Effects Of Long-term High-fat Feeding On PKC Activation In Rats Kidneys And Intervention Effect Of Compound Herbal Medicine

Objective: Diabetic nephropathy (DN) is one of the most common microvascular complications of diabetes mellitus and the leading cause of end-stage renal failure, the pathological hallmarks of DN are glomerular hyperfiltration, hypertrophy, mesangial expansion and basement-membrane thickening. The exact pathogenesis of DN is not clarified completely yet. Recent studies have identified that diacylglycerol(DAG)-Protein kinase C(PKC) signal transduction pathway play a pivotal role in the development of DN. PKC is detected in almost all types of organs, tissues and cells in the body. The pathways that activate PKC are as follows: hyperglycemia, the polyol pathway, oxidative Stress, nonenzymatic glacation , and angiotensin Ⅱ(AngⅡ). The activation of PKC can lead to development of DN by changing glomerular capillary contraction ability, penetrating and increasing the production of extracellular matrix proteins and reducing the activation of Na+-K+-ATPase. The increasing of TGFβexpression levels can stimulate the synthesis of key extracellular matrix molecules including typeⅠcollagen, type Ⅳcollagen, fibronectin and laminin. That is the major cause inducing glomerular mesangial matrix accumulation. The level of TGFβexpression is strongly correlated to DN. The activation of PKC can upregulate the level of TGFβexpression. The inhibitor of PKC, when administered orally to diabetic rats, not only prevents an elevation of glomerular filtration rate, increased albumin excretion rate, but also overexpression of TGFβ1mRNA in glomerular and accumulation of extracellular matrix proteins. All those indicated that the level of TGFβis associated with the activation of PKC. In the present studies,we observed the effect of long-term high-fat feeding on the activation of PKC and renal pathologic in rats. We also measured the content of AngⅡand the expression of TGFβ1 mRNA in kidney to explore the effect of the activation of PKC on the development of DN. We improved the traditional compound herbal medicine ---"yu ye tang"which was usually used in treating "xiao ke zheng"in traditional Chinese medicine, and got a new compound herbal medicine prescription that has the efficacy of "yi qi zi yin, gu shen xiao ke, shu xue tong luo ". Through the observation of effect of compound herbal medicine on the activation of PKC, we develop a new prescription of traditional Chinese medicine which has the effect therapy for DM and DN. Methods: Thirty nine healthy male SD rats , body weight 211±16g, age eight weeks, were provided by HeBei province experimental animal center. They were randomly divided into 4 groups and there were 9-11 rats in each group. Group 1 were the normal control group which were fed with the common feed.From group 2 to group 4 were fed with the high fat feed (which include 20% ripe porcine fat and 2% cholesterol, 0.5% cholalic salt). In the meantime, Group 3 were given with metformin. Group 4 were intervened with compound herbal medicine(yu ye tang hua cai) . Five rats were fed in each cage for 6 months. Rats were weighed and their blood samples were taken at the beginning and the end of the experiment. After an overnight fast, the blood samples were taken from the medial canthus of eyes. Serum was stored at –20℃until used. Renal tissue were taken and stored in liquid nitrogen for the determination of the activation of PKC, TGFβmRNA and the content of AngⅡ. The other renal tissue was removed and fixed in 4% neutral paraformaldehyde, processed for periodic acid schiff(PAS) and hematoxylin and eosin (HE)staining. 1. Measurements of glucose and insulin: Method of glucose oxidase for determining FBG and radio-immunity for determining FINS. HOMA-IR and insulin sensitivity index (ISI) were calculated to evaluate the resistance and sensitivity of insulin,and they were express by logarithm. 2. TG ,TC ,HDL were measured by the ratio of chrom. LDL were calculated by the Friedewald (LDL-C=TC-HDL-TG/2.2), FFAs were respectively measured by the ratio of Cu2+ chrom 3. AngⅡwere respectively measured by the radio-immunity. 4. The determination of PKC activity in kidney: 1) themembrane and the cytosol of the renal tissues were separated 2) the protein concentration were measured with some liquor of membrane and cytosol 3) the PKC activity were determined by ATP oxidative phosphated methold and presented asγ-32P-ATP pmol of incorporated per min per mg of protein. 5. Expression of TGFβ1 mRNA Rat renal tissues mRNA was extracted by the quanidinum thiocyanate. The TGFβ1 and β-actin mRNA were respectively amplified by RT-PCR. The RT-PCR products were observed by agarose gel eletrophoresis. Then we took photo. The density of TGFβ1 and β-actin cDNA band were scanned. The relative expression quantity of specific band was represented with the ratio of band densitometry unit of TGFβ1 to that of β-actin. 