Research On The Molecular Mechanism Of Panaxadiol Saponins Improving Mouse Renal Function In Lipopolysaccharide-induced Acute Kidney Injury

Background:Uncontrolled sepsis-induced systemic inflammatory responsesyndrome (SIRS) induced by sepsis is a main cause of multiple organfailure. And acute kidney injury (AKI) is one of the most seriouscomplications in SIRS with high mortality rate. However, exogenousglucocorticoids can reverse this state of crisis and save the lives of patientsthrough restore the cardiovascular system homeostasis and the organfunction. The biological activity of glucocorticoids (GC) is mediated by theglucocorticoid receptor (GR), which regulate gene expression. GR isactivated when the GC is combined with GR. Then activated GR enter intonucleus to combine with positive regulation GRE, which can induce theexpression of several inflammatory proteins such as IκB. It is believed thatthe transcriptional suppression which induced by the interaction of proteinand protein is the main mechanism of GR anti-inflammatory effects; whilemost of the main side effects of GC are induced by transcriptionalactivation. In fact, glucocorticoids are very powerful in rescuing riskdisease and a variety of refractory disease. However, the side effects ofglucocorticoids can not be ignored, for example, stress-inducedgastrointestinal bleeding can become dangerous in patients with a varietyof invisible killer. In addition, the side effects of obesity, osteoporosis,insulin resistance are difficult to control. Therefore, It is an urgent clinicalneed to find a drug with glucocorticoids similar curative effect without sideeffects of glucocorticoids. Since ancient times, ginseng is known as the "King of Herbs." The"revived" effect of Ginseng is confirmed by modern technology. Ourprevious results showed that the effect of panaxdiol saponins (PDS) issimilar to dexamethasone (Dexa) in improving cardiopulmonarymicrocirculation in hemorrhagic shock dogs. PDS and dexamethasone canupregulate the expression of IκB and reduce NF-κB of translocation intothe nucleus in acute lung injury induced by LPS in rat. So that thesecretion of cytokines was significantly reduced, the activation of theimmune system was surpressed, then the organ damage induced byendotoxin shock was reverse in rats. Particularly, PDS hadn’t side effects.Purpose:To establish acute kidney injury (AKI) induced by LPS in mouse, andexplore the effect and mechanism of PDS and dexamethasone on AKIinduced by LPS. It will provide a theoretical basis for PDS developed intoalternative glucocorticoid dexamethasone drugs.Methods and Results:1. LPS-induced AKI animal models were successfully established,PDS and dexamethasone had a similar effect in the protection of renalfunction in AKI mice1.1Group and TreatmentC57BL/6mice were randomly divided into four groups (n=8): salinegroup(Control group), LPS model group (LPS group), PDS+LPS group,Dexa+LPS group. The mice were injected intraperitoneally0.9%Nacl0.5ml in the control group, and the remaining mice were injectedintraperitoneally LPS10mg/kg. The mice of PDS+LPS group andDexa+LPS group were respectively injected intraperitoneally PDS(25.0mg/kg) or dexamethasone (2.5mg/kg) before1h of LPS injection.12h after LPS treatment, the kidney, heart and liver tissues of mice were taken under anesthesia and frozen or fixed them, prepared for proteinexpression and morphological observation. The blood were collected to testblood urea nitrogen (BUN), serum creatinine (CREA), alanineaminotransferase (ALT), aspartate aminotransferase (AST), lactatedehydrogenase (LDH) level by automatic biochemical analyzer.1.2ResultsThe blood urea nitrogen and creatinine levels in LPS group mice weresignificantly higher (P<0.01) than that of saline control group. The kidneyin LPS group had minor morphological changes in HE staining. Thissuggested that LPS-induced AKI animal models were successfullyestabalished, and is in the early stages of AKI. Serum alanineaminotransferase (ALT), aspartate aminotransferase (AST), lactatedehydrogenase (LDH) levels in LPS group were significantly higher thanthat of control group (P <0.01). It suggested that the heart and liver cells inthe LPS group was significantly damage. It proved that LPS can induce asystemic inflammatory response syndrome. Blood urea nitrogen andcreatinine levels in PDS+LPS group and Dexa+LPS group weresignificantly lower than that of LPS group (P<0.05). This suggested thatthe PDS and dexamethasone had similarly protection of renal function.2. PDS and dexamethasone have a similar mechanism in the reversalof LPS-induced AKI renal function2.1The effect of PDS and dexamethasone on apoptosis-relatedproteins in LPS-induced renal cellWe detected the expression of apoptosis-related proteins in differentgroups by immunohistochemistry and Western blot. The results showedthat the expression of Bax, Cyto C, cleaved caspase3increased in LPSgroup, while the expression in PDS+LPS group and Dexa+LPS group werelower than LPS