The effects and significances of the expression of the silent information regulator 1 protein under the influence of berberine in cholestatic kidney in rats
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Abstract:
Objective
To determinethe expression of silent information regulator 1(SIRT1), superoxide dismutase(SOD), malondialdehyde(MDA)and the glutathione(GSH), the aim of this study is to investigate the effects of the berberine to the expression of the SIRT1 protein and the protective effects to the renal function as related to cholestatic kidney injury in rats.
Methods
The animals were divided randomly into three groupls(n= 15).A rat model of cholestasis was established by bile duct ligation(BDL, B group)and compared with a Sham group receiving laparotomy without BDL(Sham, A group), and with the the BBR given respectively following BDL(BDL+ BBR, C group). All rats were sacrificed on the 7th day after BDL and the blood and kidney tissue samples were obtained.The expression of the SIRT1 proteins were analyzed by western blotting and the reverse transcriptase-polymerase chain reaction(RT-PCR)was performed to determine the mRNA expression of SIRT1 in all groups.The kits of SOD, MDA and GSH were used to detect the values in renal tissue and the apoptosis of the kidney cells was examined by Td T-mediated dUTP nick end labeling(TUNEL)staining.
Results
After the bile duct ligation, the model of cholestasis was established. Comparedto A group, the levels of the mRNA and proteins of the SIRT1,SOD and GSH were lower but the alanine transaminase(ALT), MDA and the apoptosis rate were higher in group B(P< 0.05). Compared C groups to B group, the levels of the mRNA and proteins of the SIRT1, SOD and GSH were higher but the MDA and the apoptosis rate were lower(P<0.05).The values of BUN,Cr,SOD,GSH,SIRT1 mRNA and protein and the rate of the A,Band C groups are:A group(3.14±0.53)mmol/L,(31.32±11.08)μmol/L, (19.45±2.41)nu/mg,(2.31±0.14)nmol/g,1.00±0.00, 0.27±0.01,(0.28±0.13)%; B group(8.37 ±1.57)mmol/L,(102.43±9.32)μmol/L,(5.96±1.43) nu/mg,(1.39±0.25) nmol/g, 0.51±0.05, 0.17±0.01,(19.36±2.56)%; C group(6.04±1.52) mmol/L,(78.58±13.63)μmol/L,(13.52±1.47) nu/mg,(1.97±0.19)nmol/g,0.79±0.04,0.22±0.01,(11.47±3.61)% the group A,B and C.
Conclusion
The present study demonstrates that the BBR could protect the kidney from the peroxide damage by increasing the expression of the SIRT1 which could promote the expression of the gene of SOD. And so the BBR plays a benefical role to resist peroxide damage and apoptosis in cholestatickidney injury.
Key words:
Berberine; Cholestasis; Silent information regulator 1; Superoxide dismutaseKeywords:
Malondialdehyde
Objective To investigate the effects of all-trans retinoic acid(ATRA) on manganese superoxide dismutase(MnSOD) during ischemia/reperfusion injury in rats.Methods 18 male Sprague-Dawley rats were randomized into three groups(6/group).Sham group:sham operation + no treatment;control group:liver I/R + medium(DMSO for 14 days),experimental group:liver I/R + ATRA treatment(15 mg·kg-1·d-1 for 14 days).Partial(70% volume) hepatic ischemia was established for 1 hour,followed by 24 h of reperfusion,and collected sample of liver tissue and serum.Alanine aminotransferase(ALT) and aspartate aminotransferase(AST) were measured in serum.Hepatic pathology was evaluated by hematoxylin and eosin(HE).Content of malondialdehyde(MDA) and superoxide dismutase(SOD) activity were analyzed by colorimetry.Western blot and real-time RT-PCR were used to assess the expression of manganese superoxide dismutase(MnSOD) protein and mRNA.Results Compared with the sham group,ATRA significantly decreased the levels of ALT [(138.61±54.63)U/L vs.(458.53±83.17)U/L,P=0.001]and AST[(355.57±70.92)U/L vs.(1152.48±135.86)U/L,P=0.001],improved hepatic pathological damage,reduced the activity of MDA[(0.47±0.03)nmol/mg vs.(0.77±0.04)nmol/mg,P=0.001],up-regulated the activity of SOD[(221.36±14.18)U/mg vs.(158.01±13.17)U/mg,Conclusions ATRA can significantly alleviate hepatic ischemia/reperfusion injury by increasing expression of MnSOD to increase the activity of SOD,reduce lipid peroxidation and the level of MDA.
