| Objective:Rhubarb is effective in treating chronic renal failure?CRF?.Its processed products include raw rhubarb,rhubarb steamed with wine,rhubarb stir-fried with wine and rhubarb charcoal.The purpose of this study is to determine the change rules of active components in rhubarb under different processing methods and different decoction times,to establish a rat model of CRF,and to observe the improvement of renal function of CRF rats by intragastric administration of Rhubarb Decoction with different processing methods and decocting time,to find out the best processing method and decocting time point for CRF rats,and to explore the mechanism of Rhubarb in the treatment of CRF,so as to provide theoretical basis for rational use of Rhubarb in the treatment of CRF.MethodExperiment 1:The raw rhubarb,rhubarb steamed with wine,rhubarb stir-fried with wine and rhubarb charcoal were decocted for 5,7,9,10,15,20and30minutes after boiling,and then they were made into 50ml rhubarb water decoction with concentration of 1g/ml respectively.Four kinds of rhubarb,each with 7 samples decocted at different time points.The contents of gallic acid,aloe emodin,rhein,emodin,chrysophanol and physcione in 28 kinds of rhubarb decoctions were determined by high performance liquid chromatograp-hy,and the linear relationship,precision,stability,reproducibility and added sample recovery were studied.Experiment 2:10 SD rats were randomly selected from 160 SD rats as the normal control group,and the remaining 150 rats were randomly divided into cages and fed.The rat model of chronic renal failure was established by intragastric administration of adenine solution.The concentration of the solution was 2.5%,and the model was established at 20mg·kg-1·d-11 for 28days.By observing the body weight,renal function and anatomical and pathological structure of the model rats,a method of establishing CRF model which can simulate the disease and symptoms of CRF and has low mortality rate and good repeatability was obtained,so as to provide a basis for later experiments.Experiment 3:145 CRF rats were randomly divided into model group,rhubarb water decoction treatment group?each rhubarb processed product was decocted for 5,7,9,10,15,20,30min after boiling,respectively?and normal control group,with 5 rats in each group,and a total of 30 groups.The normal group was fed with normal diet.The model group was given the same dosage of saline as the rhubarb treatment group.The 28 rhubarb treatment groups were given corresponding decoction of rhubarb by intragastric administration every day for 8 weeks.After treatment,the BUN,Cr and UA values in blood of rats in each group were measured,and the improvement of renal function was observed.Experiment 4:The first purgation time,the purgation frequency in 5 hours and the total purgation frequency of the experimental rats in one week and four weeks of treatment were observed by the method used in experiment3,the purgation effect was compared,the activity of ATPase in rat colonic tissue was measured,the renal function indexes of rats in each group at one week and four weeks after treatment were compared,and the relationship between renal function and purgation was analyzed.Experiment 5:80 CRF rats were randomly divided into model group,rhubarb steamed with wine water decoction treatment group?the rhubarb steamed with wines were decocted for 5,7,9,10,15,20,30 minutes after boiling,respectively?and normal control group,with 10 rats in each group,a total of 9 groups.The normal group was fed with normal diet.The model group was given the same dosage of saline as the rhubarb steamed with wine treatment group.The 7 rhubarb steamed with wine treatment groups were given corresponding decoction of rhubarb steamed with wine by intragastric administration every day for 8 weeks.The kidneys of rats were harvested and their morphology was observed.Obtain the rat kidney,observe its morphology,the right kidney tissue was prepared into paraffin section,and the left kidney tissue was stored in the refrigerator at-80?.Prepare pathological sections of rat kidneys,observe the pathological changes of kidneys in each group,and observe the apoptosis of kidney cells.Experiment 6:The paraffin sections of rat kidney tissues in Experiment 5were selected and the expression of LN,Col?,?-SMA,p-PI3k,PI3k,p-Akt,Akt and TGF-?1 protein in kidney tissues was detected by immunohistochemistry.Experiment 7:The fresh frozen kidney tissues of rats in Experiment 5were selected and the expression of AQP1mRNA,AQP2mRNA and TGF-?1mRNA in kidney tissues was detected by RT-PCR.Experiment 8:The fresh frozen kidney tissues of rats in Experiment 5were selected,and the expression levels of LN,Col?,?-SMA,p-PI3k,PI3k,p-Akt,Akt and TGF-?1 proteins in CRF rats of rhubarb steamed with wine group were detected by Western-blot.Result?1?In experiment 1,the contents of gallic acid,aloe emodin,rhein,emodin,chrysophanol and physcione in 28 kinds of rhubarb decoction showed good linear relationship,precision,stability,reproducibility and added sample reco-very.Among the four processed products,except rhubarb steamed with wine,the contents of the main components of the other three processed products increased with the increase of decoction time.The content of main components of rhubarb steamed with wine reached its maximum in about 10minutes.The order of total content of anthraquinones in different processed products was raw rhubarb>rhubarb steamed with wine>rhubarb stir-fried with wine>rhubarb charcoal.?2?In experiment 2,the CRF model prepared by intragastric administrati-on of adenine solution based on the relevant literature and preliminary experimental results can simulate the disease and symptoms of CRF,and this model has low mortality and good repeatability.?3?The results of Experiment 3 showed that after 8 weeks of treatment,the blood Cr,BUN and UA values in raw rhubarb group,rhubarb steamed with wine group,rhubarb stir-fried with wine group and rhubarb