| Background:Acute renal failure (ARF)is a common severe disease with an abrupt decease in kidney function that includes the increased blood urea nitrogen and serum creatinine level, disturbance of acid-base balance and water-electrolyte and systemic Complication that threats human health seriously. Although significant advances have been made in basic research along with important technical advances in the recent decades, AKI still keeps high morbidity and mortality due to complex etiopathogenisis and unclear mechanisms hitherto. Eespecially, early symptom in patients with ARF are not obvious untill it developes into some stages that manifestates increased serum creatinine level, which leads to the loss of remedy opportunity for doctor at the best time. Recently, in an attempt to earlierly diagnosis and therapy for ARF, the term "acute kidney injury (AKI) " has been proposed as the replacement of "ARF" in nephrology and emergency so that we could identify and interfere in it at the stage when kidney function begins to discrease, even before kidney tissue damage while biological marker of AKI presents alteratio. It is favourable for avoiding the occurrence of ARF. Recently, schorlars in nephrology are focusing on how to diagnosis and identify AKI earlierly, how to explain the development and progress of AKI by means of perfect theory.Studies have confirmed that ischemia-reperfusion injury (IRI) is one of the major causes of AKI. Both inflammation and apoptosis contribute to the development and progression of renal IRI injury, IRI can lead to endothelial dysfunction, increased endothelial expression of a variety of adhesion molecules that promotes the interactions between endothelial and leukocyte and monocytes, that initiates the inflammatory cascades by inflammatory corpuscle infiltration and cytokine/ chemotactic cytokine production, causes the presention of AKI. Tubular epithelial cells, injuried by hypoxia, also produce TNF-?, IL-2, IL-6, and MCP-1 to partcipate in the initiation of AKI. A growing body of evidences indicated that inhibition of IL-6, IL-1and TNF-?provided protection against IRI-induced inflammation and apoptosis, which suggests that both inflammation and apoptosis could perhaps become the target of AKI treatment.Molecular pathways, such as Notch signaling, are involved in the course of AKI. Present studies demonstrated that Notch signaling reactivated again in renal IRI is involved in the regerenation of renal tubular. Notch signaling is an evolutionarily conserved and widely used intercellular signaling pathway that influences cellular growth, proliferation and differentiation, it not only plays a key role in the dicision of life and death of cell, but also could be reactivated in the setting of disease. Reactivated notch signaling is involved in the course of apoptosis, regerenation and epithelial-to-mesenchymal transitions by the across-talking with other pathways, participating in the development of many kidney diseases. Studies demonstrated that Notch signaling is invovled in the inflammation in the asthma and arthritis by the across-talking with nucleus transcription factor kappa B pathway through mediating Thl and Th2 response. It needs to be proved whether notch signaling is involved in the course of renal ischemia reperfusion injury associated inflammation and oxidative stress.Our previous works demonstrated that Cordyceps Sinensis plays a nephroprotective role in both diabetic nephropath and hypertension nephropath rat models, there is no study whether CS have nephroprotctive effect by mediating renal IRI associated inflammation and oxidative stress, which is accomplished by repressing Notch signaling activation. Therefore, in our work, we observed the expression of IL-6, TNF-?, MCP-1, HIF-1?following renal IRI and the activation of Notch signaling by means of renal IRI rats models in vivo and ischemia /reperfusion- induced NRK-52E model that was simulated by the incubation with antimycin A in vitro. As well as we use the interventions of?-secretase inhibtor DAPT and CS to investigate as following.Objective:1. To observe whether renal IRI up-regulats the expression of IL-6, TNF-a, MCP-1 and HIF-1?, proving the exiting of renal IRI associated inflammation and oxidative stress. 