm stasis are long duration of diabetes,high FBG,high Hb Alc level,high TC and TG values,high BUA,Cys C,Hcy.2.The rats in the Liuwei Dihuang Decoction plus-flavoured group showed significant improvement in the symptoms of"three more and one less",and the blood glucose and body weight stabilized.After diabetes mellitus was established,the rats in the model group started to show crystal clouding at 6 weeks,and at 8 weeks,4 rats showed crystal clouding with protrusion of the eyeballs;2 rats in the LW-plus-flavoured group showed crystal clouding with protrusion of the eyeballs;the western medicine group and the normal control group did not show the above ocular changes.12 weeks later,8 rats in the model group showed crystal clouding with protrusion of the eyeballs;3 rats in the western medicine group showed crystal clouding with protrusion of the eyeballs;3 rats in the Liuwei Dihuang Decoction plus-flavoured group showed crystal clouding with protrusion of the eyeballs;3 rats in the Liuwei Dihuang Decoction plus-flavoured group showed crystal clouding with protrusion of the eyeballs.After 16 weeks,14 rats in the model group showed cloudy crystals and protruding eyeballs;8 rats in the western medicine group showed cloudy crystals and protruding eyeballs;9 rats in the Liuwei Dihuang Decoction plus-flavoured group showed cloudy crystals and protruding eyeballs.After simulation,the rats in the model group,western medicine group and Liuwei Dihuang Decoction plus-flavoured group drinking increased dramatically before intervention with drugs compared to the normal control group,and the difference was remarkable and valuable.（P<0.01）.After 4 weeks of pharmacological treatment,the rats in the Liuwei Dihuang Decoction plus-flavoured group consumed less water than the model group,and the difference was highly regarded as a matter of statistical value（P<0.01）.Before pharmacological intervention,the rats in the model group,the western drug group and the Liuwei Dihuang Decoction plus-flavoured group consumed significantly more food compared to the normal control group,and the difference was statistically significant（P<0.01）.4 weeks after the operation,the rats in the Liuwei Dihuang Decoction plus-flavoured group exhibited a consistent drop in food ingestion with statistically insignificant changes in food content relative to the target group（P<0.01）.Urine production was greatly expanded in the rats in the target group,the western medicine group and the Liuwei Dihuang Decoction plus-flavoured group as opposed to the target group,and the difference was considered to be highly valuable（P<0.01）.4 weeks after the interference,the urine production of rats in the Liuwei Dihuang Decoction plus-flavoured group progressively reduced compared to the modelling group,and the change was marked by a difference of great statistic meaning（P<0.01）.After the STZ intraperitoneal injection,the body weight of the rats in the model group dropped abruptly.After the pharmacological treatment,the body burden of the rats in the experimental period decreased in the modelling group,the western medicine group and the Liuwei Dihuang Decoction plus-flavoured group.At the 4th week of gavage,compared with the model group,the body weight of the rats in the Liuwei Dihuang Decoction plus-flavoured group increased and the decline trend slowed down,the variation is found to be scientifically relevant（P<0.05）.At weeks 8,12 and 16 of the pharmacological injection,the reduction in weights was more pronounced in the model and western medicine groups than in the Liuwei Dihuang Decoction plus-flavoured,while the body weight of the Liuwei Dihuang Decoction plus-flavoured group tended to stabilize,The difference is considered to have a slight impact and is found to be significant（P<0.05）.After mock-up and before pharmacological treatment,fasting blood glucose was higher in the mock-up,western medicine and Liuwei Dihuang Decoction plus-flavoured group compared with the regular controls（P<0.01）.4 weeks after the pharmaceutical treatment,the fasting blood glucose level of the rats in the Liuwei Dihuang Decoction plus-flavoured group dropped dramatically comparing with that of the modelling group,and the effect of the difference was found to be scientifically important（P<0.01）.At the 8th,12th and 16th weeks after the drug intervention,the blood glucose level of the rats in the Liuwei Dihuang Decoction plus-flavoured group decreased,but the overall trend tended to be stable.The average standardised EB seepage was shown to be greater in the modellar,Western Medicine and Liuwei Dihuang Decoction plus-flavoured group when combined with the regular test group,with the difference being considered to be statistically considerable（P<0.01）.Versus the modeller set,the mean values of standardized EB leakage in the western medicine group and the Liuwei Dihuang Decoction plus-flavoured group were significantly reduced,the variation is remarkably statistical in terms of size（P<0.01）.HE dyeing of retinal and kidney tissues revealed significant hydrops in the retina of the Rats in the modality category,with thinning thickness,abnormal cell morphology and loose and disorganized intercellular arrangement;the