6. Rat kidney pathologic The rat kiadney tissue was sectioned (3μm) and stained with HE and PAS methods. Micrograps were taken at (x400) magnification. The ratio of mesangial matrix protein area to glomerular area were calculated by Beihang Medical image analysis system. 7. Statistical methods: All data were treated with SPSS 11.0, and all continuous variable were presented as mean ±SD. The statistic significance of between means was determined by T-test. One-way ANOVA was used to compare continuous variables among groups. The significance was indicated by P. The difference which had the significance was indicated by P<0.05, and the difference which had the largest significance wasindicated by P<0.01. Results: 1. The effect of the high-fat diet on the rat weight: there was no significantly difference in weight among each group at the beginning of the experiment. At the end of the experiment, the weight of HFD group(502.7±42.9g)were significantly higher than the control group(432.2±23.9g)(P<0.01). Compared to the HFD group, the weight of compound herbal medicine group and metformin group(456.5±18.1g,438.9±24.2g respectively)were significantly decreased (P <0.01). 2. The effect of the high fat feeding and compound herbal medicine on the FBG,FINS,PBG(2h) and HOMA-IR,ISI: After feeding with normal and high-fat diet respectively for 6 months, there was no significantly difference in FBG among each group. The PBG(2h) from the HFD group(7.83±0.50mmo/L)was significantly higher than that in the normal group (5.84±0.62mmol/L). The FINS from the HFD group(30.13±50.41IU/L)was significantly higher than that in the normal group (18.02±2.51IU/L). HOMA-IR in the pure high-fat group(2.055±0. 29)was significantly higher than that in the normal group(1.39±0.23). And ISI(-5.16±0.29) of the high-fat group was significantly lower than that in the normal group(-4.51±0.23)(P<0.01). ISI among compound herbal medicine group,metformin group and normal group, there was no significantly difference. PBG(2h),HOMA-IR in compound herbal medicine group and metformin group were significantlydecreased and ISI was significantly increased (P < 0.01) compared with those in the HFD group. 3. The effect of the high fat feeding compound herbal medicine on TG, TC, LDL , HDL,FFAs After feeding for 6 months, TG (1.19±0.41mmol/L), TC (3.70±0.67 mmol/ L), LDL(1.89±0.46 mmol/ L) in the HFD group were significantly higher than those of the normal group (P<0.01). Compared with the HFD group, TG, TC, LDL in compound herbal medicine group and metformin group were significantly decreased (P<0.01). HDL in the HFD group was no significantly difference compared with that of other group. FFAs(455±45μmol/l) from the HFD group was significantly higher than that in the normal group(407±32) (P<0.01),even if the FFAs of compound herbal medicine group,metformin was not significantly lower, the tendency was up to higher. 4. Comparison of AngⅡamong groups After feeding for 6 months, there was no significantly difference of AngⅡamong each group. 5. Comparison of the activity of PKC in kidney among groups: (unit:γ-32P-ATPpmol.min-1.mg-1protein) Membrane PKC activity in the HFD group(392.75±41.71) was significantly higher than that in the normal group(103.23±15.8)(P < 0.01). Membrane PKC activity in compound herbal medicine group (153.98±18.46) and metformin group (137.27±22.69) were exhibited significantly decrease compared with that in the HFD group, but stillsignificantly higher than that in normal group (P < 0.01). Cytosolic PKC activity in the HFD group(34.742 ±10.362) was significantly lower than that in the normal group(50.584 ±11.276). However, there was no significantly difference of cytosolic PKC activity in the metformin group and compound herbal medicine group compared with normal group. 6. Comparison of the expression o TGFβ1 mRNA among groups The TGFβ1 mRNA expression in the HFD group (1.046±0.155)was significantly higher than that in the normal group(0.703±0.172) (P < 0.01), Whereas TGFβ1 mRNA expression in the compound herbal medicine group(0.767±0.130) and the metformin group(0.705±0.177)significantly lower than that in the HFD group. 7. The rat renal histopathology PAS-stained sections showed glomerulor hypotrophy, basement membrane thick, mesangial matrix expansion and part glomerulor vascular changed narrow after long-term high fat feeding. The result of Medical image analysis showed that the ECM was increased. The ratio of G/M in the HFD group (0.268±0.053) was significantly higher than that in the normal group(0.113±0.045) (P<0.01). Compared with the normal group, the ratio of G/M in the metformin group (0.213±0.027)and compound herbal medicine group(0.223±0.018) was not exihibited significantly difference but significantly lower than that in the HFD group( P<0.05).