group; the expression of anti-apoptotic protein Bcl-2in LPS group decreased and the expression in PDS+LPS group andDexa+LPS group increased compared with LPS group. This suggested thatapoptosis occurred in renal cell after12h of LPS (10mg/kg) injection, andthe degree of apoptosis reduced in PDS+LPS group and Dexa+LPS groupcompared with LPS group. it is thus clear that PDS and dexamethasoneaffect the expression of apoptosis-related proteins and reduce LPS-inducedrenal cell apoptosis.2.2PDS and dexamethasone can reduce the production and release ofcytokines through inhibiting NF-κB signaling pathway in mice AKI2.2.1The expression of IκB and NF-κB p65, p50protein was detectedby Western blotThe results showed that IκB protein expression levels in LPS groupwere significantly lower than that of control group (P<0.05). IκB proteinexpression levels in PDS+LPS group were significantly higher than that ofthe LPS group (P<0.05). Compaired with LPS group, IκB expression levelsin Dexa+LPS group also had an increasing trend. Nuclear NF-κB P65andP50expression levels in LPS group were significantly higher than that ofcontrol group (P<0.05). However, compared with the LPS group, nuclearNF-κB P50expression levels in PDS+LPS group and Dexa+LPS groupwere significantly reduced (P<0.05)，nuclear NF-κB P65expression levelshad a downward trend. So PDS and dexamethasone inhibited the activationof NF-κB signaling pathway.2.2.2The serum TNF-α and IL-6levels were tested by enzyme-linkedimmunosorbent assayThe results showed that TNF-α and IL-6levels in LPS group wassignificantly higher than that of control group (P <0.01). However, TNF-αand IL-6levels in PDS+LPS group were significantly lower than that ofLPS group (P<0.05). Compared with LPS group, TNF-α level in Dexa+LPS group had a downward trend. IL-6of Dexa+LPS group weresignificantly lower (P<0.05). It is due to NF-κB signaling pathway wassuppressed by PDS and dexamethasone.2.3PDS and dexamethasone can inhibit nitrogen-oxygen free radicalsproduction and increase SOD activity in kidney tissueiNOS protein expression and NO levels in LPS group weresignificantly higher than that of control group (P<0.05). However, iNOSprotein expression and NO levels in PDS+LPS group and Dexa+LPS groupwere significantly lower than that of LPS group (P<0.05).MDA levels in LPS group were significantly higher than that ofcontrol group (P<0.05), while the SOD levels and Mn-SOD enzymeprotein expression which scavenging oxygen free radicals decreasedobviously (P<0.05). Oxidative stress induced by LPS aggravated kidneydysfunction in AKI mice. However, PDS and dexamethasone protectedkidney function of LPS-induced AKI through resistance to oxidative stress.3. PDS and dexamethasone have a similar effect of increasing nuclearGR expression. PDS may be a functional ligand of GR3.1PDS significantly increased the expression of GR in the nucleusThe expression of glucocorticoid receptor was detected by Westernblot. The results showed that total GR（t-GR） in control group was thelowest and the t-GR expression of remaining three groups weresignificantly increased, no difference between the groups. The nuclear GR(n-GR) expression levels in LPS group was the lowest in each group, then-GR expression levels in PDS+LPS group was highest, Dexa+LPS groupfollowed. It is speculated that for GR, PDS has dexamethasone-like GRligand effects.3.2PDS may be a functional ligand of the glucocorticoid receptor andincreases transcriptional activity of GR in a dose-dependent The low fluorescence intensity can be found in containingglucocorticoid response element luciferase reporter gene (GRE-Luciferase)and GR co-transfected293cells. When adding dexamethasone(100μmol/L), the transcription activity of GR increased by36.5times.When replacing dexamethasone with different doses of PDS, GRtranscription activity was as follows:①When adding PDS (25μg/ml), thetranscriptional activity of the GR was not affected;②W henadding PDS(50μg/ml), the transcriptional activity of GR increased by5.5times;③When adding PDS(100μg/ml), the transcriptional activity of GRincreased by12times. So, PDS can bind with GR just like dexamethasoneand increase GR transcriptional activity in a dose-dependent. Thus, wethink that PDS may be a functional ligand of GR.Conclusion:1. Panaxdiol saponins and dexamethasone had similarly reversedeffect on LPS-induced AKI mice. Moreover, this reversed effect wasachieved by the same mechanisms.2. PDS may be the functional ligand of GR: PDS and dexamethasonehave the same effect of enhancing the nuclear glucocorticoid receptor (GR)expression. In the reaction system of containing glucocorticoid responseelement luciferase reporter gene (GRE-Luciferase) and GR co-transfected293cells, PDS and dexamethasone have similar effect of binding with GRand PDS increases transcriptional activity of GR in a dose-dependent. Thuswe think that PDS may be a functional ligand of GR. Further research isneeded to confirm the finding.3. PDS has potential clinical application prospect.