肾衰2号方对慢性肾衰大鼠肾皮质环氧化酶2及环氧化酶1 mRNA表达的影响

　　　　　　 作者：蒲冠军， 王琛， 郑平东， 何立群

【摘要】 目的：观察肾衰2号方对慢性肾衰（chronic renal failure，CRF）大鼠肾皮质环氧化酶2（cyclooxygenase2，COX2）及环氧化酶1（cyclooxygenase1，COX1）mRNA表达的影响。方法：采用左肾动脉的2/3分支结扎，右肾摘除（ablation/infarction， A/I）法制作大鼠CRF模型。造模成功后，将造模大鼠随机分为模型组、西乐葆（塞来昔布）组、肾衰2号方组。选用10只正常大鼠作为正常对照组。治疗2个月后，苏木精和伊红染色观察大鼠肾组织形态学改变，检测治疗前后大鼠血清肌酐（serum creatinine，SCr）、尿素氮（blood urea nitrogen，BUN）水平，并用逆转录实时聚合酶链反应检测肾组织中COX2和COX1 mRNA的表达。结果：肾衰2号方能明显降低CRF大鼠SCr和BUN水平，改善肾功能及肾组织形态，并能明显降低CRF大鼠肾皮质COX2 mRNA表达量，但对COX1 mRNA表达无明显影响。结论：肾衰2号方可通过抑制COX2 mRNA的过度表达改善肾功能，减轻肾小球硬化和纤维化。

【关键词】 慢性肾功能衰竭; 环氧化酶2; 肾小球硬化; 肾脏纤维化; 大鼠

Cyclooxygenase2 (COX2) and cyclooxygenase1 (COX1) are the ratelimiting enzymes in synthesizing prostaglandins (PGs). According to studies abroad， COX2 metabolites implicated in residual glomerular damage［1］ and abnormal PGs of COX2 involve in changes of glomerular hemodynamics， which， to some extent， have connections with occurrence and development of glomerular sclerosis and the deterioration of renal function［2］. Therefore， inhibiting overexpression of COX2 may be one of the effective ways to prevent and treat chronic renal failure (CRF). At present， COX2 and its inhibitors have become the research hotspot in chronic kidney diseases. COX2 and its catalytic metabolites play important roles in many aspects such as kidney development and regulation of renal hemodynamics. In recent years， thanks to the continuous efforts of exploration and practice， traditional Chinese medicine has achieved preliminary results in preventing and treating CRF. Based on the previous research， we observed the efficacy of No.2 Renal Failure Recipe (No.2RFR) in rats with CRF and explored its mechanism from the point of view of biochemistry， pathology and molecular biology.

　　1 Materials and methods

　　1.1 Experimental materials

　　1.1.1 Experimental animal Fifty SD male rats， SPF， weighing (170±10) g， were provided by Shanghai SuperB&&K Laboratory Animal Corp. Ltd. The animal certification number is SCXK(Hu)20030002.

　　1.1.2 Drugs and reagents The No.2RFR consists of Dangshen (Radix Codonopsis) 15 g， Yinyanghuo (Herba Epimedii Brevicornus) 15 g， Danshen (Radix Salviae Miltiorrhizae) 15 g， prepared Dahuang (Radix et Rhizoma Rhei) 15 g， Zisu (Perilla frutescens L.) 15 g， Huanglian (Rhizoma Coptidis) 15 g， Dongchongxiacao (Cordyceps) hyphae 5 g， Taoren (Semen Persicae) 15 g and Chuanxiong (Rhizoma Chuanxiong) 15 g， which were provided by Manufacturing Laboratory of Shuguang Hospital affiliated to Shanghai University of Traditional Chinese Medicine. After soaking， boiling and concentrating， the herbs were made into decoction with the crude herb concentration of 1.6 g/mL. Celebrex (also celecoxib， batch number J20030099， 100 mg/tablet) was provided by Pfizer Pharmaceutical Co.， Ltd. Celebrex was dissolved in pure water at 5 mg/mL. TRIzol， MMuLV (200 U/μL) and RNAsin (40 U/L) reagents were purchased from Invitrogen Corporation. DNA marker was purchased from Tianwei Shidai Biological Science and Technology Co.， Ltd. Diethyl pyrocarbonate (DEPC)， Taq polymerase (5 U/μL)， dNTP (10 mmol/L) and random primers (100 μmol/L) were purchased from Bocai Biotechnology Development Company.

