| 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 analysisSPSS17was 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. |
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