2. To observe whether renal IRI activates Notch signaling that mediates the transcription of IL-6, TNF-?, MCP-1 and HIF-1?,proving whether Notch signaling regulates renal IRI associated inflammation and oxidative stress.3. To observe whether CS represses the activation of Notch signaling, through which mediates the transcription of IL-6, TNF-?, MCP-1 and HIF-1?, plays a nephroprotective role by the way of anti-inflammation and anti- oxidative stress, investigating the mechanism and target of CS.Method:1. After 60 healthy male Sprague-Dawley(SD) rats were anesthetized with intraperitoneal hydral (0.35ml/100g), Unilateral flank incisions were made, and right kidneys were excised and collected as the normal group kidneys after 10 min stabilization period, the 60 healthy male Sprague-Dawley(SD) rats were randomly divided into four groups: sham group(sham, n=15), ischemia-reperfusion (I/R)group(I/R, n=15), DAPT treatment group(DAPT, n=15), and CS treatment group(CS, n=15), and the rats in each group were divided into 24h,48h,72h time point respectivly. The left kidney in the I/R group was subjected to 60min of ischemia with atraumatic vascular clamp, then the clamps were removed to anastate reperfusion, Rats in the I/R group received NS (an equivalent volume with DAPT group and CS)by intraperitoneal injection. The rats in the DAPT treatment group received DAPT (500?g.100g-1) intragastric administration. The rats in the CS treatment group received CS (5g/kg.d)by intragastric administration. Sham group rats, subjected to the same surgical procedure without clamping the left renal vessels, received the same volume of NS. Both blood and urine in all the rats were collected before sacrificed at the time point aboved. Kidney function was evaluated by measurement of Scr and BUN level with a Biochemical Autoanalyzer, and the concentrations of serum TNF-?and IL-6 were measured by Enzyme-linked immunosorbent assay (ELISA) and urine NAG level was measured with Microplate colorimetric assay, Serial sections were stained with hematoxylin and eosin (HE) for histopathological analysis, and the degree of tubular damage was valuated in terms of semi-quantitative score, the mRNA expression of Notch2 and hes-1 were detected with with methods of reverse transcription-Polymerase chain reaction(RT-PCR). and the protein expression of Notch2, hes-1, MCP-1and HIF-?were measured by means of immune confocal laser microscope and western blot.2. Observ the effect of different concentration DAPT (0.1?mol/L, 1?mol/L, 10?mol/L,100?mol/L) and CS(10mg/L,20mg/L,40mg/L, 80mg/L) on the proliferation of NRK-52E by means of MTT so that we can identify the saturation of DAPT fluid and CS fluid in the next experiment.3. Rat renal tubular epithelial cells (normal rat kidney cells, NRK-52E) cultured in complete low-glucose DMEM was randomly divided into four groups:normal group (n=5), renal ischemia and reperfusion model group (I/R, n=15), treatment group with DAPT (DAPT group, n=15); treatment group with Cordyceps Sinensis group (CS, n=15). The I/R-induced NRK-52E models were established as following:the NRK-52E were cultured in no serum- D-Hanks fluid and antimycin A (10?mol/L) for three hour to simulate ischemia. There was no treatment in NRK-52E cells in normal group cultured under noral condition; NRK-52E cells in DAPT group were treated with DAPT fluid(10?mol/L) at 24h before operation and following reperfusion; NRK-52E cells in DAPT group were treated with CS fluid(40mg/L) at 24h before operation and following reperfusion; In additon to normal group, NRK-52E cells in the other groups were collected at 24h,48h 72h, after reperfusion respectively. Protein expressions of Notch2, hes-1, MCP-1, HIF-1?in the NRK-52E in all the groups were detected by means of cell immunofluorescence and Western blot.Results:1. Our results in vivo showed that, renal I/R induced the increase of serum Scr and BUN level, the elevation of urinary NAG, and tubulointerstitial injury including focal areas of proximal tubular dilation and distal tubular casts, effacement and loss of proximal tubule brush border, and interstitial inflammatory cell infiltration, which suggests an successful renal IRI associated AKI model. Renal I/R induced the increase of the concentrations of serum TNF-a, IL-6 (P<0.01), the up-regulation in mRNA and/or protein expression of Notch2, hes-1, MCP-1 and HIF-1?