Malondialdehyde
Dismutase
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Objective To investigate the effect of oleanolic acid pretreatment on hepatic ischemiareperfusion (I/R) injury in rats. Methods One hundred and twenty-eight male SD rats weighing 230-250 g were randomly divided into 4 groups (n = 32 each): sham operation group (group S), I/R group, 0.5% sodium carboxymethyl cellulose group (group CMC) and oleanolic acid preconditioning (group OA). Partial liver ischemia was produced by clamping hepatic portal vein and hepatic arteries for 60 min with atraumatic mini-clamp, followed by 12 h of reperfusion in group I/R, CMC and OA. Oleanolic acid suspension 100 mg/kg was infused intragastrically in group OA, while the equal volume of 0.5% CMC-Na (in group CMC) and drinking water (in group S and I/R) was infused intragastrically instead once a day for 7 days, and then hepatic I/R was performed at day 8. The left liver was removed and blood sample was taken from inferior vena cava at 0, 3, 6 and 12 h ofreperfusion for determination of serum alanine amino transferase (ALT) activity, superoxide dismutase (SOD)activity, malondialdehyde (MDA) and glutathione (GSH) content, and expression of phospho-phosphatidylinositol3-kinase (p-PI3K), Akt, p-Akt, Bcl-2, Bax, p-Bad and Bad in the liver, and microscopic examination. Results Serum ALT activity and MDA content in the liver were significantly increased, SOD activity and GSH content in the liver were significantly decreased, expression of p-PI3K, p-Akt, Bax, Bad and p-Bad was up-regulated, and Bcl-2 expression was down-regulated during reperfusion in group I/R, CMC and OA as compared with group S (P ＜0.05). Compared with group I/R, serum ALT activity and MDA content in the liver were significantly decreased, SOD activity and GSH content in the liver were significantly increased, expression of p-PI3K, p-Akt,Bcl-2 and p-Bad was up-regulated, and expression of Bad and Bax was down-regulated during reperfusion in group OA (P ＜ 0.05), but no significant change was found in the indexes mentioned above in group CMC (P ＞ 0.05).Serum ALT activity and MDA content in the liver were significantly lower, SOD activity and GSH content in the liver were significantly higher, expression of p-PI3K, p-Akt, Bcl-2 and p-Bad was significantly higher, and expression of Bad and Bax was significantly lower during reperfusion in group OA than in group CMC (P ＜ 0.05).The pathological changes in the liver were milder in group OA than in group I/R. Conclusion Oleanolic acid pretreatment can alleviate hepatic I/R injury by activating PI3K/Akt signaling pathway and inhibiting apoptosis.
Key words:
Oleanolic acid; Reperfusion injury; Liver
Malondialdehyde
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Objective To investigate the effect of L-camitine on renal ischemia-reperfusion (IR) injury (IRI) and Nrf2-ARE signaling pathway in rats.Methods Rats were randomly separated into the following experimental groups:control group (group C),IRI group (group I) and L-carnitine group (group L).Rats accepted no treatment of ischemic reperfusion in group C.In groups I and group L,the renal IRI model was established.L-carnitine was injected through the tail vein in group L,while the equal volume of saline was injected in group C and group I.Rats were killed at 3,6,and 24 h after IR.The levels of serum creatinine (Cr) and blood urea nitrogen (BUN),the activity of superoxide dismutase (SOD) and the content of malonaldehyde (MDA) in serum were measured.The histopathological lesions were observed in renal tissues after 24-h IR.RT-PCR was used to detect the levels of Nrf2,HO-1 and γ-GCS mRNA.Western blotting and immunohistochemistry were used to detect the levels and localization of Nrf2 protein in renal tissues after 6-h IR.Results The levels of Cr and BUN in group I and group L were higher than those in group C at 3 h after IR.At 6 h after IR,the levels of Cr and BUN in group L were lower than those in group I (P＜0.01 ).At 24 h after IR,the levels of Cr and BUN in group L were still lower than those in group I though both of them were reduced (P＜0.05).At all time points,the activity of SOD in group L was higher and the content of MDA was lower than those in group I (P＜ 0.05). As compared with group I,the renal histopathological lesions were alleviated in group L at 24 h after IR.At 6 h after IR,levels of Nrf2,HO-1,γ-GCS mRNA and Nrf2 protein in group I were increased as compared with group C,but decreased as compared with group L.Beyond that,the expression of nuclear Nrf2 protein in group L was higher than that in group I.Conclusion L-carnitine can protects the kidney against IRI significantly,which may be due to the up-regulated expression of antioxidant genes by activating the Nrf2-ARE signaling pathway.
