Study Of Compound Huang Gan In Slowing The Progression Of Chronic Renal Failure In 5/6 Nephrectomy Rats

1.Background and objectiveChronic kidney disease(CKD)poses higher risks on public health issue for high morbidity and mortality and its treatment remains as major challenge in clinical practice.According to the statistics of epidemiology and nationwide registries,the incidence of CKD in general population in China reaches 10%on average.Currently,the annual costs of dialysis has exceeded RMB 9.6 billion yuan.Chronic renal failure(CRF),a common clinical syndrome,occurs on the basis of various CKDs with slowly renal-function decline and renal failure in the end.Being the major pathological feature of CRF,renal fibrosis is the unstoppable and irreversible end-result of various CKDs.The primarily,renal interstitial fibrosis originates from the massive accumulation of extra cellular matrix(ECM)due to unbalanced synthesis and degradation.Meanwhile,oxidative stress also exists in the incipient and end stage of various kidney diseases,which is considered as the common pathway for various pathological damages of CKD.Oxygen free radicals can be produced by a wide range of renal cells,including mesangial cells,renal interstitial cells and renal tubular epithelial cells,etc.Oxygen free radicals stimulates these cells to secrete various inflammatory cytokines and adhesion molecules,e.g.,fibronectin(FN),intercellular adhesion molecule-1(ICAM-1)and monocyte chemotactic protein-1(MCP-1),which may accelerate the extracellular deposition of ECM meanwhile inhibit its degradation and lead to renal fibrosis eventually.Researchers have attached great importance to the new anti-inflammatory and anti-oxidative pathway of stress due to the close association between chronic inflammation and oxidative stress and the development of CKD.Recent research indicates that Nrf-2 activator,Bardoxolone methyl,is able to repair the dynamic redox equilibrium of the inflammatory site,however,the phase III clinical trial with this Nrf-2 activator was suspended due to severe side effects.Some other studies on PPAR-a receptor activator,Palmitoylethanolamide(PEA),provide evidence that it can suppress inflammation and stabilize mast cells,but its role requires a further study.In addition,powerful evidence from clinical research has demonstrated the renin-angiotensin-aldosterone system(RAAS)is a possible potent blocker in delaying the progress of CRF,yet it fails to act frequently.Other unsatisfactory therapies include endothelin-receptor antagonist,glycosaminoglycans,advanced glycation end-products inhibitors,erythropoiesis stimulating factor,HMG-CoA reductase inhibitors,etc.Therefore it is of great significance to strengthen the early prevention of CRF and to reduce the morbidity of end-stage renal disease(ESRD).In the paradigm of traditional Chinese medicine,CKD consists of two perspectives:primary asthenia syndromes(deficiency of vital energy of spleen and kidney,deficiency of vital energy and yin,yin deficiency of liver and kidney,yang deficiency of spleen and kidney,deficiency of yin and yang)and secondary sthenia syndromes(turbid dampness,damp-heat,blood stasis,water vapor,turbid toxin),yet there still lack of convincing arguments in view of the recognition of its morbidity and even CKD itself.For this reason,the clinical symptoms of CKD can be treated in TCM through the following approaches:1)Improve the microinflammatory state commonly existing among patients using oral administration of compound Danshen tablets and intravenous transfusion of Astragalus injection:2)Improve azotemia using the components of tannin,especially rhatannin in rhubarb because it can reduce the intestinal absorption of amino acid,increase the concentrations of essential amino acid in blood,thereby reduce the synthetic urea in liver and kidney tissue and restrain the decomposition of proteins using the ammonia to synthesize proteins in order to promote the excretion of urine from kidney.3)Improve the body's immune function by using such medicines as Jinshuibao capsule containing Cordyceps sinensis mycelium.4)Inhibit glomerular sclerosis and interstitial fibrosis using Danshen injection,Ligustrazine injection,Erigeron injection,Shuxuetong injection,etc.Currently,the Chinese patent medicine for the treatment of CRF in clinical application mainly includes Niaoduqing granules,Shenshuaining capsule,Shenkang injection,etc.whose curative effect still leave much to be desired.Among these prescriptions,Niaoduqing granules developed by Nanfang Hospital have been proved from our original research to be effective in anti-CRF for its significant effective rate of 20.67%and total effective rate of 69.33%.Not only does this medicine still get room for improvement in terms of curative effects,but its mechanism of action also remains to be further discussed in pursuit of experimental data for more effective medicines.Based on the original prescription of Niaoduqing granules,our research focuses on its combination with other prescriptions using a CRF rat model by gavage with adenine under a hypothesis that the medicine may reduce serum Scr,BUN and the oxidative stress in CRF rats.In this research,the CRF rat model was established using the 5/6 nephrectomy.Different combinations with selected prescriptions were administered on the model to