　　1.2 Experimental methods

　　1.2.1 Methods of model making Forty rats were used for model building. A rat model of CRF was established by ligation of two branches of the left renal artery and ablation of 5/6 right kidney (ablation/infarction， A/I)［3］. After separation of the renal capsule and exposure of left kidney under sterile conditions， ligation of 2/3 branch of left renal artery was conducted with separate ligation of posterior and anterior descending arteries. After suturing， the 1/6 blood supply of the residual renal tissue was retained and the right kidney was removed after 1 week. Content of serum creatinine (SCr) was detected 2 weeks after nephrectomy. CRF was successfully induced if the level of SCr in CRF rats was significantly higher than that in the normal rats.

　　1.2.2 Grouping and drug administration A total of 30 rats survived from model building. The rats with the higher or lower SCr content were selected out and then equally pided into untreated group， No.2RFR group and celebrex group according to the SCr level. Another 10 normal rats were selected as normal control. Rats in the No.2RFR group were intragastrically administered with 20 g/kg No.2RFR decoction from the next day after grouping; rats in the celebrex group were administered with 33 mg/kg celebrex suspension. And 3 mL pure water was administered to rats in the normal control group and the untreated group. The treatment was lasted for 60 days. In the process of the treatment， 1 rats died in the normal control group in the 10th day， 3 rats in the untreated group on the 16th day and 2 rats in the No.2RFR group because of the injury caused by the administration. And there was 1 rats died in the celebrex group with unknown reason.

　　1.2.3 Parameters and methods of detection

　　1.2.3.1 Biochemical indicators Blood was taken from the tails of rats before the administration and from abdominal aorta after twomonth treatment respectively. Contents of SCr and blood urea nitrogen (BUN) were detected by Jaffe rate method with Aeroset2000 automatic biochemical analyzer (Abbott Company， U.S.A.).

　　1.2.3.2 Renal morphology The renal tissue was stored in 10% formaldehyde solution for 24 hours after 60day administration and bloodtaking. The 3 μm paraffin sections were made after paraffinembedding， then conventional dyeing methods such as dewaxing， dehydration and hematoxylin dyeing were taken to observe the histopathology of the renal tissues under a light microscope (×400).

　　1.2.3.3 Expressions of COX2 and COX1 mRNAs in renal tissues With 50 mg freezing fresh renal cortex， the expressions of COX2 and COX1 mRNAs in renal tissues were detected by reverse transcriptionpolymerase chain reaction (RTPCR). With the method provided by TRIzol， a total RNA was extracted to detect its purity and content. With a total RNA at 4 g， cDNA was transcripted reversely by reverse transcription kit. COX2 and COX1 primers were synthesized by Shanghai Yingjun Biotechnology Co.， Ltd. Upstream primer sequence of COX2 was 5'AAT GAG TAC CGC AAA CGC3'; downstream primer sequence was 5'TAG TCT GGA GTG GGA GGC3'. Upstream primer sequence of COX1 was 5'ATC ACT GGC ATC CGT TCA3'; downstream primer sequence was 5'AGT TGG GCT GGC ACT TCT3'. The sample volume of RNA was monitored by glyceraldehyde phosphate dehydrogenase (GAPDH). The upstream primer sequence was 5'TCC CTC AAG ATT GTC AGC AA3' and the downstream primer sequence was 5'AGA TCC ACA ACG GAT ACA TT3'. With a total of 30 cycles， the amplification conditions of COX2 PCR were 94 ℃ for 3 min， 94 ℃ 40 s， 55 ℃ 1 min， 72 ℃ 1 min and the amplification products were 417 bp in length. The amplification conditions of COX1 and GAPDH were the same with COX2 and the amplification products were 393 bp and 308 bp in lengths respectively. Finally， the amplified products were put into 1.6% agarose gel electrophoresis， with onehole plus DNA marker， 80 V voltages， electrophoresis 1 h. After taking pictures in ultraviolet light， the value of gray scale was scanned and calculated through FR2000type bioelectrophoresis image analysis system. The relative levels of COX2 and COX1 mRNAs were the band density ratio of COX2， COX1 to GAPDH.

　　1.3 Statistical methods Measurement data were expressed as x±s. Oneway ANOVA was used to compare differences among multigroups and Post Hoc Test was used to compare the differences between two groups. All the statistical methods employed the statistical software of SPSS 12.0 for Windows.

　　2 Results

　　2.1 SCr and BUN contents in each group before and after treatment After the operation， there were no statistical differences in the contents of SCr and BUN among the untreated group， No.2 RFR group and celebrex group (P＞0.05). After 60day treatment， there were statistical differences among the untreated group， the No.2RFR group and the celebrex group (P＜0.01， P＜0.05). The SCr content in the No.2RFR group was lower than that in the celebrex group (Table 1).