Key words:
Levocarnidine; Kidney; Ischemia reperfusion injury; NF-E2-related factor 2
Blood urea nitrogen
Renal ischemia
Group A
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Objective To investigate the effect of berberine preconditioning on the intestinal injury caused by liver cold ischemia/reperfusion (I/R) in rats.Methods Twenty-four pathogen-free male Sprague-Dawley rats,weighing 220-250 g,were randomized into 3 groups (n =8 each) using a random number table:sham operation group (S group),I/R group,and berberine preconditioning group (B group).The animals were anesthetized with 5％ chloral hydrate 60 mg/kg.In B group,berberine 200 mg/kg was administered through a gastric tube once a day for 7 consecutive days starting from 7 days before operation.The equal volume of normal saline was given instead of berberine in group Ⅰ/R.At 8 h after operation,the rats were sacrificed and the intestines were harvested for determination of wet/dry lung weight ratio (W/D ratio),levels of malondialdehyde (MDA) and superoxide dismutase (SOD),expression of cytochrome C,Bax and Bcl-2 and apoptotic cell count,and for microscopic examination.Intestinal damage was assessed and scored according to Chiu.Results Compared with group S,Chiu's score,W/D ratio,MDA content and apoptotic cell count were significantly increased,SOD activity was decreased,the expression of cytochrome C and Bax was up-regulated,and the expression of Bcl-2 was downregulated in I/R and B groups.Compared with group I/R,Chiu's score,W/D ratio,MDA content and apoptotic cell count were significantly decreased,SOD activity was increased,the expression of cytochrome C and Bax was down-regulated,and the expression of Bcl-2 was up-regulated in B group.Conclusion Berberine preconditioning can reduce the intestinal injury caused by liver cold I/R in rats,and the mechanism is related to inhibition of lipid peroxidation and cell apoptosis.
Key words:
Berberine ; Reperfusion injury ; Liver; Intestines
Malondialdehyde
Chloral hydrate
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Objective:To evaluate the role of Adenosine 5′-monophosphate (AMP)-activated protein kinase (AMPK) in berberine-induced reduction of renal fibrosis in a mouse model of renal ischemia-reperfusion (I/R) and the relationship with ferroptosis.Methods:Twenty-four SPF healthy adult male C57BL/6 mice, aged 8-10 weeks, weighing 20-25 g, were divided into 4 groups (
n=6 each) by a random number table method: sham operation group (group S), renal I/R group (I/R group), berberine group (B group), and berberine plus AMPK inhibitor Compound C group (BC group). The renal I/R model was established by clamping the renal pedicle of the left kidney for 45 min followed by reperfusion in anesthetized mice.In B and BC groups, berberine 100 mg/kg was given for continuous intragastric administration at 14 days before surgery, Compound C 0.2 mg/kg was intraperitoneally injected continuously at 3 days before surgery in group BC, and the equal volume of normal saline was given instead in S and I/R groups.Blood samples were collected from the orbital vein on the 14th day of reperfusion to measure serum Cr and BUN concentrations.Then the mice were sacrificed and kidney tissues were removed for determination of the Fe
2+ and malondialdehyde (MDA) contents and superoxide dismutase (SOD) activity, interleukin-1beta (IL-1β) and tumor necrosis factor-alpha (TNF-α) contents (by enzyme-linked immunosorbent assay), and expression of phosphorylated AMPKα (p-AMPKα), Acyl-CoA synthetase long-chain familymember4 (ACSL4) and recombinant glutathione peroxidase 4 (GPX4) (by Western blot) and for examination of the pathological changes (with a light microscope) and ultrastructure of cell mitochondria (with a transmission electron microscope). The damage to the renal tubules was scored.
