| Objective1.The study of pharmacodynamics of San Huang Tang Shen Kang granular to earlydiabetic nephropathy rats.2.The study of drug mechanism of San Huang Tang Shen Kang granular to earlydiabetic nephropathy rats.3.Observe clinical curative effect of San Huang Tang Shen Kang granular inpatients with early diabetic nephropathy.Materials and methods1. Trial Study1.1Methods of modelingWith high fat, high sugar diet combined with intraperitoneal injection ofstreptozotocin(STZ),we duplicate diabetes model and lead insulin resistance,hyperglycemia, and miner albuminuria.1.2Method of grouping and oral medication administeringSPF Wistar rats (90,280+20g weight). Only according to the weight, wenumbered90rats of1-90. By using random numbers,90rats were randomly dividedinto normal group and model group, when diabetes model was succeeded and thenthey were grouped according to their blood sugar levels, the total is6groups,respectively A: normal group; B: the model group; C: Chinese medicine preventiongroup; D1: low dose group; D2: the high dose group of TCM; E: the western medicinegroup. In addition to the normal group, the rest of the group using high fatand sugar feed in combination with small dose of STZ (42mg/Kg bw) method copyof diabetic nephropathy (DN) rats model, the model was copied in success after2weeks. In addition to the normal group and Chinese medicine prevention group,respectively we give the corresponding drug intervention to fill the stomachthrough the mouth, for eight consecutive weeks. Chinese medicine precautiousgroup rats from model replication which starts from the date of delivery, for10weeks in succession. According to the normal adult dose, Chinese medicine precautious group and low dose group of Chinese traditional medicine give6.3times dose. High dose group according to the3times of low dose group. Groupwas given western medicine that split lavage and dosage according to the normaladult dosage equivalent conversion into adult rats dosage, namely,0.9mg/kg.d.1.3Observing index and measuring methodWe should mainly observe pharmacodynamics indexes and San Huang Tang ShenKang particles to the influence of inflammation in diabetic nephropathy ratsincluding the experimental ended, separately for each group of rats serumglycosylated hemoglobin, blood urea nitrogen, creatinine, beta2-microglobulin,blood glucose and urine trace albumin content in urine test. Between groups ratswere randomly selected3only, after death, harvesting kidneys, fixed in4%paraformaldehyde solution, using the method of HE staining and PAS staining,observing their kidneys pathological changes. Using fluorescence quantitativepolymerase chain reaction (PCR), NF-κB, MCP-1and CCR2mRNA expression levelsin the kidney tissue of each group rats were detected. Using western blot method,NF-κB,MCP-1and CCR2protein expression levels in the kidney tissue of eachgroup rats were detected. Through the above effect index detection, we furtherdiscuss the San Huang Tang Shen Kang particles on the curative effect of diabeticnephropathy in rats and its mechanism.2. Clinical researchChoosing40patients of blood deficiency type with early diabetic nephropathy,adopting the method of randomly contrast, patients were randomly divided intotraditional Chinese medicine treatment group and western medicine control groupfor20cases each. On the basis of foundation treatment, Chinese medicinetreatment group patients with San Huang Tang Shen Kang particles, a daily pay,two oral; On the basis of foundation treatment, western medicine control grouppatients give lotensin oral treatment,10mg each time, once per day, courseof8weeks. After completion of treatment on two groups of patients with generalmedical examination, cereal third transaminase (ALT), serum creatinine (Scr),urea nitrogen (BUN), fasting and postprandial2hours blood glucose, glycosylated hemoglobin,24h urine trace albumin (U-mAlb), TCM symptomscurative effect index, we observe San Huang Tang Shen Kang particles clinicalcurative effect in patients with early diabetic nephropathy.Results1. Results of animal experiments1.1The affection on general situation of ratsIn experiment process, the mental state of normal group rats is good andno death; Model group rats eat less, drink quantity obviously increased, anda total of6cases died. The western medicine group rats died in1case.Traditional Chinese medicine high dose group died in1case. Low dose group diedin1case. Model group in all3cases died.1.2The affection to microalbuminuria on ratsCompared with normal group, urine trace albumin levels of the rest of thegroup rats increased significantly (p <0.01)and statistically different. Urinetrace albumin content of model group is the highest, compared with the group,the treatment group and prevention group decreased significantly (p <0.05).In Comparison among different treatment, prevention group is lower than thetreatment group. Compared with western medicine group, preventive group waslower than western medicine group and the difference was statisticallysignificant (p <0.01). Low dose group was higher than western medicine groupand the difference was statistically significant (p <0.05). Compared with thewestern