Curing Effect Of Baicalin Bai On DN Rats At The Advanced Stage And Influence Of Baicalin Bai On Kidney Micro-environment

Diabetic Nephropathy (DN) refers to the pathological changes of glomerulus, tubule, renal interstice, and renal vessel when Diabetes Mellitus (DM) occurs. It is one of the most serious micro-vessel complications of DM, and often results in End Stage Renal Failure. In the recent years, DN incidence increases every year, and becomes one of the serious diseases which impair people's health and living quality. The study on DN mechanism and looking for effective prevention and treatment ways becomes the common topic of DM researchers.The main manifestations of DN are renal dysfunction and renal structural lesions, which promotes each other. Many researches show that high glucose environment and glycation-endproducts produced by metabolism disorder cause the body to be in an oxidative stress state, activate part of Renin Angiotensin System, cause some renal inflammatory changes, abnormal renal hemodynamics, large release of some cell factors, and therefore result in renal structural lesions and dysfunction, including hyperplasia of extracellular matrix (incrassation of glomerulus basement membrane, expansion of mesangium, incrassation of tubule basement membrane, interstitial fibrosis), podocyte injury (decrease of podocyte, structural lesion), decrease of glomerulus filtration, and increase of urine protein.Based on the above mentioned mechanism, many helpful explorations of clinical and animal experiment on the treatment of DN have been conducted. However, they cannot be used practically because of unsatisfactory results or side effects. Currently treatment of DN is mainly to control blood sugar, blood pressure, and diet. In the recent years prevention and treatment of DN by Chinese medicine has shown some advantages: renal function has been improved, and clinical symptoms have been alleviated. As more and more researches have been done on inflammation and oxidation lesions of DN mechanism, Chinese medicine with anti-inflammatory and anti-oxidation function is in the list of drugs curing DN. Baicalin Bai has anti-bacterial, anti-inflammatory, and anti-infective pharmacological effect. In the recent years as the development of new usage of traditional Chinese medicine, Baicalin Bai has been further studied, and it has been found that Baicalin Bai plays an important role in the prevention and treatment of DN.Clinical experiments reveal that Baicalin Bai can improve excretion function of the kidney, and inhibit AR activity. However, few researches have been done on the anti-inflammatory and anti-oxidation effect of Baicalin Bai. Based on the relationship among some cell factors, inflammation factors, and oxidative stress reaction in the kidney of DN rats, this study makes an attempt to find the acting point of Baicalin Bai by the level of protein and molecule, discusses the treatment mechanism, and provides new perspective and experimental foundation for the prevention and treatment of DM by Chinese medicine.1 Material and Methods1.1 Establishment of DN Rat ModelSPF male SD rats with the weight of 230-300g were randomly divided into normal control group and DN model group. Rats in the control group were not handled after they were weighed, and blood was drawn by needles from the tails, and blood sugar was monitored. After fasting for 10 hours, rats in the DN model group were weighed, blood was drawn by needles from the tails, blood sugar was monitored, and 2% STZ by 55mg/kg was injected into the abdomen. One week later, postprandial blood sugar was 16mmol/L or more of the rats in the model group was diagnosed as DM. Excretion amount of urine protein and creatinine in DM model rats were monitored for 24 hours. Six weeks later, if urine protein was stably higher than 500rag/L, the rats could be diagnosed as DN. Weight, blood sugar, urine creatinine, and urine protein were monitored.1.2 Experiment of Baicalin Bai Improving Kidney Function in DN RatsAccording to weight, blood sugar, and urine creatinine and protein, DN model rats are divided into three groups: model group, Baicalin Bai low dose group, and Baicalin Bai high dose group. There is no significant difference (13＞0.05) among the three groups as to weight, blood sugar, urine creatinine and protein. Rats with significant difference as to the above indices were not taken into the experiment. The result was: normal control group 5 rats, model group7 rats, Baicalin Bai low dose group 7 rats, and Baicalin Bai high dose group 7 rats. Their food was controlled: 25g/rat/day, but they were free to drink. Rats in the normal group were not handled; rats in the model group were injected water 1ml/rat/day abdominally; rats in the Baicalin Bai low dose group were injected Baicalin Bai solution 40mg/kg/day abdominally; rats in the Baicalin Bai high dose group were injected Baicalin Bai solution 60mg/kg/day abdominally. Six weeks later, weight, blood sugar, urine creatinine, blood creatinine, and urine protein were monitored and compared before and after treatment and between groups.1.3 Experiment of Baicalin Bai Improving Kidney Structure in DN RatsRats were divided into groups and treated the same as the above. Six weeks later, the weight of their kidneys was monitored. Several slices of cortices were fixed in 3% glutaraldehyde solution, and electronic scopy was performed. Other kidney tissues were fixed in 10% formalin solution, and pathological scopy was performed.1.4 Experiment of Baicalin Bai Adjusting Some Kidney Bio-active Substances in DN RatsRats were divided into groups and treated the same as the above. Six weeks later, blood and kidneys were drawn, AngⅡwas measured by radioimmunoassay, and TGF-β1 and NF-κB were measured by immunohistochemical way, also PKC activity were monitored by the same way.1.5 Experiment of Baicalin Bai interering in Kidney Oxidative Stress Reaction in DN RatsRats were divided into groups and treated the same as the above. Six weeks later, kidneys of the rats were drawn, and antioxidase SOD and GSH-PX were monitored after homogenate of some renal tissue.2 Results2.1 Establishment of DN Rat ModelDN rats had a decreased weight, increased blood sugar, and increased urine protein, and there was significant difference compared to the normal group, p＜0.05. Excretion amount of urine creatinine decreased compared to the normal group, but there was no significant difference. According to DN process, GFR increased at the early stage, decreased at the clinical stage, and this showed that the rats were in clinical DN stage.2.2 Experiment of Baicalin Bai Improving Kidney Function in DN RatsAfter treatment with Baicalin Bai, DN rats in the low dose and high dose group had a