| Part One The effects of Huayujiedu herbs on the infiltration of renal inflammatory cells and the expression of inflammatory factorsin rats with obstructive nephropathyObjective: To observe the general conditions,renal pathological changes and the inflammatory cell infiltration in obstructive kidney of rats with obstructive nephropathy,and to observe the changes of inflammatory factors(IL-1β,IL-18,TNF-α and MCP-1)in the serum and the obstructive kidney of rats,so as to explore the effect of Huayujiedu herbs on the renal inflammatory cells infiltration and the inflammatory factors expression in rats with obstructive nephropathy,thereby providing experimental basis for antagonizing inflammatory injury of Huayujiedu herbs.Methods:1 Animal grouping and model preparation: A total of 40 healthy SD rats were randomly divided into four groups(10 rats in each group): the sham-operation group(Sham group),the unilateral ureteral obstruction group(UUO group),the Eplerenone treatment group(EPL group)and the traditional Chinese medicine group(TCM group).Obstructive nephropathy rat models were established by unilateral ureteral obstruction.Ureter of the rats in the Sham group was freed without ligation.2 Drug intervention: Eplerenone was administered orally at daily dose of 100 mg/kg to rats in the EPL group.Chinese medicine was administered orally at daily dose of 17.3g/kg to rats in the TCM group.An equal volume of saline was given to rats in both Sham and UUO groups.All medications were given once daily for 10 consecutive days.After 10 days,all rats were sacrificed to collect specimens of blood and harvest left kidney.3 Test indicators and methods: The general conditions of rats were observed.Food and water intake was recorded.The body weight and the kidney weight were weighed,and kidney index was calculated.The serum creatinine,urine creatinine and serum urea were detected by automatic biochemical analyzer.The inflammatory cell infiltration in renal tissue was examined by HE staining.The expression F4/80 in macrophages was examined by immunohistochemistry(SABC method).The serum IL-1β,IL-18 and TNF-α were detected by Radioimmunoassay.The serum MCP-1 was examined by enzyme linked immune-sorbent assay(ELISA).Real time-PCR was used to detect the expression of IL-1β,TNF-α and MCP-1 mRNA.SABC and Western blot were employed to examine the expression of IL-1β protein.4 Statistical analysis: SPSS13.0 software was used for data analysis.The experimental data were expressed as mean ± SD.One-way analysis of variance(ANOVA)was performed for multiple comparison,and LSD tests for comparison among groups.All results were considered statistically significant at P<0.05.Results:1 The general condition,body weight,kidney weight and kidney index:Rats in the Sham group were quick in movement,with stable food and water intake as well as body weight growth,whereas those in the other 3 groups became slow in action,with decreased food and water intake and body weight growth.On the tenth day after UUO,the body weight was decreased(P<0.05),as accompanied by increased the left kidney weight(P<0.01)and elevated KI on the obstruction side(P<0.01)in rats from UUO group,as compared with Sham group.Compared with UUO group,the left kidney weight of rats was decreased in EPL and TCM group(P<0.05),the body weight was increased and the KI on the obstruction side decreased without statistical significance(P>0.05).2 Renal function: Compared with Sham group,there was no significant increase in the SCr and SUr or remarkable decrease in Ccr in UUO group(P>0.05).Various indicators did not change notably,as compared with UUO group(P>0.05).3 The inflammatory cell infiltration in renal interstitium: HE staining showed normal renal structure of rats in the Sham group,with occasional inflammatory cell infiltration.The infiltrated inflammatory cells were increased significantly in the kidneys of rats in the UUO group,and renal tubular epithelial cells(TECs)were denatured,with cloudy swelling and even necrosis.Compared with UUO group,the renal TECs in the kidneys of rats from EPL and TCM group were also denatured,with cloudy swelling,whereas the infiltrated inflammatory cells were decreased significantly.4 Expression of F4/80 in renal tissue: The expression of F4/80 was not obvious in renal tissue of rats from Sham group,which,however,was notable in UUO group with a lot of interspersed,flaky or fused tawny granules deposited.Nevertheless,the expression of F4/80 was significantly lower in EPL and TCM group than in UUO group.5 The content of IL-1β,IL-18,TNF-α and MCP-1 in serum: Compared with the Sham group,the serum IL-1β,IL-18,TNF-α and MCP-1 were increased in UUO group(P<0.01).The serum IL-1β,TNF-α and MCP-1 were decreased in EPL and TCM group(P<0.01;P<0.05)whereas IL-18 was reduced slightly,as compared with the UUO group(P>0.05).6 The expression of IL-1β,TNF-α and MCP-1 mRNA in kidney of rats: Compared with the Sham group,the expression of IL-1β,TNF-α and MCP-1 was increased notably in the