(P<0.01). Our work also indicates that expression of Notch2, hes-1, MCP-1 and HIF-1?mRNA and/or protein expression reaches a peak at 24h after reperfusion, a time point that coincides with the peak of renal tubular injury, urinary NAG levels, BUN, Scr, serum TNF-?and IL-6 and semi-quantitative score of renal tubular cells damage. Inhibtion of the protein expression of Notch2 and hes-1 by DAPT treatment could attenuate the severity of renal tubule damage, improve kidney function, lower the concentration of pre-inflammation factors(TNF-a and IL-6), and down-regulate protein expression of MCP-1 (P<0.01), up-regulate the protein expression of HIF-1?(P<0.01), which suggests renal I/R may induce the activation of Notch2/hes-1 signaling in the renal tubular cells that mediates the transcription of IL-6, TNF-a, MCP-1 and HIF-1?. Notch2/hes-1 signaling maybe is involved in the course of renal IRI associated inflammtion and oxidative stress. DAPT plays a nephronprotective role, and Notch signaling may be considered as a promising pharmacological target for AKI.2.Our results in vivo showed that, CS treatment could attenuate the severity of renal tubule damage, improve kidney function, lower the concentration of pre-inflammation factors(TNF-?and IL-6), down-regulate the protein expression of Notch2, hes-1 and MCP-1 (P<0.01), up-regulate the protein expression of HIF-1?(P<0.01) in the rats with renal IRI, which suggests that CS could protect against renal IRI by the inhibition of Notch2/hes-1 signaling activation, then represse the transcription of inflammation factors such as IL-6, TNF-?, MCP-1, upregulate the transcription of HIF-1?, inhibiting of the course of renal IRI associated inflammation and oxidative stress, Notch signaling maybe is its target.3. MTT examination showed that there was no significant difference in the integrated optical density (IOD) between normal group and DAPT group (from 0.1?mol/L to 10?mol/L in concentration) and CS group (from 10 mg/L to 40mg/L in concentration). However, DAPT (100?mol/L) and CS (100?mol/L) could significantly inhibt the proliferation of NRK-52E(P<0.01). Therefore, we choosed 10?mol/L and 40mg/L as treatment concentration of DAPT and CS respectively in the next experiment.4. Cell immunofluorescence and Western blot results in vitro showed that, I/R could induce the up-regulation in protein expression of Notch2, hes-1, MCP-1 and HIF-1?that reached a peak at 24h after reperfusion, then gradually decreased at 48h and 72h. However, DAPT treatment could down-regulate the protein expression of Notch2, hes-1 and MCP-1 (P<0.01), up-regulate the protein expression of HIF-la(P<0.01), which suggests that I/R may induce the activation of Notch2/hes-1 signaling in NRK-52E cells that mediated the transcription of inflammation factors such as MCP-1 HIF-1?. Notch2/hes-1 signaling maybe is involved in the course of renal IRI associated inflammation and oxidative stress.5.Our results in vitro showed that, CS treatment could down-regulate the protein expression of Notch2, hes-1, NF-?B2 and MCP-1 (P<0.01), up-regulate the protein expression of HIF-la(P<0.01), which suggests that CS could inhibit the activation of Notch2/hes-1 signaling, then represse the transcription of inflammation factors such as MCP-1, Notch2/hes-1 signaling could be its target, and its nephroprotective mechanism is associated with the inhibition of inflammation and oxidative stress.Conclusion:1. Renal IRI up-regulates the expression of IL-6, TNF-?, MCP-1 and HIF-1?. Our work confirms that there are inflammation and oxidative stress following renal IRI.2. Renal I/R may induce the activation of Notch2/hes-1 signaling in the renal tubular cells that mediates the transcription of IL-6, TNF-?, MCP-1 and HIF-1?.3. CS treatment could attenuate the severity of renal tubule damage and kidney function, inhibit the activation of Notch2/hes-1 signaling down-regulate the transcription of inflammation factors such as IL-6, TNF-a, MCP-1, upregulate the transcription of HIF-la. Our work also confirms CS could provide a protection against renal IRI by anti-inflammation and anti- oxidative stress, and Notch signaling may be considered as its target. |
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