decreased blood sugar, and there was significant difference compared to the model group, p＜0.05; however, the blood sugar in these two groups were still higher than normal, and there was significant difference compared to the normal group, p＜0.05; the blood sugar decreased greater in the low dose group. The weight of rats in the low dose and high dose group increased, and there was significant difference compared to the model group, p＜0.05; there was also significant difference compared to the normal group, but this was because the difference already existed before the treatment. However, during the treatment, the increase of weight in the low dose and high dose group was similar to that of the normal group, and there was no significant difference. Urine protein decreased in the low dose and high dose group, and there was significant difference compared to the model group, p＜0.05; there was no significant difference between the low dose group and normal group, and there was significant difference between the high dose group and normal group. This showed that the low dose group had better effect on decreasing urine protein. Urine creatinine decreased in the model group, there was significant difference compared to the normal group, p＜0.05. Urine creatinine increased in the low dose group, there was significant difference compared to the model group, p＜0.05; but there was no significant difference between the high dose group and the model group, p＞0.05; and there was significant difference between the high dose group and the normal group, p＜0.05. As to blood creatinine, there was significant difference between the three DN groups and the normal group, and this showed that there was metabolic product in the body. Blood creatinine was quite low in the low dose group, and there was significant difference compared to the model group, p＜0.05, and there was also significant difference compared to the high dose group, p＜0.05. There was no significant difference as to blood creatinine between the high dose group and the model group, p＞0.05.The results showed that Baicalin Bai could reduce blood sugar, improve excretion function of kidney in DN rats, and the treatment effect of the low dose group was better than that of the high dose group.2.3 Experiment of Baicalin Bai Improving Kidney Structure in DN RatsAfter treatment with Baicalin Bai, the kidney index decreased in the low dose and high dose group, and there was significant difference compared to the model group, p＜0.05; there was also significant difference compared to the normal group, p＜0.05, and the kidney index in the low dose group was closer to that in the normal group. This showed that Baicalin Bai can decrease kidney hypertrophy. Under microscope, compared to the model group, glomerular hypertrophy was alleviated in the low dose and high dose group, there was a decrease in the area of PAS dye positive substance, and there was a decrease in FN andⅣexpression; under electric scopy, incrassation of basement membrane was alleviated, injured podocytes were repaired, and normal foot process array could be seen in some areas; cell edema could still be seen in tubule epithelium, and tubuleⅣexpression decreased; interstial fibrosis and inflammatory cell soakage decreased. The data showed that there was significant difference as to glomerular PAS positive substance and FN in the low dose and high dose group compared to the model group, p＜0.05; there was no significant difference compared to the normal group, p＞0.05, and glomemlar PAS positive substance and FN in the low dose group was closer to that in the normal group. Compared to the model group, there was significant difference as to tubuleⅣexpression decrease in the low dose group, p＜0.05; no significant difference in the high dose group and model group, p＞0.05.The results showed that Baicalin Bai could decrease FN andⅣdeposit in the glomerulus and tubule, alleviate basement membrane hypertrophy, and thus alleviate kidney hypertrophy. The treatment effect of the low dose group was better than that of the high dose group.2.4 Experiment of Baiealin Bai Adjusting Some Kidney Bio-active Substances in DN RatsAfter treatment with Baicalin Bai, AnglI in the blood decreased in the low dose and high dose group, and there was significant difference compared to the model group, p＜0.05; there was no significant difference of AngⅡin the kidney tissue. Tubule TGF-β1 expression in the low dose and high dose group decreased significantly compared to the model group, and the TGF-β1 expression in the low dose group was closer to that in the normal group. PKC's expression on tubule epithelial cell membrane increased in the model group. Tubule epithelial cell membrane PKC expression in the low dose and high dose group decreased, PKC in the low dose group was closer to that in the normal group. NF-κB was weak positive expression, and there was no significant difference among the four groups.The results showed that Baicalin Bai could reduce AngⅡin the blood, and was helpful in improving renal hemodynamics. Baicalin Bai could also decrease the increased TGF-β1 and PKC in renal tissue, and thus adjust deposit and degrading of ECM.2.5 Experiment of Baicalin Bai interering in Kidney Oxidative Stress Reaction in DN RatsAfter treatment with Baicalin Bai, SOD in the renal tissue increased in the low dose and high dose group, and there was significant difference compared to the model group, p＜0.05; there was no significant difference compared to the normal group, p＞0.05. There was no statistical significance of GSH-PX among the four groups, but GSH-PX in the model group decreased, and GSH-PX in the low dose and high dose group increased and was similar to that in the normal group.The results showed that Baicalin Bai could increase the decreased antioxidase SOD and GSH-PX when DN occurred, and prevent some oxidative stress reaction in the kidney.3 ConclusionsThe above results show that Baicalin Bai has the effect of curing DN, including: 1) decreasing blood sugar, thus improving rats' general state; 2) increasing GFR, decreasing protein urine, and improving kidney function; 3) decreasing ECM deposit, and improving kidney structure. The mechanism of Baicalin Bai curing DN is probably related to its reduce AngⅡin the blood, decrease of TGF-β1 and PKC activity, and increase of antioxidase SOD and GSH-PX activity.By analyzing the negative results, the reason why there was no significant difference as to AngⅡin the renal tissue was probably related to dilution of AngⅡcaused by homogenate of renal tissue; NF-κB was weak positive expression, and there was no significant difference among the four groups, and these were probably related to insensitiveness of immunohistochemistry to this index.