Results:Compared with group S, serum Cr and BUN concentrations, renal tubular damage score and renal fibrosis score were significantly increased in I/R, B and BC groups, the contents of MDA, IL-1β and TNF-α were significantly increased, and SOD activity was decreased in I/R and BC groups, the expression of p-AMPKα and ACSL4 in I/R and BC groups, ACSL4 in BC group and GPX4 in B group was significantly up-regulated, and the expression of GPX4 was significantly down-regulated, and the Fe
2+ content was increased in I/R and BC groups (
P<0.05). Compared with I/R group, serum Cr and BUN concentrations, renal tubular damage score and renal fibrosis score were significantly decreased in B and BC groups, the contents of MDA, IL-1β and TNF-α were significantly decreased, and the activity of SOD was increased in group B, the expression of p-AMPKα and GPX4 in B group and ACSL4 in BC group was up-regulated, and the expression of p-AMPKα and GPX4 in BC group and ACSL4 in B group was down-regulated (
P<0.05). Compared with group B, serum Cr and BUN concentrations, renal tubular damage score and renal fibrosis score were increased, the contents of MDA, IL-1β and TNF-α were increased, SOD activity was decreased, the expression of p-AMPKα and GPX4 was down-regulated, the expression of ACSL4 was up-regulated, and the content of Fe
2+ was increased in BC group (
P<0.05). The pathological and ultrastructural damage of kidney tissues was more serious in group BC than in group B.
Conclusion:Berberine can significantly reduce renal fibrosis in a mouse model of renal I/R, and the mechanism is related to the activation of AMPK and further inhibition of ferroptosis.
Malondialdehyde
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Objective
To evaluate the role of thioredoxin-interacting protein(TXNIP)/oligomerization domain-like receptor family pyrin domain-containing 3(NLRP3)signaling pathway in renal ischemia-reperfusion(I/R)injury in diabetic rats.
Methods
Pathogen-free healthy male Sprague-Dawley rats, aged 8-12 weeks, weighing 200-220 g, were used in the study.Diabetes mellitus was induced by intraperitoneal injection of 1% streptozotocin 65 mg/kg and confirmed by blood glucose ≥16.7 mmol/L 3 days later.Twenty-four diabetic rats were divided into 3 groups(n=8 each)using a random number table: sham operation group(group S), renal I/R group(group I/R)and resveratrol(TXNIP inhibitor)group(group R). Resveratrol 10 mg/kg was intraperitoneally injected every day for 7 consecutive days starting from 3rd week after successful establishment of the model in group R. At 4th week after successful establishment of the model, renal I/R was produced by occlusion of bilateral renal pedicles for 25 min followed by reperfusion in anesthetized rats in group R. The animals were sacrificed at 48 h of reperfusion, and renal specimens were obtained for microscopic examination of pathologic changes and for measurement of malondialdehyde(MDA)content, superoxide dismutase(SOD)activity and superoxide anion scavenging capability(using colorimetric method), interleukin-1beta(IL-1β)and IL-18 contents(by enzyme-linked immunosorbent assay), cell apoptosis(using TUNEL)and expression of TXNIP, NLRP3 and caspase-1 in renal tissues(using Western blot). Blood samples were obtained from the left ventricle for determination of serum urea nitrogen(BUN)and creatinine(Cr)concentrations.
Results
Compared with group S, the serum Cr concentration and apoptosis index were significantly increased, superoxide anion scavenging capability in renal tissues was decreased, and the expression of TXNIP, NLRP3 and caspase-1 was up-regulated in I/R and R groups, and the serum BUN concentration and contents of MDA, IL-1β and IL-18 in renal tissues were increased, the SOD activity was decreased(P<0.05), and the pathological changes of renal tissues were aggravated in group I/R.Compared with group I/R, the serum BUN and Cr concentrations were significantly decreased, the contents of MDA, IL-1β and IL-18 and apoptosis index were decreased, the SOD activity and superoxide anion scavenging capability were increased, the expression of TXNIP, NLRP3 and caspase-1 was down-regulated(P<0.05), and the pathological changes of renal tissues were significantly attenuated in group R.
