Effect Of Matrine On Peritoneal Fibrosis Indueced By Lipopolysaccharide Through TGFβ1/Samd3 Signal In Uremia Rats

Aim Using two hemostatic mathods in 5/6 nephrectomy to establish rats uremia model.To compare the effects of two hemostatic methods including 3M vetbond tissue adhesive and electrocoagulation in a rat uremia model.Then through molecular level to investigate the effects of matrine on peritonieal fibrosis induced by LPS in uremia rats.Method（1）Male SD rats were divided into groups A and B.To induce the uremia model,all rats underwent right and 2/3 left kidney resection;3M vetbond tissue adhesive was used in group A for hemostasis,while electric coagulation was used in group B.The amount of bleeding and the duration of left renal blood flow blockage during operation were assessed.Moreover,the postoperative survival rates of the two groups were compared.The creatinine values of all rats after operation were measured,and rats for which the creatinine value was two to three times higher than normal were further regarded as a uremic model.Kidney tissues were stained with HE and Masson.（2）The uremia rat model was determined according to the 3M vetbond tissue adhesive in experiment（1）.Prepare 30 uremic rats that were treated 3 months after nephrectomy of 5/6,and place the catheter into enterocoelia after they are anesthetized with isoflurane.After successful catheterization,the rats were randomly divided into 5 groups,each group of 6.Intraperitoneal injection via catheter with 2ml saline and peritoneal dialysis fluids（100ml/Kg）,LPS（2mg/Kg,2ml in total）+peritoneal dialysis fluids（100ml/Kg）+matrine（12.5mg/Kg）,LPS（2mg/Kg,2ml in total）+peritoneal dialysis fluids（100ml/Kg）+matrine（25mg/Kg）,LPS（2mg/Kg,2ml in total）+peritoneal dialysis fluids（100ml/Kg）+matrine（50mg/Kg）,one time/every two days×7 times.Seven days after the end of peritoneal dialysis,peritoneal equilibrium test was performed to evaluate peritoneal function.Rats were killed by carbon dioxide method and peritoneal tissuewascollected.TissueswerestainedwithMassonand immunohistochemical of COL I,thickness of peritoneal tissue in each group was analyzed.The expressions ofα-SMA、Vimentin、COLI、TGF-β1、E-cadherin and Smad3 are tested by PCR or WB.Results（1）The serum creatinine after the application of both hemostatic methods after 5/6 renal resection was two to three times higher than the normal level.Left renal blood flow blockage of group the A was 59.97±7.56 s and that of group B was 174.1±15.28 s（P<0.05）.The blood loss of group A during left renal resection was 0.11±0.037 ml,and that of groupB was 0.24±0.056 ml（P<0.05）.HE staining of renal tissues in both groups indicated glomerular ischemic shrinkage and tubular interstitial fibrosis.The postoperative survival rate of group A was significantly higher than that of the group B（P<0.05）.（2）Pathology indicated that the mean peritoneal thickness of the normal saline group was 24.38±5.91μm,the group of LPS stimulation was 199.48±23.40μm,the group of matrine 12.5mg/Kg was 48.41±5.21μm,the group of matrine25mg/Kg was 42.40±5.83μm,and thegroup of matrine 50mg/Kg was81.62±7.14μm.The peritoneal tissue of LPS stimulation group was significantly thickened,and the difference was statistically significant with P<0.05,compared with that of normal saline control group.When compared with LPS stimulation group,the group of matrine 12.5mg/Kg,25mg/Kg and 50mg/Kg was statistically significant with P<0.05.The epithelial cells of the peritoneum in saline stimulation group were flattened and continuous,but in LPS stimulation group were terete or elliptic and partially exfoliated.Peritoneal injure in matrine intervention group was lower than that in LPS group.Peritoneal equilibrium test indicated that D/P_urearea in normal saline control group was 0.67±0.68,and D/P_Crr was 0.66±0.07,which was a high average transport type.The D/P_urearea in LPS stimulation group was 1.07±0.11,and D/P_Crr was 1.0±0.09,which was a high transport type.Compared with normal saline control group,the difference was statistically significant.D/P_urearea in group of matrine 12.5mg/Kg group was0.92±0.10,and D/P_Crr was 0.84±0.10,which was a high average transport type,compared with normal saline control group and LPS stimulation group,the differences were both statistically significant.D/P_urearea in group of matrine25mg/Kg was 0.78±0.13,and D/P_Crr was 0.75±0.07,which was a high average transport type,and the difference was statistically significant compared with LPS group.Compared with normal saline control group,the difference was no statistically significant.D/P_urearea in group of matrine 50mg/Kg was 1.00±0.61,and D/P_Crr was1.01±0.05,which was a high average transport type,and compared with normal saline group,P<0.05,the difference was statistically significant.However,compared with LPS stimulation group,the difference was no statistically significant.Fluorescence quantitative PCR indicated the mRNA expression of TGF-β1、Smad3、α-SMA and COL I was low in normal saline control group.After being stimulated by LPS,its expression was rising evidently.Compared with normal saline control group,P<0.05,the differences were statistically significant.Compared to LPS stimulation groups,the expression of matrine groups were decreased,P<0.05,the differences were statistically significant.The expression of E-cadherin decreased after LPS stimulation,then increased after matrine intervention.The increase was most obvious when matrine was 25mg/Kg,but when the concentration reached 50mg/Kg,the expression of E-cadherin was almost as same as that in LPS stimulation group.The mRNA expression of Vimentin was low in normal saline control group,but after it was stimulated by LPS,its expression was rising,its difference was no statistically significant.When matrine was added,it rise higher,but there was no statistically significant.WB results showed that,α-SMA was rising after being stimulated by LPS,Compared with normal saline control group,P<0.05,the difference was statistically significant.After intervention with matrine,the protein expression decreased.Compared with LPS stimulation group,P<0.05,the the difference was statistically significant.Compared with normal saline control group,only the difference of matrine 12.5mg/kg group was statistically significant,with p=0.039.TGF-β1 is rising after being stimulated by LPS.After intervention with matrine,the protein expression decreased.Meanwhile,the protein expression of vimentin rose evidently after LPS stimulation.Compared with normal saline control group,the difference was statistically significant with P<0.05.However,after invention of matrine,there was no noticeable decrease in its expression.Compared with LPS stimulation groups,there was no significance in statistics.Conclusion 1.Both 3M vetbond tissue adhesive and electrocoagulation can made a rat uremia model.But 3M vetbond tissue adhesive is more effective than electrocoagulation to shorten the bleeding time,decrease of the bleeding amount,or increase of the postoperative survival rate.2.Intraperitoneal injection LPS can induced uremia rats peritoneal fibrosis,which probably associated with EMT.3.Matrine at a certain concentration can alleviate LPS-induced peritoneal fibrosis,TGF-β1/Smad3 may play an important role in this process.