further look into the possible mechanisms of action in delaying CRF progression in order to provide sound experimental basis for the clinical treatment of CRF.2.Methods:2.1 Preparation of Compound Huanggan Extracts Part of the herbs were soaked with 7 times amount of 70%ethanol for 30 min,water bathed and then decocted 1.5h twice,the filtrated decotions mixed.The other part of the herbs were soaked with 7 times amount of water for 30 min and decocted 2h twice,the filtrated decotions mixed and concentrated.In the following,the mixed decotions were subject to alcohol precipitation for 24h at 4? with ethanol at the final concentration of 80%and then the supernatants were filtrated to obtain extracted solution.The precipitated was washed twice with 80%ethanol to obtain extracted solution as well.Then,the two extracted solutions obtained were mixed and concentrated by rotary evaporation.Finally,the prepared extracted solutions were further concentrated through vacuum drying for three times into pure extracts ultimately.The extracts were prepared with distilled water and solubilized with 0.5%CMC-Na before gavage.The HPLC was used for tests with the chromatographic column of ZORBAX Eclipse Plus-C18 Columns(4.6mm,150 mm,5 ?m)and mobile phase(acetonitrile:water).Linear gradient elution method was used as aqueous phase from 90%-10%for 90min with wavelength of 274nm,flow rate of 0.8 mL-min-1,column temperature of 30? and injection volume of 20?L.2.2 Construction of Rat CRF Models for the 5/6 Nephrectomy The operating room was exposed to ultraviolet radiation for 30 minutes for disinfection.10%chloral hydrate anesthesia was administered by intraperitoneal injection with the dose of 0.7mL/200g before operation.Sector resection was used to remove 1/3 of the upper and lower pole of the left kidney.The section was covered with absorbable gelatin sponge for hemostatic use.The remnant kidney was returned to the abdominal cavity and the abdomen was closed with double layer suture.Nephrectomy(right kidney)was performed after 1 week.Only the renal capsules were peeled away in the sham-operation group.Two weeks after operation,60 rats with 5/6 nephrectomy were divided into 6 groups(10 each)by weight stratified randomized block method,including Model Group,Niaoduqing Group,Losartan Group,three Compound Huanggan Groups of high,middle and low dosages as well as Sham-operation Group,All rats were fed by standard diet and had free access to water.2.3 Drug Administration and Detections of Biochemical Indexes In the sham-operation group and rat model groups distilled water solution containing 0.5%CMC-Na was administered by gavage(1mL/100g).In the Niaoduqing group,Niaoduqing suspension(3.6g/kg/d)was administered by gavage.Corresponding Compound Huanggan suspension was administered to Compound Huanggan groups of high(14.4g/kg/d),middle(7.2g/kg/d)and low(clinical equivalent dose 3.6g/kg/d)dosages by gavage once per day.During the experiment,the rats of each group were weighed weekly and the dieting,drinking,behavior,mental state,hair,urine and stool were under close observation.12 weeks after drug administration,the rats of each group were anesthetized to collect its blood serums from the abdominal aorta.Scr,BUN,Ucr and 24h UPr were detected with the automatic biochemical analyzer.The pathological changes were observed with HE and Masson Staining of the remnant renal tissues.2.4 Detection of oxidative stress and fibrosis index in each group 12 weeks after drug administration,the serum and renal tissues of rat models of all groups were collected.The serum was placed at 4? for 0.5h and then centrifuged at 3000r/min for 15min to obtain the supernatant.The indexes including SOD,MDA,GSH-PH,etc.in the serum of all groups were determined following the instruction of test kit.Part of the renal tissues were fixed in 4%formalin,dehydrated and embedded and the expressions of FN,ICAM-1 and MCP-1 in the renal tissues were then detected.In the following,part of renal cortical tissues were obtained for extraction of total RNA.The expressions of FN,ICAM-1 and MCP-1 in the tissues were then detected.3.Results3.1 Content Determination of Compound Huanggan Extracts 1g of Compound Huanggan Extracts was equivalent to 4g of natural herbs.The components and contents of extracts from the extractions of three times were relatively stable.According to HPLC analysis,the main components and contents of the extracts included Liquiritin(0.06%),Paeonol(1.06%),Aloe-emodin(0.1%),Rhein(0.47%),Rheum emodin(0.19%),Chrysophanol(0.41%)and Physeion(0.13%),of which Paeonol was the highest in content and Rhubarb took up the most of the components.3.2 Vital Sign Changes of Rat Models 12 weeks after drug administration,rats in model groups were characterized by dim and sparse hair,and pale ears and tails,and weighed a little less than those in other groups.Comparatively,the rats in other groups were characterized by smooth hair,healthy shape and gradual weight gain.However,there were no statistically significant differences in weight between the groups(P>0.05).3.3 Differences in Biomedical Indexes There were no statistically significant differences in weight of the remnant kidneys between the groups.Except for Niaoduqing Group,the renal indexes(RI)of other treatment groups were significantly lower than those of the Model Group(P<0.05).The