摘要
背景：
腹膜功能衰竭已成为终末期肾功能不全(ESRD)患者腹膜透析技术性失败的最重要的原因,并成为制约腹膜透析(PD)发展的“瓶颈”。研究表明完整的间皮细胞层可以显著延缓腹膜超滤衰竭的进程,组织病理学也证实腹膜功能衰竭早期的重要特征表现为腹膜间皮细胞(PMC)的损伤和腹膜上皮-间皮细胞转分化(EMT)。因此,防治腹膜纤维化的重要靶点是阻止或逆转腹膜间皮细胞损伤的发生或治疗EMT所导致的后果如细胞的移行、细胞外基质(ECM)的合成增多。转化生长因子-β(TGF-β)是EMT过程中关键的细胞因子,如果能抑制TGF-β的过度表达,就能抑制其下游相应的细胞因子,从而减缓EMT的发生和发展,维护PMC的结构和功能,维持正常的腹膜功能。
目的：
通过观察高糖腹透液诱导的大鼠腹膜纤维化模型,揭示其对腹膜超滤功能的影响及对PMC的形态、数量及功能变化规律,以及TGF-β1、碱性成纤维细胞生长因子(bFGF)、带状封闭蛋白-1(ZO-1)、水通道蛋白-1(AQP-1)等细胞因子在腹膜组织中的表达情况。并通过中药复方制剂参芎注射液进行干预,探讨在大鼠腹膜纤维化过程中参芎注射液对PMC保护的可能机制。并通过临床研究,观察参芎注射液对腹透患者超滤量的影响及其对PMC的保护作用。
方法：
1、大鼠腹膜透析模型的建立及超滤量的观察：SD大鼠分A、B两组,均给予4.25%腹透液(PDF)透析,A组透析剂量为100ml/Kg,B组透析剂量为200ml/Kg；分别测定两组在1h、2h、3h、4h超滤量。
2、动物实验部分：建立高糖诱导的大鼠腹膜纤维化模型,40只大鼠随机分为空白组、模型组、低剂量组和高剂量组,每组10只。空白组：不作任何处理和干预；模型组：4.25%PDF100ml/Kg腹腔注射,每日1次；低剂量组：4.25%PDF87.5ml/Kg+参芎注射液12.5ml/Kg腹腔注射,每日1次；高剂量组：4.25%PDF75ml/Kg+参芎注射液25ml/Kg腹腔注射,每日1次。实验4周后处死大鼠作检测。行4h腹膜平衡试验(PET),测定透析液肌酐浓度(Dcr),血浆肌酐浓度(Pcr),初始腹膜透析液葡萄糖浓度(DO),4h透析液葡萄糖浓度(D4),计算D/Pcr、D4/D0。计量各组大鼠PD平均超滤量。大鼠腹膜行HE和VG染色,采用Image-Pro Plus6.0图像分析系统测量间皮下红色的纤维组织厚度。大鼠腹膜标本行超薄切片,透射电子显微镜观察病理改变。电化学发光法检测腹透引流液中CA125浓度,计算CA125表达率。免疫组织化学法检测腹膜TGF-β1、bFGF、Z0-1、AQP-1表达水平。选择背景清晰且阳性表达好的切片作统计学分析,每张切片随机选择10个高倍视野(200×),计数阳性细胞,以10个高倍视野总数作为阳性细胞数。
3、临床研究部分：选择武汉市中西医结合医院64例病情稳定,透析时间超过3个月的腹透患者,予参芎葡萄糖注射液50ml加入每袋(2L)腹透液,行标准CAPD,疗程为7天。治疗前后行标准腹膜平衡试验,生化指标检测,腹透引流液CA125浓度检测,计算透析前后的尿素清除指数(KT/V),肌酐清除率(Ccr)。
4、统计学方法
统计软件为SPSS17.0,实验结果均为定量数据,用均数±标准差(x±s)表示,各组间差异性比较采用单因素方差分析并进行两两比较(LSD法),P<0.05(双侧)为差异有显著性意义。
结果：
1、大鼠超滤量实验部分：A、B两组在留腹1h、2h、3h、4h的超滤量均逐渐增加,A组2h平均超滤量为10.46ml,较1h的平均超滤量9.57ml增加无显著差异(p>0.05),A组3h、4h平均超滤量12.85ml和13.84ml较1h和2h超滤量增加均有显著差异(p<0.05)。B组2h平均超滤量11.14ml,较1h的9.15ml增加有显著差异(p<0.05)；B组3h、4h的超滤量15.09ml和16.1ml较1h、2h增加均有显著差异(p<0.05)。B组第3h、4h超滤量显著高于A组同时间段的超滤量(p<0.05)。
2、空白组、模型组、低剂量组和高剂量组之间超滤量的比较,空白组的平均超滤量为13.75±1.17ml,较模型组4.53±1.10ml明显高,低剂量组的超滤量有明显改善,达到7.46±1.23ml,高剂量组有更为显著的改善,为9.73±1.11ml。经统计学处理各组间均有显著性差异(P<0.05)。