charcoal group were significantly lower than those in model group?P<0.01?.Among them,raw rhubarb decreased most significantly at the 9th minute?P<0.05?,rhubarb steamed with wine and rhubarb charcoal at the 10th minute?P<0.05?,and rhubarb stir-fried with wine at the 15th minute?P<0.05?;Among the four processed rhubarb groups,the rhubarb steamed with wine group decreased most significantly?P<0.05?.?4?The results of Experiment 4 showed that each rhubarb treatment group had a purgation effect compared with the normal group and the model group at the first week of treatment?P<0.01?;The purgation effect of 10-minutes raw rhubarb group was the most obvious?P<0.05?;The levels of blood Cr,BUN and UA in rhubarb treatment group were significantly decreased?P<0.01?;Among them,the decrease of raw rhubarb group was the most obvious?P<0.05?.At the fourth week of treatment,the purgation effect of rhubarb in each group was not obvious,and there was almost no diarrhea effect,which could not be counted.Compared with the model group,the blood Cr,BUN and UA values of the rhubarb treatment group in the four rhubarb processing groups decreased significantly?P<0.01?;among them,rhubarb steamed with wine group decreased most significantly,and there was statistical significance?P<0.05?.Compared with the normal group,each rhubarb processed products treatment group had inhibitory effect on the activity of Na+-K+-ATPase in rat colon?P<0.01?,and raw rhubarb group had the most obvious inhibitory effect?P<0.05?.?5?The results of Experiment 5 showed that the ratio of glomerular mesangial area to glomerular area in the rhubarb steamed with wine treated group was significantly lower than that in the model group?P<0.01?,and the most significant decrease was observed at the 10th minute?P<0.05?;the apoptotic rate of cells in the rhubarb steamed with wine treated group decreased significantly?P<0.01?,and the most significant decrease was observed at the 10th minute?P<0.05?.?6?The results of Experiment 6 showed that the average optical density of LN,Col?,?-SMA,TGF-?1,p-PI3k,PI3k,p-Akt and Akt in rat kidney tissue of model group was significantly higher than that of normal group?P<0.01?;while the average optical density ratio in rhubarb steamed with wine group was significantly lower than that in model group?P<0.01?,among them,the10-minutes rhubarb steamed with wine group decreased most significantly?P<0.05?.?7?The results of Experiment 7 showed that compared with model group,the expression of AQP1mRNA and AQP2mRNA in normal group was significantly increased?P<0.01?,especially in the 10-minutes rhubarb steamed with wine group.The expression of TGF-?1mRNA in kidney tissue of model group was significantly higher than that of normal group?P<0.01?;Compared with the model group,the expression of rhubarb steamed with wine treated group was significantly decreased?P<0.01?,especially in the 10-minutes rhubarb steamed with wine group?P<0.05?.?8?The results of Experiment 8 showed that the average gray ratio of PI3k,Akt,LN,Col?,?-SMA and TGF-?1 in kidney tissue of model group was significantly higher than that of normal group?P<0.01?;while the average gray ratio of rhubarb steamed with wine treatment group was significantly lower than that of model group?P<0.01?,and the decrease was the most obvious at the 10th minute?P<0.05?.The average gray ratio of p-PI3kt,p-Akt in kidney tissue of model group was significantly lowerr than that of normal group?P<0.01?,while the average gray ratio of cooked rhubarb treatment group was significantly higher than that of model group?P<0.01?,and the increase was the most obvious at the 10th minute?P<0.05?.Conclusion?1?High performance liquid chromatography can determine the compo-nents of rhubarb decoction steadily and accurately.The order of total content of anthraquinones in different processed products was raw rhubarb>rhubarb steamed with wine>rhubarb stir-fried with wine>rhubarb charcoal.The content of main components of rhubarb steamed with wine reached maximum in about10 minutes.?2?The CRF rat model prepared by intragastric administration of adenine solution at a concentration of 2.5%according 200mg·kg-1·d-11 was most stable and had a low mortality.?3?The kidney function of CRF rats was improved most obviously in the rhubarb steamed with wine group with 10 minutes of decoction after boiling.Rhubarb steamed with wine decocted for 10 minutes after boiling has the best effect on CRF rats,which is superior to processed products with other decoction time.?4?The mechanism of rhubarb in treating CRF is related to its purgation effect,and the purgation effect of raw rhubarb is stronger than that of other processed rhubarb.Raw rhubarb has certain advantages in early recovery of renal function indicators,and rhubarb steamed with wine is the best in late treatment.?5?The mechanism of rhubarb in treating CRF rats is to delay the process of CRF by inhibiting the proliferation of glomerular mesangium,inhibiting cell apoptosis and reducing the degree of glomerular sclerosis.?6?The mechanism of rhubarb steamed with wine in treating CRF rats is to reduce water retention and toxin accumulation by regulating AQP1 and AQP2,and to reduce the degree of glomerulosclerosis by inhibiting the expression of TGF-?1mRNA,thus delaying the progress of renal function.?7?The mechanism of rhubarb steamed with wine in treating CRF rats may be through inhibiting p-PI3k and p-Akt,thereby inhibiting the TGF-?1/PI3k/Akt pathway,and ultimately alleviating renal fibrosis in CRF rats.?8?Through the above seven points,we believe that rhubarb can effectively improve renal function and fibrosis in CRF rats.In the processed products of rhubarb,the rhubarb steamed with wine with decocting time of about 10 minutes after boiling is more suitable for the treatment of CRF rats.We explored its possible mechanism,and provide a theoretical basis for better use of rhubarb in the treatment of CRF. |
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