levels of Scr,BUN,Ccr and UPr of the Model Group were all significantly higher than Sham-operation Group(P<0.01).Except for Losartan Group,the Scr levels of other treatment groups were significantly lower than that of the Model Group(P<0.05)with Compound Huanggan Groups of high,middle and low dosages even significantly lower than Losartan Group(P<0.05).The Ccr levels of all drug administration groups were significantly higher than that of the Model Group(P<0.01)with Compound Huanggan Group of high dosage higher than Losartan Group(P<0.05).The Upr levels of all drug administration groups were significantly lower than the Model Group(P<0.01).The biomedical indexes of Compound Huanggan Groups of high,middle and low dosage and Niaoduqing Group had no statistical difference(P>0.05).HE Staining showed that there were no signs of pathological changes in renal tissues or abnormal changes in glomerulus,renal interstitium and renal tubules in the sham-operation group.In the model group,however,there were large amount of infiltrated inflammatory cells in glomerulus and renal interstitium,sclerosis,multi-focus and capsular synechia and proliferation of mesangial cells in the glomerulus,and atrophy in the renal tubules presenting protein casts and indicating severe fibrosis.In the Compound Huanggan Groups,the glomerulus,renal interstitium and renal tubules improved in varying degrees and the glomerular sclerosis and renal interstitial fibrosis were greatly improved.Masson Staining showed clear glomerular and renal tubular structure with normal morphology and no sign of proliferation and basically normal renal structure with little proliferation of fibrous tissue in Sham-operation Group;dialed tubules and large amount of green deposits in renal capsule,glomerulus,renal tubules and renal interstitium suggesting the proliferation of a considerable amount of collagen fibers in Model Group;and improvement of glomerulus,renal tubules and renal interstitium in varying degrees and marked improvement in the glomerular sclerosis and renal interstitial fibrosis in the Compound Huanggan Groups.3.4 Determination of oxidative stress indexes in serum of each group MDA contents in the serum of the Model Group were significantly elevated compared with Sham-operation Group(P<0.05).MDA contents in the serum of Compound Huanggan groups of high,middle and low dosage all dropped compared with the Model Group(P<0.05).SOD contents in the renal tissues of Compound Huanggan Groups of high,middle and low dosage were significantly increased compared with Model Group.There were no statistical differences in MDA and SOD contents between Compound Huanggan Groups of high,middle and low dosage,Niaoduqing Group and Losartan Group(P>0.05).GSH contents in Compound Huanggan Groups of high,middle and low dosage were significantly higher compared with the Model Group(P<0.05),with Compound Huanggan Groups of high and middle dosages higher than Niaoduqing Group(P<0.05)and Compound Huanggan Group of high dosage higher than Losartan Group(P<0.05).3.5 FN,ICAM-1 and MCP-1 gene and protein expression in renal tissue FN mRNA expression in renal issues in all drug administration groups were significantly lower than the Model Group(P<0.01),but higher than Sham-operation Group(P<0.05).ICAM-1 expression in renal issues in all drug administration groups were significantly lower than the Model Group(P<0.01),and there were no statistical differences between drug administration groups and Sham-operation Group(P>0.05).MCP-1 expression in renal issues in all drug administration groups were significantly lower than the Model Group except for Niaoduqing Group(P<0.05),with Compound Huanggan Groups of high,middle and low dosages higher than Sham-operation Group(P<0.05),but with no statistical difference when compared with Niaoduqing Group(P>0.05).The results by immunohistochemistry showed that FN expressed at a higher level in glomerulus,and at a lower level in renal tubules and renal interstitium.IOD of FN positive expression was analyzed semiquantitatively to find that FN of the Model Group expressed significantly higher than that of Sham-operation Group(P<0.01)as well as other drug administration groups(P<0.05),and there was no significant statistical difference in the expression among the drug administration groups(P>0.05).However,ICAM-1 expressed at a higher level in renal tubules and renal interstitium,and at a lower level in glomerulus.IOD of ICAM-1 positive expression of the Model Group expressed significantly higher than that of any other group(P<0.05),with Niaoduqing Group,Losartan Group and Compound Huanggan Group of low dosage expressed at even a much lower level(P<0.01).MCP-1 expressed at a relatively low level in renal tissues of all groups.However,IOD of MCP-1 positive expression of Niaoduqing Group,Losartan Group and Compound Huanggan Group of high dosage still expressed at a lower level than that of the Model Group(P<0.01).Immunohistochemistry results were roughly consistent with PCR results.4.ConclusionCompound Huanggan Extracts can improve the renal function,reduce renal fibrosis and delay renal failure of the CRF.Compound Huanggan Extracts may reduce the oxidative stress level in CRF rats and inhibit the expression of chemokines,thereby lowering the level of renal fibrosis and delaying the progress of CRF in rat models.