3、空白组、模型组、低剂量组和高剂量组之间腹膜转运功能的比较,D/Pcr以模型组升高最为显著,达到0.84±0.07,较空白组0.53±0.03升高明显。低剂量组和高剂量组D/Pcr分别为0.76±0.08,0.65±0.12,均能减缓D/Pcr的上升,以高剂量组更为明显。经统计学处理各组间均有显著性差异(P<0.05)。D4/D0的比较,四组分别为0.59±0.04,0.38±0.06,0.45±0.05,0.51±0.04,各组间均有显著性差异(P<0.05)
4、采用电化学发光法检测各组腹透引流液CA125浓度及表达率,空白组CA125浓度为11.99±1.37U/ml,CA125表现率为2.19±0.30U/ml,模型组有显著下降,分别为3.53±0.51U/m1,0.51±0.12U/ml。低剂量干预后上升为6.46±0.89U/ml和1.01±0.15U/ml；高剂量干预后为7.96±0.97U/ml,1.32±0.16U/ml。各组组间比较均有统计学差异(P<0.01)。
5、PMC形态及各组腹膜厚度比较,空白组为22.2±5.1μm,模型组明显增厚为97.7±18.3μm,低、高剂量组有不同程度增厚,分别为57.9±7.8μm,46.1±7.2μm。各组组间比较有显著性差异(P<0.05)。光镜下,空白对照组腹膜由一层扁平间皮细胞覆盖,间皮细胞基本完整、连续分布,间皮下覆有疏松结缔组织,结缔组织未见明显增生；模型组及低剂量、高剂量组壁层腹膜明显增厚、疏松,间皮细胞脱落,间皮下有大量的胶原纤维沉积,尤以模型组明显。
6、超微结构的比较。空白对照组：腹膜间皮细胞结构完整,细胞无皱缩、变形；细胞游离面微绒毛丰富,分布均匀；细胞器清晰,见吞饮泡、线粒体、内质网等,细胞核结构清晰；相邻间皮细胞间紧密连接完整。模型组：偶见少量间皮细胞,大部分脱落,细胞变形,微绒毛稀疏可见,细胞内线粒体等细胞器明显肿胀,结构破坏。间皮下纤维组织增生明显。低剂量组：间皮细胞疏松,细胞表面不连续,微绒毛明显减少,细胞器肿胀,胞核染色质边集、凝固,相邻间皮细胞紧密连接消失。高剂量组：间皮细胞疏松,细胞表面不连续,微绒毛分布稀疏,胞浆内可见吞饮泡、线粒体、内质网等细胞器,细胞核染色质边集,相邻间皮细胞少见紧密连接。
7、腹膜间皮细胞TGF-β1、bFGF、ZO-1、AQP-1的表达。空白组腹膜组织中PMC阳性表达TGF-β1的细胞数为36.21±3.53,较模型组62.24±4.46低,低剂量组和高剂量组分别为56.43±5.00,48.15±8.72。各组间比较有显著性差异(P<0.05)。四组中bFGF阳性表达的细胞数分别为13.46±1.41,24.54±3.12,19.20±3.36,16.94±2.54,各组间比较有显著性差异(P<0.05)。腹膜组织中ZO-1阳性表达的细胞数分别为33.24±2.21,4.36±2.53,10.44±3.11,17.86±2.93。各组间比较也有显著性差异(P<0.05)。腹膜组织中AQP-1阳性表达的细胞数分别为21.88±1.17,14.36±2.16,16.86±1.51,17.90±0.81。各组间比较也有显著性差异(P<0.05)。
8、临床观察中发现治疗后患者超滤量由治疗前320±65ml/d明显提高到662±85ml/d,统计学处理有非常显著性差异(P<0.01)。腹透引流液CA125平均浓度由治疗前的14.6±3.6U/ml提高到17.8±3.2U/ml,统计学处理有显著性差异(P<0.05)。而患者的残余尿量治疗前为360±116ml/d,治疗后为365±118ml/d；KT/V治疗前为1.88±0.38,治疗后为1.92±0.41；D/Pcr治疗前为0.63±0.18,治疗后为0.66±0.17。均未见明显改变,统计学处理P>0.05。
结论：
1、高糖腹透液能损伤PMC,促进纤维化。高糖腹透液诱导PMC过度表达TGF-β,使bFGF的表达上调,促进纤维化的发生和发展,导致超滤功能的障碍；高糖腹透液在导致腹膜纤维化的发生和发展的同时,还使PMC间的正常连接松散或破坏,细胞凋亡加速,使水通道蛋白的减少,也导致超滤功能的障碍。
2、在高糖诱导的PMC损伤过程中,通过在腹透液中加入参芎注射液可以起到干预EMT过程,保护PMC之间的细胞连接,减少细胞凋亡,维持腹膜组织的完整性。
3、参芎注射液也能通过拮抗TGF-β、bFGF过度表达,从而减缓腹膜纤维化的发生；通过保护PMC,上调ZO-1、AQP-1表达,达到阻止、延缓腹膜纤维化的进程,增加腹透超滤量,提高腹膜透析效能。
4、临床观察表明PD患者经参芎注射液干预后,能显著改善超滤量,不影响残余肾功能和溶质清除率；能有效保护腹膜功能,减少PMC的凋亡
Background:
Failure of peritoneal membrane function has been the most important reason for the technique fail of dialysis of end-stage renal disease patient. It has become the bottle-neck which limited development of peritoneal dialysis. It has been proved that the intact mesothelial cell layer could significantly slow down the process of peritoneal fibrosis. It has also been proved that the important character of peritoneal function failure are the lost of peritoneal mesothelial cell(PMC) and peritoneal epithelial to mesothelial transition(EMT) in histopathology. So the key to prevent peritoneal fibrosis is to stop or reverse the consequences of EMT such as the cell migration and increased synthesis of extracellular matrix(ECM). TGF-(3is the key cell cytokine in EMT process. If the over-expression of TGF-(3could be inhibited, the downstream cell cytokine could also be inhibited. In that way, the progression of EMT could be slowed-down, the construction and function of PMC could be protected, the normal peritoneal function could be maintained.
Objective:
According to high glucose induced rat peritoneum fibrosis model, the peritoneum ultrafiltration function, the changes of morphology, number and function of PMC and the expression of TGF-β1、AQP-1、ZO-1、bFGF in peritoneum were observed. After interfere with compound formulation Shenqiong injection, the possible mechanism of Shenxiong injection protecting PMC in rat peritoneal fibrosis was discussed. Through clinical research, the effect of Shenxiong injection on peritoneal dialysis patient ultrafiltration and its protect effect on PMC were observed.
Methods:
1. The establishment of rat peritoneal dialysis model and observation of ultrafiltration volume:SD rats were divided into group A and group B, both with4.25%peritoneal dialysis fluid (PDF), but the dialysis dose of group A was100ml/kg and group B was200ml/kg. The ultrafiltration rate was tested in lh,2h,3h and4h respectively.
