| [Object]The aim of our clinical study was to evaluate the clinical effectiveness and safety of Yiqi Yangyin Tongluo Sanjie Decoction (YYTSD) on Early Diabetic nephropathy (DN) patients with deficiency of both Qi and Yin and blood stasis obstructing. In animal experiment, our aim was to investigate the effects of YYTSD on the KKAy mice nephropathy, and the regulatory effect on ACE-Ang Ⅱ-AT1 axis and ACE2-Ang-(1-7)-Mas axis in renal RAS system.[Methods]In Clinical research:Based on the principle of random and control, we recruited 64 patients with early DN of deficiency of both Qi and Yin and blood stasis obstructing. They were randomly allocated into the YYTSD group (33 cases) or the Control group (31cases). Patients in YYTSD group were treated with basic treatment and YYTSD; meanwhile Patients in Control group were treated with basic treatment and irbesartan.The treatment lasted for 12 weeks. During the treatment, we recorded the vital signs, blood glucose. To evaluate the effectiveness, Glycosylated hemoglobin (HbA1c), total cholesterol (TC), triglyceride (TG), low density lipoprotein cholesterol(LDL-C), high density lipoprotein cholesterol (HDL-C), the 24h urine microalbumin (24h U-MA), blood β 2-microglobuline(β 2-MG), blood Uric Acid (UA) were detected before and after treatmertt.The traditional Chinese medicine syndrome were observed in our study. In addition, we detected the blood routine, urine routine, feces routine, liver function (includingAST, ALT, ALB) and renal function (including Scr, BUN) as the safety indexes, and monitored adverse reaction.In Experimental Research, Spontaneously diabetic KKAy mice (male,10-11 weeks of age) and C57BL/6J mice were used. After acclimating to new surroundings for 1 week, KKAy mice with fasting blood glucose level>13.9mmol/L twice were randomly divided into model group and YYTSD group with 24 mice in each group. While 24 male C57BL/6J 10-11 week-old mice were used as normal control. And then, each group were divided into 3 minor groups including 4-week-group,8-week-group and 12-week-group. Finally,8 mice were in each group. The general condition, body weight and blood glucose of mice were recorded in the experiment. Mice were sacrificed at 4th,8th and 12th weeks after treatment respectively. Renal morphological changes were observed by Hematoxylin and Eosin (HE), Masson’s Trichrome (Masson), Periodic Acid-Sliver Methenamine(PASM). The concentration of Ang II in serum were detected by ELISA. Location and semiquantitative protein expression of ACE, ACE2, Ang II were detected by Immunohistochemical Staining. The protein expression of ACE, ACE2, MAS was detected by Western Blot. The mRNA expression of Ang II were detected by real-time PCR.[Results]In clinical research, after 12 week treatment, compared with the control group, YYTSD obviously alleviated the symptoms of early DN patients with syndrome of deficiency of both Qi and Yin and blood stasis obstructing(P<0.05), and can effectively reduce the 24h U-MA (P<0.05). The improvement of HbAlc, TG and LDL-C in YYTSD group were better than those in the control group (P< 0.05). The total effective rate in of YYTSD group was 84.8%, which were better than that of the control group 61.2%. There was no abnormal blood routine, urine routine, feces routine, liver function, blood abnormalities, and serious adverse events in two groups before and after the treatment.In animal study, the body weight, FBS, TC, TG levels of the KKAy mice were significantly increased compared with the normal group. After the treatment, compared with the model group, YYTSD group decreased the blood glucose and blood lipid in different degrees after the treatment. At the 8th week, HE staining showed that the Pathological lesions of KKAy mice in model group were glomerular hypertrophy, glomerular blsement membrane expansion and increased extra-cellular matrix, as well as part of renal tubular epithelial cell swelling. Masson staining showed that aizen area increase, suggesting increased collagen fibers. And PASM staining showed that the thickening of basement membrane. At the 12th week, KKAy mice in model group showed obvious thickening of glomerular basement membrane and capillary expansion, renal tubular enlargement, epithelial hypertrophy, vacuolar change in the cytoplasm, protein tube in part of renal tubes. And the bowman’s capsule cavity were expansion and adhesion, and the semiquantitative analysis showed that grade of renal tissue pathological injury was raised. After therapy, pathological lesions of the renal were improved effectively in YYTSD group. From 4th weeks, compared with normal group, the expression of Ang II in serum and mRNA expression of Ang II and the protein expression of ACE in renal tissue were significant increased in the model group and YYTSD group(p<0.05 or p<0.01). At 12th week, compared with control group, the expression of Ang II in serum decreased significantly in YYTSD group(p<0.05), as well as the mRNA expression of AngⅡand the protein expression of ACE. At 4th weeks, the protein expression of ACE2 in the model group and YYTSD group were higher than normal group(p>0.05), while, from 8th weeks, the protein expression of ACE2 were significant decreased in the model group and YYTSD group (p<0.05 or p<0.01). At 12 th weeks, the protein expression of ACE2 in the YYTSD group were higher than model group. From 8 weeks, the protein expression of MAS were significant increased in the model group and YYTSD group, while at 12w the protein expression of MAS in the YYTSD group were significant higher than model group.[Conclusion]1.It has been proved that YYTSD is effective and safety on early Diabetic nephropathy patients with deficiency of both Qi and Yin and blood stasis obstructing syndrome.2. YYTSD alleviates the blood glucose and lipids metabolism in KKAy mice, mitigates the renal lesion and prevent the progression of DN. YYTSD attenuates the renal lesion by reducing the expression of serum Ang Ⅱ,mRN A expression of AngⅡ and the protein expression of ACE and Ang Ⅱ in renal tissues of KKAy mice, and up-regulating ACE2 and MAS expression to regulate the imbalance of ACE-Ang Ⅱ-AT1 axis and ACE2-Ang-(1-7)-Mas axis in renin-angiotensin system.
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