inner boundary membrane was swollen and thickened,the number of ganglion cells was reduced,loosely arranged and vacuolated;the gaps in the arrangement of cells in the inner and outer nuclear layers were enlarged and the structure was disorganized;capillaries were obviously dilated,the diameter of vessels was thickened,the lumen was broken and collapsed,a small number of red blood cells leaked out of the vessels The endothelial cells were proliferated;the optic nerve was loosened and detached from the pigment epithelium,while the pigment epithelium showed different degrees of hyperplasia.Compared with the model group,the retinal pathological changes in the Liuwei Dihuang Decoction plus-flavoured group and the western medicine group were less severe.The structure of the retinal layers was clearer than that of the model group,the cells were regularly arranged,the ganglion cells were more neatly arranged,the endothelial cells were mildly hyperplastic,the inner boundary membrane was mildly edematous,the swelling and thickening were less severe,the cells in the inner and outer nuclear layers were more neatly arranged,and there was no obvious thickening or breakage of capillaries.In the kidneys of the model group,dilated glomeruli,localized atrophy and sclerosis of the glomeruli,proliferation of thylakoid cells,marked expansion of the thylakoid matrix,adhesion of the glomerular capsule wall and vacuolation of the tubular epithelial cells were observed,whereas the renal pathological changes in the western medicine group and the Liuwei Dihuang Decoction plus-flavoured group were less than those in the model group.3.Determination of the concentration of the inflammation factors in blood serum and piss of rats by ELISA.The outcome indicated that the manifestation of the inflammation factors IL-1β,IL-6,IL-8 and TNF-αin the blood fluids of the western medicine group and the Liuwei Dihuang Decoction plus-flavoured group were decreased dramatically according to the models,and the results were highly regarded as valuable（P<0.01）.The apoptosis of rat retinal and kidney cells was detected by TUNEL method.The number of tan chromogenic cells in the retinal tissue slides of rats in the Western medicine group and Liuwei Dihuang Decoction plus-flavoured group was decreased in comparison with the sample group,and the quantity of green phosphor nuclei in the kidney tissue slides was decreased,and the percentage of defective cells was obviously reduced,the discrepancies were found to be remarkably statistically important（P<0.01）.Apoptotic proteins caspase-1 and caspase-3were labeled by IHC method in rat retinal and kidney tissues,and the nuclei of hematoxylin-stained cells were blue,and the positive expression revealed by DAB was brownish-yellow.Compared with the model group,the staining of the rat retina and kidney tissue sections was relatively lighter in the western medicine group and the Liuwei Dihuang Decoction plus-flavoured.By analyzing the mean optical density values,the results suggested that the positive expression of caspase-1 and caspase-3 protein in the rat retina and kidney was significantly reduced in the western medicine group and the Liuwei Dihuang Decoction plus-flavoured group compared with the model group,the variance is effectively subject to statistical significance（P<0.01）;In contrast to the rat sample,the expression of TLR4,My D88and NF-κBp65 in the retina and kidney tissues was markedly lower,with striking difference in the analysis（P<0.01）.Analysis of protein expressed levels of TLR4,My D88 and NF-κBp65in rat retina and kidney through Western Blot.The findings indicated that the TLR4,My D88and NF-κBp65 protein profiles were substantially declined in the retina and kidney of the western medicine and Liuwei Dihuang Decoction plus-flavoured group versus the target group（P<0.01）.The q-PCR obtained indicated that the contents of TLR4,My D88 and NF-κBp65m RNAs were dramatically less in the retinal and kidney tissues of the rats in the western medicine and Liuwei Dihuang Decoction plus-flavoured groups respectively comparing to the target group（P<0.01）.Conclusion:1.Scr,BUN,BUA,Cys C,Hcy in renal function indexes,FBG,Hb Alc,TC,TG,LDL-C in glycemic and lipid metabolism indexes,SBP in blood pressure,and diabetes course can provide certain objective basis for TCM syndrome differentiation and classification of DR,and also provide reference for prevention and treatment of DR By integrated traditional Chinese and Western medicine.2.Liuwei Dihuang Decoction plus-flavoured can obviously improve the fundus vascular status of diabetic rats,reduce the pathological changes of retina and kidney caused by high sugar,prevent diabetic microvascular complications,and delay the progression of DR.It can improve the function of BRB and reduce the damage of BRB.3.Liuwei Dihuang Decoction plus-flavoured can inhibit the excessive activation of TLR4/My D88/NF-κBp65 inflammatory pathway,reduce the release of inflammatory factors,reduce microvascular inflammatory damage,and then inhibit cell apoptosis,protect microvascular,and treat DR Through anti-inflammatory and anti-apoptotic effects. |
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