2. Animal model part:Establishment of rat peritoneal fibrosis induced by high glucose.40rats were randomly divided into control group, model group, low-dose intervention group and high-dose intervention group, with10rats for each group. Control group:no management or intervention was done; model group:4.25%PDF100ml/kg was intraperitoneal injected, once per day; low-dose group:4.25%PDF87.5ml/kg+Shenxiong Injection12.5ml/kg intraperitoneal injection, once per day; high-dose group:4.25%PDF75ml/kg+Shenxiong Injection25ml/kg intraperitoneal injection, once per day.4weeks after the start of the experiment, the rats were sacrificed and the blood samples were tested. Before sentenced them to death, peritoneal equilibrium test(PET) were tested, the creatine concentration in dialysis fluid(Dcr), creatine concentration in plasma(Pcr), glucose concentration of initial peritoneum dialysis fluid(DO), glucose concentration of peritoneum dialysis fluid in4th hour(D4). D/Pcr and D4/D0were calculated. Record the average PD ultraflltration volume of every group. Rat peritoneum was stained with HE and VG dyeing.10different views were taken under microscope with200×magnification. The thickness of the fibrous tissue in submesothelial area was tested with Image-Pro Plus6.0image analysis system. The super-thin section sample was prepared. The morphology change, shape of PCM and tight junction were observed with a transmission electron microscope. Concentration of CA125in peritoneal fluid was tested with electrochemiluminescene method. The expression rate of CA125was calculated. The expression level of TGF-β1, AQP-1, ZO-1and bFGF were detected with immunohistochemistry. The protein expression level of them were observed under light microscope. The cytoplasm and membrane of PMC were dyed positive. Those with good positive expression and clean background were chosen to statistics analysis. The number of positive cells were sumed in10different high powered field.
3.The clinical research part:we selected64stable peritoneal dialysis patients from Wuhan first hospital whose dialysis age were over3months. Shenxiong Injection50ml was added into every bag(2L) with peritoneal fluid. And standard CAPD was performed for7days. PET was performed before and after PD treatment, as well as biochemistry index, concentration of CA125in peritoneal fluid, KT/V and Ccr.
4.Statistic analysis
SPSS17was used for statistic analysis. The data of this experiment was quantitative data and was expressed as average±standard deviation(x±s). The difference between every two groups was analyzed with single factor analysis of variance and the comparison among groups was performed with LSD method. P<0.05was considered as with significant difference.
Results:
1. Rat ultrafiltration volume:The ultrafiltration volume of group A and B at lh,2h,3h and4h after the dialysis fluid was kept in peritoneum cavity were gradually increased. The average ultrafiltration volume of group A at2h was10.46ml. There was no significant difference compared with the average ultrafiltration volume at1h which was9.58ml(p>0.05). The average ultrafiltration volume of group A at3h (12.85ml) and4h(13.84ml) were significantly different compared with the average ultrafiltration volume at1h and2h(p<0.05). The average ultrafiltration volume of group B at2h was11.14ml. There was significant difference compared with the average ultrafiltration volume at1h which was9.15ml(p<0.05). The average ultrafiltration volume of group B at3h(15.09ml),4h(16.1ml) were significantly different compared with the average ultrafiltration volume at lh and2h(p<0.05). But the average ultrafiltration volume of group B at3h and4h were significantly higher than group A at3h and4h(p<0.05).
2. The ultrafiltration volume in each group were as follows:The average ultrafiltration volume of blank group was13.75±1.17ml which was significantly higher than the model group4.53±1.10ml. The ultrafiltration volume of Low-dose intervention group had significant improvement and had reached to7.46±1.23ml. The ultrafiltration of High-dose intervention group were significantly upregulated and had reached to9.73±1.11ml. And there was significant difference after statistic analysis(P<0.05).
3. Comparison of peritoneal transportation function in each group:The increase of D/Pcr in model group was the highest and had reached to0.84±0.07. And the control group was0.53±0.03which was significantly lower than the model control group. D/Pcr of low-dose intervention group and high-dose intervention group was0.76±0.08and0.65±0.12which could slow down the increase of D/Pcr with the high-dose group having more obvious effect. The statistic analysis showed there was significant difference between the groups(P<0.05). The results of D4/D0of four groups were as follows:0.59±0.04,0.38±0.06,0.45±0.05,0.51±0.04, with significant difference(P<0.05).
4. The test of the concentration and expression of CA125using electrochemiluminescene immunoassay methods. The concentration of CA125of the control group was11.99±1.37U/ml. The expression rate of CA125was 2.19±0.30U/ml. The model control group had significantly decreased results which were3.53±0.51U/ml and0.51±0.12U/ml respectively. The results of low-dose intervention group increased to6.46±0.89U/ml and1.01±0.15U/ml respectively. And the results of high-dose intervention group increased to7.96±0.97U/ml and1.32±0.16U/ml respectively. The comparison between every two groups had significantly difference (P<0.01)
5. Measure the thickness of peritoneum in each group:The blank group was22.2±5.1μm, the model group was significantly thicker than other groups with the thickness of peritoneum was97.7±18.3μm. The low-dose and high-dose group had relatively thicker peritoneum with the results of57.9±7.8μm and46.1±7.2μm. There was significant difference between them(P<0.01). Under the light microscope, A single layer of flat mesothelial cells with intact structure and continuous distribution covered peritoneum in blank group. Under the mesothelial cells, there was connective tissue with no significant proliferation. While the obvious increase and loosen of peritoneum, the fall of mesothelial cells, massive collagen fiber deposition under mesothelial cells could be seen in model group, low-dose group and high-dose group, especially the model group.