　　2.2 Morphological changes of renal tissues of rats The glomerular structure of rats in the normal control group was normal， with no expansion of the lumen， no thickening of capillary basement membrane. Bowman’s capsule cysts were clear with no expansion. No inflammatory cells were infiltrated in renal interstitium and no hyperplasia of fibra tissue was found. The glomerular structure of rats in the untreated group was in disorder， with severe mesangial hyperplasia， obvious thickening of capillary basement membrane， serious pressure on capillary lumen， diffusing glomeruluar fibrosis， hyperplasia of mesenterium base， reduced number of cells， adhesions of Bowman’s capsule， fibrosis of surrounding glomerular and inflammatory cell infiltration of renal interstitium. After treatment， the glomerular structure in the No.2RFR group was in minor lesions， with mild mesangial hyperplasia， mild thickening of part of capillary basement membrane， basically normal morphology of Bowman’s capsule with no obvious adhesion， no significant expansion of the lumen， obvious decrease in protein cast and a small amount of inflammatory cell infiltration of renal interstitium. The glomerular structure of the celebrex group was clear， with mild lobule in glomerular， reduced mesangial hyperplasia and inflammatory substances， mild thickening of capillary basement membrane， reduced hyperplasia of mesenterium base in glomerular， more complete cysts structure and less glomerular fibrosis than those in the untreated group (Figure 1).

　　Table 1 Serum contents of SCr and BUN in four groups（略）

　　**P＜0.01， vs normal control group; △P＜0.05， △△P＜0.01， vs untreated group; ▲▲P＜0.01， vs celebrex group.

　　Figure 1 Nephridial tissue characteristics of rats in different groups observed by light microscopy (HE staining， ×400（略）

　　A: Normal control group; B: Untreated group; C: No.2RFR group; D: Celebrex group.

　　2.3 Expressions of COX2 and COX1 mRNAs in renal cortex of rats The expression of COX2 mRNA in renal cortex was little in the normal control group， while the expression of COX2 mRNA in the untreated group was significantly higher than that in the normal control group (P&<0.01). The expressions of COX2 mRNA in renal cortex of rats in the No.2RFR group and the celebrex group were significantly lower than that in the untreated group (P&<0.01) and higher than that in the normal control group. The COX2 mRNA in the No.2RFR group was lower than that in the celebrex group (P&<0.01). There were no statistical differences in the expression of COX1 mRNA in renal cortex among these groups (Table 2 and Figure 2).

　　Table 2 Expressions of COX2 and COX1 mRNAs in renal cortex tissues in four groups（略）

　　**P＜0.01， vs normal control group; △△P＜0.01， vs untreated group; ▲▲P＜0.01， vs celebrex group.

　　Figure 2 Expressions of COX1 and COX2 mRNAs in renal cortex of rats in different groups（略）

　　1: Marker; 2: Normal control group; 3: Untreated group; 4: No.2RFR group; 5: Celebrex group. Each group includes three strips.

　　3 Discussion

　　Kidney is a very important organ to synthesize PGs by COX and a target organ for PGs. PGs metabolism is very active in kidney and is ratelimited by COX. At present， there are two kinds of isozymes for COX (COX1 and COX2)［4］. Some scholars have reported that COX2 and COX1 could be increased in experimental glomerular disease， and the expression of COX2 was increased in rats with 5/6 renal ablation［1］. Kong et al［5］ found that expression of COX2 in rats with subtotal renal cortex ablation was about 11.35 times of that in the control group. COX2 might be involved in glomerular injury during inflammatory diseases and the expression of COX2 was increased in experimental and human glomerular injury. PGs， especially TXA2， played an important pathogenic role and the inflammatory cells of infiltration， glomerular macrophages and mesangial cells were the source of COX products［6］. In this study， the expression of COX2 mRNA in renal tissues in rats with CRF was upregulated which was in corresponding with previous research［7］.This study also suggested that there were no statistical differences in the expression of COX1 mRNA in renal cortex among all groups.