UUO group(P<0.01).As compared with the UUO group,the expression of IL-1β,TNF-α and MCP-1 was decreased in the EPL and TCM group(P<0.01).7 The expression of IL-1β protein by SABC: The weak expression of IL-1β protein was observed in the Sham group.IL-1β was expressed in the cytoplasm of renal TECs in UUO group.Compared with the Sham group,the expression of IL-1β was increased obviously in the UUO group rats.The expression of IL-1β was decreased in the EPL and TCM group than in the UUO group.8 Expression of IL-1β protein by Western blot: Compared with the Sham group,the expression of IL-1β was increased significantly in the UUO group rats(P<0.01).As compared with the UUO group,the expression of IL-1β was decreased in the EPL and TCM group rats(P<0.01).Part Two The effects of Huayujiedu herbs on pyroptosis and NLRP3 inflammasome signaling pathway in rats with obstructive nephropathyObjective: To observe the renal cellular pyroptosis in rats with obstructive nephropathy,and to observe the effect of Huayujiedu herbs on the NLRP3-Caspase-1-IL-1βaxis and the NLRP3-Caspase-1-Pyroptosis pathway in rats with obstructive nephropathy.In the meantime,the relationship between the NF-κB pathway and the NLRP3 pathway as well as the effect of Huayujiedu herbs was explored,thereby clarifying the molecular mechanism of Huayujiedu herbs antagonizing the inflammatory injury and pinpointing their target.Methods:1 Animal grouping,model preparation,drug intervention and statistical analysis were performed,as previously described in Part One.2 Test indicators and methods: TUNEL staining was used to detect the cellular DNA damage of renal cells.HE staining was utilized to observe the morphology of renal damaged cells.Real time-PCR was applied to detect the expression of renal NLRP3,Caspase-1,IL-1β and NF-κB mRNA.SABC and Western blot were employed to examine the expression of NLRP3,Caspase-1 and IL-1β protein in renal tissue cells.Results:1 The TUNEL positive rate of renal cells in rats: A small number of TUNEL positive cells were seen in the Sham group rats.Compared with the Sham group,the positive rate was increased in the UUO group(P<0.01),predominated in distal tubule epithelial cells.The positive rate was decreased in the EPL and TCM group compared to the UUO group(P<0.05,P<0.01).2 The morphology of the renal damaged cells: Pycnotic or fractured nucleus and complete membrane were characteristics of the apoptotic cells.The pyroptosis of cells were characterized by pycnotic or fractured nucleus and incomplete membrane.The necrosis cells were characterized by swollen or dissolved nucleus and incomplete membrane.3 Expression of NLRP3 and Caspase-1 mRNA in kidney of rats: Compared with the Sham group,the expression of NLRP3 and Caspase-1 mRNA was increased obviously in the UUO group rats(P<0.01).Compared with the UUO group,the expression of NLRP3 and Caspase-1 mRNA was reduced in the EPL and TCM group rats(P<0.01).4 Expression of NLRP3 and Caspase-1 protein in kidney of rats by SABC: The expression of NLRP3 protein was weak in the Sham group rats but strong in renal interstitial macrophagocytes and renal TECs in the UUO group,as accompanied with significantly weaker expression in the EPL and TCM group than in the UUO group.The expression of Caspase-1 protein was weak in the Sham group rats but strong in the cytoplasm of TECs in the UUO group,and remarkably weaker in the EPL and TCM group than in the UUO group.5 Expression of NLRP3 and Caspase-1 protein in kidney of rats by Western blot: Compared with the Sham group,the expression of NLRP3 and Caspase-1 was increased obviously in the UUO group rats(P<0.01).As compared with the UUO group,the expression of NLRP3 and Caspase-1 was lowered in the EPL and TCM group rats(P<0.01).6 Expression of renal IL-1β mRNA and protein: See Part Two.7 Expression of NF-κB mRNA and protein in kidney of rats: Compared with the Sham group,the expression of NF-κB mRNA was elevated in the UUO group rats(P<0.01).The expression of NF-κB mRNA was decreased in the EPL and TCM group rats,compared to the UUO group(P<0.01).Western blot results showed notably increased expression of NF-κB in UUO group rats compared with the Sham group(P<0.01),and the expression was reduced in EPL and TCM group compared with the UUO group(P<0.01).Part Three The effects of Huayujiedu herbs containing serum on pyroptosis and NLRP3 inflammasome signaling pathway in mDCT cellsObjective: To observe the expression of NLRP3,Caspase-1,IL-1β and NF-κB in mDCT cells and to observe the DNA damage and membrane damage simultaneously,in order to explore the effect of Huayujiedu herbs on NF-κB-NLRP3-Caspase-1-IL-1β/IL-18 signaling pathway and pyroptosis,thus pinpointing the molecular mechanisms of Huayujiedu herbs antagonizing inflammatory injury and the drug target.Methods:1 Drug serum preparation: A total of 10 healthy SD rats were randomly divided into the control group and traditional Chinese medicine group,with 5 rats in each group.Chinese medicine was administered at