medicine group, high dose group was no statistically significantdifference (p>0.05).1.3The affection on blood sugarExcept for normal group, the blood sugar of other groups’ rats were higherthan16.7tendency for L. Compared with normal group, the rest of the group ratsblood glucose was significantly increased (p <0.01). Compared with model group,western medicine group, preventive group, high dose group, low dose group bloodglucose values were less than model group, and the difference was statistically significant (p <0.01). Prevention group had lowest blood sugar levels.Comparedwith the rest of the group, the difference was statistically significant (p <0.05). Compared with traditional Chinese medicine low dose group, preventivegroup and high dose group were lower than low dose group, and the differencewas statistically significant (p <0.01, p <0.05); Between the high dose groupand western medicine, there was no statistically significant difference (p>0.05).1.4The affection on glycosylated hemoglobinSerum glycosylated hemoglobin content of groups of rats determinated, modelgroup and prevention group were significantly higher than normal group (p <0.01)and was statistically significant. Compared with model group, preventive groupand each treatment group were significantly lower (p <0.01) and wasstatistically significant. In comparison between western medicine group,preventive group, high dose group and low dose, there was no statisticallysignificant difference (p>0.05).1.5The affection on blood β2-micro globulinCompared with normal control group, the rest of the group rats blood β2-micro globulin content increases, and the difference was statisticallysignificant (p <0.01). Blood β2-micro globulin content of model control groupis the highest. Compared with the group, the treatment group and prevention groupdecreased significantly (p <0.01) with statistical significance.1.6The affection on serum creatinine and urea nitrogenSerum creatinine content of each group of rats was no significant difference(p>0.05), no statistical significance. Serum urea nitrogen content of modelgroup rats increased significantly. Compared with model group, the rest of thegroup in the rat serum urea nitrogen content decreased significantly (p <0.01),and there was statistical significance. Compared with normal control group, onlyChinese medicine low dose was statistically difference (p <0.05). In comparisonbetween western medicine group, preventive group, high dose group and low dose,there was no statistically significant difference (p>0.05). 1.7The pathological changes of kidney tissue in each groupHE staining: At high magnification shows, model group of glomerularsclerosis, mesangial cell and mesangial matrix proliferation of renal tubularepithelial cell cavity deformation. Each treatment group and Chinese traditionalmedicine prevent group rats kidney pathological changes degree obviously lighterthan the control group. Chinese traditional medicine prevent group rats renalglomerulus and renal tubule increased slightly than normal group rats. Thewestern medicine group and high dose of Chinese medicine in the rat kidneyglomerular volume had the similar size, but bigger than the normal group.PAS staining: Kidney tissue of model group rats is a wide range of PASpositive substances, mainly in the centrilobular, glomerular basement membranethickening, mesangial matrix increased obviously, glomerular diffuse lesions,arteriole hyaline changes, glomerulus internal occasional round, oval or conicaltubercles, individual glomerular waste, and each treatment group and Chinesetraditional medicine prevent group rats kidney PAS staining showed weaklypositive. Western medicine group is visible for diffuse glomerular hard,mesangial matrix and extracellular matrix increased obviously, and renal tubularatrophy. Chinese medicine prevention increased glomerular mesangial matrix setof visible light, the glomerular basement membrane and renal tubular lesion;Chinese medicine high dose group of glomerular mesangial matrix and mesangialcells increased, glomerular basement membrane changed. Chinese medicine low dosegroup is visible for section break in rats, increased glomerular mesangial matrix,and thickening of basement membrane.1.8The affection on CRP levelsGroups of CRP in serum and kidney tissue of rats have the same change trend.Compared with normal group, model group rats CRP content in serum and kidneytissue increased significantly (p <0.01), and there was statisticalsignificance. Compared with model group, Chinese medicine prevention and thetreatment group rats CRP content in serum and kidney tissue decreasedsignificantly (p <0.01), and there was statistical significance. Compared with western medicine group, Chinese medicine prevention group, the Chinese medicinehigh, low dose group rats serum CRP levels decreased significantly (p <0.01)with statistical significance.1.9The affection on Nuclear transcription factors in kidney tissue NF-κBprotein and gene expression levelKidney tissue of model group rats in the NF-κB mRNA and protein expressionlevels were significantly raised in the normal group (p <0.01), and there wasstatistical significance. Compared