Conclusion
The pathophysiological mechanism of renal I/R injury is associated with the activation of TXNIP/NLRP3 signaling pathway in diabetic rats.
Key words:
Thioredoxins; NLR family, pyrin domain-containing 3 protein; Diabetes mellitus; Kindey; Reperfusion injury
Malondialdehyde
Intraperitoneal injection
Renal ischemia
Blood urea nitrogen
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Objective To investigate the protective effect of calpain inhibitor E-64 on cyclosporine A-induced nephrotoxicity in KM mice.Methods Thirty male KM mice were divided into three groups:control group ( group N,n =10),model group ( group C,n =10 ) and E-64 pretreatment group ( group CE,n =10).Serum CRE,blood urea nitrogen (BUN) and NO levels,kidney malondialdehyde ( MDA),superoxide dismutase (SOD),CAT and calpain activities in mice were measured.The cytosolic protein expression levels of kidney inducible nitric oxide synthase (iNOS) and 3-NT were detected by using Western blotting with the use of specific antibodies.Results There was significant difference in the serum content of CRE,BUN,NO,the kiney content of MDA,and SOD,calpain activity between group CE and group C (P ＜ 0.05 ).No significant difference was found in the CAT activity between group CE and group C (P ＞0.05 ).The expression of inducible nitric oxide synthase and 3-nitrotyrosine was significantly decreased in group CE as compared with group C ( P ＜ 0.01 ).Conclusion Pre-administration of E-64 can oblviously alleviate the nephrotoxicity of KM mice induced by cyclosporine A.
Key words:
Kidney injury; Cyclosporine A; E-64; Calpain
Nephrotoxicity
Blood urea nitrogen
Malondialdehyde
Dismutase
Nitrotyrosine
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Objective
To evaluate the effect of hydrogen on the endoplasmic reticulum stress during liver cold ischemia-reperfusion(I/R)in rats.
Methods
Twenty-four SPF healthy adult male Sprague-Dawley rats, weighing 200-250 g, were divided into 3 groups(n=8 each)using a random number table: sham operation group(group S), liver cold I/R group(group I/R)and hydrogen-saturated lactated Ringer′s solution group(group HL). After occlusion of the liver blood flow, livers were perfused through the portal vein with 4 ℃ hydrogen-saturated lactated Ringer′s solution 6-8 ml/min for 30 min.The liver blood flow was restored after the end of perfusion.At 6 h of reperfusion in I/R and HL groups or at 6 h after peritoneum closure in group S, blood samples from the inferior vena cava were collected for determination of serum alanine transaminase(ALT)and aspartate transaminase(AST)concentrations.After blood sampling, liver tissues were obtained for examination of pathological changes and for determination of cell apoptosis(using TUNEL), malondialdehyde(MDA)content(by thiobarbituric acid method), superoxide dismutase(SOD)activity(using xanthine oxidase method), expression of endoplasmic reticulum protein 46(ERP46), immunoglobulin heavy chain binding protein(BiP)and caspase-12(by immunohistochemistry), and expression of ERP46, BiP and caspase-12 mRNA(using quantitative real-time polymerase chain reaction). The pathological changes were scored.Apoptosis rate was calculated.
Results
Compared with group S, the serum ALT and AST concentrations, pathological scores, apoptosis rate and MDA content were significantly increased, the SOD activity was decreased, and the expression of ERP46, BiP and caspase-12 protein and mRNA was up-regulated in I/R and HL groups(P<0.05). Compared with group I/R, the serum ALT and AST concentrations, pathological scores, apoptosis rate and MDA content were significantly decreased, the SOD activity was increased, and the expression of ERP46, BiP and caspase-12 protein and mRNA was down-regulated in group HL(P<0.05).
Conclusion
The mechanism by which hydrogen reduces cell apoptosis may be related to inhibition of endoplasmic reticulum stress during liver cold I/R in rats.
