Effects Of Uremic Toxins In The Expression Of Inflammatory Factor And D1on Human Hepatocellular Carcinoma Cells

Objectives: To investigate the effect of uremic toxins in the expression of D1, IL-1β,IL-6and TNF-α mRNAon human hepatocellular carcinoma cells (HepG2). To searchthe causal relationship between nonthyroidal illness syndrome and microinflammation state in chronic renal insufficiency patients.Methods:(1) HepG2cells which cultured in vitro were divided into several groupsrandomly:①blank control group: untreated HepG2cells;②low concentration ofuremic toxins stimulated group (the mixture concentration:20mmol/L urea,100umol/L creatinine,40umol/L uric acid,0.2pg/ml parathyroid and1×10-5mol/Lspermidine);③medium concentration of uremic toxins stimulated group (themixture concentration:50mmol/L urea,200umol/L creatinine,80umol/L uric acid,2pg/ml parathyroid and1×10-4mol/L spermidine);④high concentration of uremictoxins stimulated group (the mixture concentration:80mmol/L urea,500umol/Lcreatinine,200umol/L uric acid,20pg/ml parathyroid and1×10-3mol/L spermidine).Each group of stimulation for12hour,24hour. Reverse transcription polymerasechain reaction (RT-PCR) was used to evaluate the mRNA levels of IL-1β, IL-6,TNF-α and D1.(2) Collect the blood samples and clinical data from patients withchronic renal insufficiency and the people conducted health examination. ELISA wasused to measure the levels of IL-6proteins in supernatant.Results:(1)①The expression of IL-1β, IL-6and TNF-α mRNA were significantlyincreased in the low concentration of uremic toxins stimulated group after12h(IL-1β,P=0.016; IL-6, P=0.042; TNF-α, P=0.015) and24h(IL-1β, P＜0.01; IL-6, P＜0.01; TNF-α, P＜0.01) compared with blank control group. The expression ofIL-1β, IL-6and TNF-α mRNA were significantly increased in the mediumconcentration of uremic toxins stimulated group after12h and24h compared withblank control group(IL-1β, P＜0.01; IL-6, P＜0.01; TNF-α, P＜0.01). Theexpression of IL-1β, IL-6and TNF-α mRNA were significantly increased in the highconcentration of uremic toxins stimulated group after12h and24h compared withblank control group(IL-1β, P＜0.01; IL-6, P＜0.01; TNF-α, P＜0.01). Compared with blank control group, the expression of D1mRNA was significantlydecreased in the low concentration of uremic toxins stimulated group after12h and24h(P＜0.01). Compared with blank control group, the expression of D1mRNAwas significantly decreased in the medium concentration of uremic toxins stimulatedgroup after12h and24h(P＜0.01). Compared with blank control group, theexpression of D1mRNA was significantly decreased in the high concentration ofuremic toxins stimulated group after12h and24h(P＜0.01).②The expression ofIL-1β, IL-6and TNF-α mRNA were significantly increased in the mediumconcentration of uremic toxins stimulated group after12h(IL-1β, P＜0.01; IL-6, P=0.012; TNF-α, P＜0.01) and24h(IL-1β, P＜0.01; IL-6, P＜0.01; TNF-α, P＜0.01) compared with the low concentration of uremic toxins stimulated group.Compared with the low concentration of uremic toxins stimulated group, theexpression of D1mRNA was significantly decreased in the medium concentration ofuremic toxins stimulated group after12h(P=0.013) and24h(P＜0.01).③Theexpression of IL-1β, IL-6and TNF-α mRNA were significantly increased in the highconcentration of uremic toxins stimulated group after12h and24h compared with thelow concentration of uremic toxins stimulated group(IL-1β, P＜0.01; IL-6, P＜0.01; TNF-α, P＜0.01). Compared with the low concentration of uremic toxinsstimulated group, the expression of D1mRNAwas significantly decreased in the highconcentration of uremic toxins stimulated group after12h and24h(P＜0.01).④The expression of IL-1β, IL-6and TNF-α mRNA were significantly increased inthe high concentration of uremic toxins stimulated group after12h(IL-1β, P＜0.01;IL-6, P＜0.01; TNF-α, P＜0.01) and24h(IL-1β, P＜0.01; IL-6, P=0.035;TNF-α, P=0.013) compared with the medium concentration of uremic toxinsstimulated group. Compared with the medium concentration of uremic toxinsstimulated group, the expression of D1mRNAwas significantly decreased in the highconcentration of uremic toxins stimulated group after12h(P=0.011) and24h(P=0.036).⑤The expression of IL-1β, IL-6and TNF-α mRNA were significantlyincreased in the low concentration of uremic toxins stimulated group after24h than12h(IL-1β, P＜0.01; IL-6, P＜0.01; TNF-α, P＜0.01). The expression ofIL-1β, IL-6and TNF-α mRNA were significantly increased in the medium concentration of uremic toxins stimulated group after24h than12h(IL-1β, P＜0.01;IL-6, P＜0.01; TNF-α, P＜0.01). The expression of IL-1β, IL-6and TNF-αmRNA were significantly increased in the high concentration of uremic toxinsstimulated group after24h than12h(IL-1β, P=0.011; IL-6, P＜0.01; TNF-α, P=0.042). The expression of D1mRNA was significantly decreased in the lowconcentration of uremic toxins stimulated group after24h than12h (P=0.038). Theexpression of D1mRNA was significantly decreased in the medium concentration ofuremic toxins stimulated group after24h than12h (P=0.017). The expression of D1mRNA was significantly decreased in the high concentration of uremic toxinsstimulated group after24h than12h (P=0.038).(2) ELISA method and the databaseswere searched for clinical showed there was positive correlation between theexpression of eGFR and the serum concentrations of FT3(r=0.465, P＜0.01),there was negative correlation between the expression of eGFR and the serumconcentrations of IL-6(r=-0.764, P＜0.01), there was negative correlationbetween the serum concentrations of IL-6and FT3(r=-0.643, P＜0.01).Conclusions: Within a certain range, the higher concentration of mixed uremic toxins,the longer of stimulation time, the bigger effect of stimulation on IL-1β, IL-6andTNF-α mRNA expression, the bigger inhibitory effect on D1mRNA expression. AseGFR value is reduced and the serum levels of IL-6increased of patients with chronicrenal insufficiency, the serum FT3concentration decreased.