with model control group, Chinese medicineprevention group and each treatment group in the rat kidney tissue in the NF-κB mRNA and protein expression levels were significantly lower (p <0.01).Compared with traditional Chinese medicine prevention group, normal group, highdose group, low dose group in the rat kidney tissue in the NF-κB protein andgene expression level were significantly increased (p <0.01) with statisticalsignificance. Western medicine group had significant difference in low doseChinese traditional medicine and Chinese traditional medicine prevent group (p<0.01) with statistical significance. Western medicine group and high dose groupof Chinese medicine has no significant difference (p>0.05).1.10The affection on MCP-1and CCR2in kidney tissue protein and gene expressionlevelExpression level determination results of each group MCP-1mRNA and protein,CCR2mRNA and protein in the kidney tissue, compared with normal control group,model group rats were significantly raised (p <0.01), and there was statisticalsignificance. Compared with model control group, Chinese medicine preventiongroup and each treatment group rats MCP-1and CCR2gene and protein expressionlevels were significantly lower (p <0.01). CCR2and MCP-1in the rat kidneytissue protein in Chinese medicine prevention group and gene expression levelwas significantly lower than western medicine group and Chinese medicine highand low dose group (p <0.01or0.05). CCR2and MCP-1protein and gene expressionlevel in western medicine group was lower than those of high and low dose ofChinese medicine (p <0.05). Low dose group and high dose of Chinese medicine in rats of CCR2protein and gene expression level has no significant difference,but the MCP-1mRNA and protein expression level was significant difference(p <0.05).2. Result of clinical observation2.1The affection on effect of indicatorsTwenty-four hours urine micro protein in patients between traditionalChinese medicine treatment group and western medicine control group has nosignificant difference. Before treatment (0weeks), urine trace albumin betweenChinese medicine treatment group and western medicine control groupsignificantly reduced (p <0.01), and there was statistical significance.Patients’ urine trace albumin between traditional Chinese medicine treatmentgroup and western medicine control group gradually reduce with the extensionof treatment time.Before the treatment, patients symptoms between Chinese medicine treatmentgroup and western medicine control group were no significant difference (p>0.05);6weeks and8weeks treatment, TCM syndrome integral treatment group wassignificantly lower than western medicine control group (p <0.05). Thecomparison between the two groups was statistically significant; Compared withbefore treatment,4,6,8weeks in both groups of symptom scores weresignificantly lower (p <0.05or p <0.01) with statistical significance.In Chinese medicine treatment group, there were4patients markedlyeffective,8patients effective,8non-effective, and the total effective ratewas60%. In western medicine control group there were4patient markedlyeffective,9patients effective,7non-effective, and the total effective ratewas65%.2.2The affection on blood sugar and glycosylated hemoglobinTwo groups of FBG,2HPG and HbA1c levels in patients has no significantdifference. Compared before and after treatment, FBG,2HPG and HbA1c levelsin patients between Chinese medicine treatment group and western medicinecontrol group has no significant difference (p>0.05). 2.3The affection on Security indicatorsTwo groups of patients before and after treatment, serum creatinine, ureanitrogen, cereal third transaminase levels were no significant difference (p>0.05). In each group before and after treatment, serum urea nitrogen,creatinine, alanine aminotransferase levels in Chinese medicine treatment groupand western medicine control group were no significant difference (p>0.05).Conclusion：1. San Huang Tang Shen Kang particles can effectively reduce the DN rats’ bloodglucose, glycosylated hemoglobin, urine trace albumin, blood beta2-microglobulin, and urea nitrogen levels.2. San Huang Tang Shen Kang particles can effectively reduce the DN model rats’CRP content in serum and kidney tissue.3. San Huang Tang Shen Kang particles can effectively reduce the DN model rats’renal cortex of the NF-κB and CCR2, MCP-1mRNA and protein expression level.4. San Huang Tang Shen Kang particles on prevention and cure of DN rats may bemediated by inhibiting the NF-κB pathway, thus inhibite MCP-1and its receptorCCR2combination.5. San Huang Tang Shen Kang particles can effectively reduce the patients withearly diabetic nephropathy in24hour urine micro protein and symptom integrallevel.6. San Huang Tang Shen Kang particles on early diabetic nephropathy patientsin serum urea nitrogen, creatinine, alanine aminotransferase, FBG,2HPG andHbA1c levels had no significant effect.7. In the treatment of6weeks, San Huang Tang Shen Kang particles can achievethe best curative effect of traditional Chinese medicine, and it has the idealcontrol effect for patients with early diabetic nephropathy. |
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