daily dose of 17.3g/kg to rats in the traditional Chinese medicine group.An equal volume of saline was given to rats in the control group.All medications were given once daily for 10 consecutive days.At 1h after the final administration,all rats were sacrificed to collect specimens of blood from the aorta abdominalis.2 Cell culture: The mDCT cells were cultured in DMEM medium containing 100U/mL penicillin and 100U/mL streptomycin in a humidified 5% CO2 at 37℃.The single-cell suspension made from cells in logarithmic phase was seeded in six-well plates or new culture bottles for incubation for 24 h.Subsequently,the serum-free medium was used to culture for 24 h for cell synchronization.The resulting supernatants or the cells were collected 24 h after medication administration.3 Cell grouping: The cells were divided into 5 groups: the control group(Cont group),the aldosterone group(ALD group),the eplerenone group(EPL+ALD group),the normal serum group(NS+ALD group)and the drug-containing serum group(DS+ALD group).The drug-free solution was added in the Cont group,whereas the solution containing ALD(1μM)was added in the other groups.EPL(10μM)was first added to act on the cells for 30 min,followed by supplementation of ALD(1μM)-containing solution in the EPL+ALD group.10% normal serum and 10% drug serum were added in the NS+ALD group and the DS+ALD group respectively.In Western blot tests,cells were divided into 4 groups: the Cont group,the ALD group,the EPL group and the TCM group.The drug-free culture solution and that containing ALD(1μM)were added in the Cont group and the other groups respectively.EPL(10μM)was first added to act on the cells for 30 min,and then supplemented with ALD(1μM)in the EPL group.10% drug serum and 10% normal serum were added in the TCM group and in the Cont,ALD and EPL group respectively.4 Test indicators and methods: Immunofluorescent staining and laser scanning confocal imaging system were used to observe the expression of NLRP3,Caspase-1 and IL-1β protein in mDCT cells induced by aldosterone.Lactate dehydrogenase(LDH)test was used to examine the membrane damage of mDCT cells.TUNEL staining was utilized to detect the DNA damage of mDCT cells.Western blot were employed to examine the expression of NLRP3,Caspase-1,IL-1β and NF-κB(p65)protein in mDCT cells.5 Statistical analysis is performed,as described previously in Part One.Results:1 Immunofluorescent staining and laser scanning confocal imaging system: mDCT cells nuclei were labeled with DAPI(blue).NLRP3,Caspase-1 and IL-1β were predominantly expressed in the cytoplasm of mDCT,exhibiting reddish orange fluorescence.It was confirmed that NLRP3,Caspase-1 and IL-1 β were expressed in the cytoplasm of mDCT cells stimulated by aldosterone.2 The LDH content in cultural supernatants: The LDH content in supernatants was higher in ALD group than in Cont group(P<0.01),and lower in EPL+ALD group than in ALD group(P<0.01).The LDH content was lower in DS+ALD group than in NS+ALD group,which showed no significant difference.3 The positive rate of mDCT cells by TUNEL staining: The number of positive cells by TUNEL staining were larger in ALD group than in Cont group(P<0.01).Compared with ALD group,the positive rate was decreased in EPL+ALD group(P<0.05).The positive rate was decreased in DS+ALD group,as compared with NS+ALD group(P<0.05).4 Expression of NLRP3,Caspase-1 and IL-1β protein in mDCT cells by Western blot: Compared with Cont group,the expression of NLRP3,Caspase-1 and IL-1β was increased obviously in ALD group(P<0.01).The expression of NLRP3,Caspase-1 and IL-1β was decreased in EPL and TCM group,as compared with ALD group(P<0.01).5 Expression of NF-κB(p65)protein in mDCT cells by Western blot: Weak expression of NF-κB(p65)was found in Cont group,whereas the expression was increased significantly in ALD group(P<0.01).Compared with ALD group,the expression of NF-κB(p65)was reduced in EPL and TCM group(P<0.05).Conclusions:1 Inflammatory injury plays a pivotal role in the progression of obstructive nephropathy.Inflammatory factors such as IL-1β,IL-18,TNF-α and MCP-1 are involved in this process.2 Pyroptosis is an important pathological change of obstructive nephropathy,which is associated with NLRP3 activation,and induced cell damage through NLRP3/Caspase-1/IL-1β pathway.3 The mechanism of Huayujiedu herbs relieving the inflammatory injury of obstructive nephropathy is associated with inhibiting the secretion of inflammatory factors,and suppressing the Caspase-1-mediated pyroptosis by down-regulating the expression of NLRP3.4 Inflammatory cell infiltration is one of the cytological bases of nephropathy “turbidity toxin”;inflammatory factor increase is one of the molecular bases of nephropathy “turbidity toxin”;NF-κB-NLRP3-Caspase-1 signaling pathway is one of the molecular mechanisms of forming nephropathy “turbidity toxin”.NLRP3 may be one of the targets of Huayujiedu herbs antagonizing inflammatory injury. |
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