Key words:
Hydrogen; Endoplasmic recticulum; Stress; Liver transplantation; Reperfusion injury
Malondialdehyde
Thiobarbituric acid
Aspartate transaminase
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Objective To investigate the change of caspase-3 in rabbits after lung ischemia-reperfusion injury(LIRI) and the effect of puerarin(葛根素).Methods Thirty healthy rabbits used for unilateral lung ischemia-reperfusion model were randomly divided into 3 groups(each n=10): control group(C group),lung ischemia-reperfusion group(I/R group) and puerarin group.The activity of serum superoxide dismutase(SOD),the contents of serum malondialdehyde(MDA) and nitric oxide(NO),the wet to dry weight(W/D) ratio of lung tissue and the index of quantitative assessment of histological lung injury(IQA) were measured respectively in different groups;the pneumocyte apoptosis index(AI) was achieved by terminal deoxynucleotidyl transferase mediated dUTP nick end labeling(TUNEL);caspase-3 protein and mRNA expression were studied by using in situ hybridization(ISH) and immunocytochemistry(IHC) techniques in the groups mentioned above.Results The activity of SOD and content of NO were significantly lower in I/R group than those in C group(both P0.01);while the content of MDA,the value of W/D,IQA and AI,together with the expression of caspase-3 mRNA and protein were evidently higher(P0.01,respectively).In the comparisons between puerarin group and I/R group,it was shown that the content of MDA,the value of W/D,IQA,AI and expression of caspase-3 were decreased(P0.01,respectively),while the activity SOD and the content of NO were increased in the former group than those in the latter group(P0.01,respectively).Result of correlation analysis: AI was significantly negatively correlated with the activity SOD and the content of NO,and significantly positively correlated with the content of MDA,caspase-3 mRNA and caspase-3 protein(all P0.01).Caspase-3 mRNA was significantly negatively correlated with SOD activity and NO content,and markedly positively correlated with MDA content(all P0.01).Conclusion Puerarin may elevate the level of NO in the body,lower the level of oxygen free radicals,alleviate the lipoperoxide reaction and down regulate the expressions of caspase-3 mRNA and protein,consequently,the abnormal apoptosis of the pulmonary tissue cells after lung I/R can be inhibited and LIRI injury can be ameliorated.
Puerarin
Malondialdehyde
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Objective
To investigate the protective effects of L-carnitine on liver warm ischemia-reperfusion injury (WIRI) in rats and to explore its possible role of L-carnitine in affecting Nrf2/HO-1 pathway.
Methods
Twenty-four male Sprague-Dawley (SD) rats were randomly divided into three groups including sham-operated (SO) group, liver warm ischemia and reperfusion (WIR) group and L-carnitine pretreatment (LC) group. L-carnitine was injected intraperitoneally in LC group while the equal volume of saline was injected in SO group and WIR group. The levels of ALT and AST in serum as well as the activities of superoxide dismutase (SOD) and the content of malondialdehyde (MDA) in liver tissues were detected. Meanwhile, pathologic change of liver tissue was observed. And then, the mRNA expression of NF-E2-related factor 2(Nrf2) and heme oxygenase 1(HO-1) in liver tissues was determined.
Results
Compared with the SO group, ALT and AST level in serum were significantly increased (P<0.05) in the WIR group; the activity of SOD in liver tissues was significantly reduced in the WIR group [(66.6±12.3) U/mgprot vs. (25.1±9.0) U/mgprot, P<0.05]; and the content of MDA in liver tissues was significantly increased in the WIR group [(1.5±0.3) nmol/mgprot vs. (3.6±0.8) nmol/mgprot, P<0.05]; the mRNA expression of Nrf2 and HO-1 were significantly increased (P<0.05) in the WIR group. Compared with the WIR group, ALT and AST level in serum were significantly reduced (P<0.05) in the LC group; the activity of SOD in liver tissues was significantly increased in the LC group [(25.1±9.0) U/mgprot vs. (86.0±11.4) U/mgprot, P<0.05)]; and the content of MDA in liver tissues was significantly reduced in the LC group [(3.6±0.8) nmol/mgprot vs. (1.4±0.2)nmol/mgprot, P<0.05]; the mRNA expression of Nrf2 and HO-1 were significantly increased (P<0.05) in the LC group.
Conclusion
L-carnitine may attenuate liver WIRI in rats, and this protective effect may be through activating the Nrf2/HO-1 pathway.
Key words:
L-Carnitine; Liver warm ischemia-reperfusion injury; Nrf2; HO-1; Oxidative stress
Malondialdehyde
Group A
Liver tissue
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