　　There are different factors which could lead to the renal injury. When the kidney diseases develop to end stage renal failure， the glomerular sclerosis and tubulointerstitial fibrosis would develop［8］. Mechanisms of CRF are mainly owned to damage of glomerular capillary and development of microthrombosis induced by reduction of renal parenchyma and changes of glomerular hemodynamics， capillary obstruction induced by blood circulation of micromolecule into mesangial region because of increase of mesangial matrix and expansion of mesenterium base， and transparent fibra material deposit under endodermis. The pathological metabolites of COX2 source were closely related to development of glomerular sclerosis and deterioration of renal function. There were evidences that the inflammation of kidney was caused by COXmediated products which may be involved in the injury of immune and nonimmune cells of kidney［9］. There were no expressions of COX2 protein or mRNA in normal renal macula densa， however， the COX2 immune response had been found in renal parenchyma in a variety of kidney diseases［10］. At present， COX2 may be an important factor which leads to the development of CRF.

　　Celebrex (celecoxib) is the selective inhibitor for COX2 which can inhibit the synthesis of COX2. Studies had shown that selective COX2 inhibitors could actually inhibit deterioration of kidney in rats with subtotal renal ablation［9］. Selective COX2 inhibitors (Mobic) could inhibit the catalytic activity of COX2 to block the formation of pathological prostaglandin， reduce gene transcription of COX2 and correct the abnormal state of degradation of extracellular matrix， which confirmed that selective COX2 inhibitors could protect the kidney by regulating local reninangiotensin system of kidney in rats with unilateral obstruction of ureter［11］. As celebrex at therapeutic dose does not affect the synthesis of prostaglandin substances activated by COX1， it does not interfere with the normal physiological progression related to COX1， especially in stomach and intestine， blood platelet and renal tissues. Mobic has a weak inhibition to the synthesis of prostaglandin in stomach and kidney. Therefore， there are fewer adverse effects to the stomach and kidney by using COX2 inhibitor (celebrex). The experimental results showed that celebrex could inhibit the overexpression of COX2 in the kidney， thus improve renal function.

　　In theory of TCM， pathological characteristics of CFR are deficiency in root and excess in the branch， and the treatment principle is reinforcing deficiency and reducing excess， and simultaneous treatment of the branch and the root. Deficiency in spleen and kidney is the fundamental factor that leads to CRF， while most doctors believed that pathogenic excess would include dampness， dampness and heat， dampness and toxin， blood stasis， phlegmdamp. The therapeutical principle of No.2RFR was strengthening the spleen qi， tonifying the kidney yang， activating blood circulation and removing blood stasis and turbidity. In the prescription， Dangshen is to strengthen the spleen qi and warm the kidney yang， which recuperates spleen and kidney simultaneously， secures the root and cultivates the origin. Danshen is used to activate and nourish the blood， Dahuang to clear away the pathogenic heat， remove the turbidity， activate blood circulation and help metabolism and Taoren to activate blood circulation and remove blood stasis. Zisu is used to enter the blood aspect and disperse blood vessel， dry the dampness， Huanglian to strengthen yin with bitter cold， dry the dampness and purge the fire， clean away pathogenic heat and dissolve toxin. Cordyceps hyphae is to warm the fire of the life gate， nourish essence and marrow and tonify the spleen and kidney［12］. The experimental results showed that the expression of COX2 mRNA was declined significantly in the No.2RFR group and more than those in the untreated group and the celecoxib group， which suggested that No.2RFR can decrease the ratelimiting enzymes which synthesize prostaglandins， reduce pathological metabolites of COX2， thus improve the renal structure.

　　The experimental results also showed that the levels of SCr and BUN in the untreated group were significantly higher than those in the normal control group， which indicates that the rat model of CRF was successfully established. After sixtyday treatment， the contents of SCr and BUN were decreased significantly， and the SCr content was less than that in the celebrex group which indicates that No.2RFR could improve renal function and retard the process of CRF. The glomerular structure of the untreated group was in disorder， with severe mesangial hyperplasia， diffusing glomeruluar fibrosis， hyperplasia of mesenterium base， fibrosis of surrounding glomerular and inflammatory cell infiltration of renal interstitium. After treatment， the glomerular structure of the No.2 RFR group was in minor lesions， with mild mesangial hyperplasia， no significant expansion of the lumen and a small amount of inflammatory cell infiltration of renal interstitium. The glomerular structure of the No.2RFR group was obviously better than that of the untreated group， and that of the celebrex group was also better than that of the untreated group to some extent. The experimental results suggested that No.2RFR and celebrex could improve renal function by inhibiting the overexpression of COX2 mRNA and the effect of No.2RFR was better than that of celebrex.

　　In conclusion， No.2RFR can inhibit the overexpression of COX2 mRNA in renal cortex and reduce glomerular sclerosis and renal fibrosis， which is the pharmacodynamic basis to treat CRF by strengthening the spleen qi， tonifying the kidney yang， activating blood circulation and removing blood stasis， purging turbidity.

　　

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