6. The comparison under electron microscope. The control group:Intact structure of peritoneum mesothelial cells were observed. No shrink or deformation of the cells were observed. On the free surface of the cell with abundant and uniform distribution microvilli was existed. Pinosome, mitochondrial and endoplastic reticulum was clear. Tight junction between mesothelial cells was intact. The model group:Few mesothelial cells, most exfoliated, deformation of cells, rarely seen microvilli, sweelling and destruction of cell organs inside cytoplasm such as mitochondral, Obvious proliferation of fibrous tissue under mesothelial cells was observed. Low-dose group:loosen of mesothelial cells, discontinuous distribution of cell surface, obvious decrease of microvilli, swelling of cell organ, margination and solidification of nucleus chromatin, disappear of tight junction of neighbour mesothelial cells were observed. High-dose group:loosen of mesothelial cells, discontinuous distribution of cell surface, rare distribution of microvilli, cell organs such as pinosome, mitochondrial and endoplastic reticulum could be seen, margination of nucleus chromatin, disappear of the tight junction of neighbour mesothelial cells were observed.
7. The test of expression of TGF-β1、bFGF、ZO-1、AQP-1using immunohistochemistry method. The number of peritoneal cells in blank group with expression of TGF-β1among PMC positive cells were36.21±3.53which was lower than model group which was62.24±4.46. And the number of low-dose group and high-dose group was56.43±5.00and48.15±8.72respectively. There was significant difference between the groups(P<0.05). The number of cells with expression of bFGF was13.46±1.41,24.54±3.12,19.20±3.36,16.94±2.54respectively, with significant difference (P<0.05).The number of cells with expression of ZO-1was33.24±2.21,4.36±2.53,10.44±3.11,17.86±2.93respectively, with significant difference (P<0.05). The number of cells with expression of AQP-1was21.88±1.17,14.36±2.16,16.86±1.51,17.90±0.81respectively, with significant difference (P<0.05)
8. In clinical research part, the ultrafiltration volume, KT/V and average concentration of CA125of peritoneal dialysis patient were observed. The observation indicated that the ultrafiltraion volume after treatment had significantly increased from320±65ml/d to662±85ml/d (P<0.01). The average concentration of CA125in peritoneal draining fluid had increased from14.6±3.6U/ml to17.8±3.2U/ml with significant difference(P<0.05). But the residual urine volume of the patient before and after treatment were360±116ml/d and365±118ml/d. KT/V before and after treatment were 1.88±0.38and1.92±0.41. D/Pcr before and after treatment were0.63±0.18and0.66±0.17. All of them with no significant difference(P>0.05)
Conclusion:
1. High glucose peritoneal dialysis fluid could injure PMC, enhance fibrosis. High glucose peritoneal dialysis fluid induce PMC over-express TGF-P, up-regulate expression of bFGF, promote the development and progression of fibrosis, leading to ultrafiltration failure. At the same time, high glucose peritoneal dialysis fluid could loosen and broken the normal cell junction between PMC, accelerate cell apoptosis, decrease the number of aquaporin and also induce ultrafiltration failure.
2. In the process of high-glucose induced PMC injury, adding Shenxiong Injection into the peritoneal fluid could interfere EMT, protect the cell junction between PCM, decrease cell apoptosis, maintain the intact of peritoneum.
3. Shenxiong Injection could slow down fibrosis through the effect of inhibit over-expression of TGF-β and bFGF. Shenxiong Injection could also stop and slow-down peritoneum fibrosis, increase peritoneum ultrafiltration volume and raise peritoneum dialysis effect through the effect of protecting PMC, up-regulating the expression of ZO-1and AQP-1.
4. Clinical observation demonstrated that after Shenxiong Injection interference, PD patient could have better ultrafiltration volume, with no effect on residual renal function and solution clearance, could protect peritoneal function, and reduce PMC apoptosis.
引文
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