Efek Pemberian Sekretom Sel Punca Mesenkimal terhadap Ekspresi NF-kB, Kadar TNF-α, IL-6, IL-10, IL-4 dan Ekspresi PD-1, PD-L1 serta Survival Rate Tikus Model Sepsis

Abstract

Introduction: Currently worldwide, the mortality rate due to sepsis is still high. The uncontrolled inflammatory response with the imbalance of pro-inflammatory and anti-inflammatory as well as the increased release of immune checkpoints in the form of PD-1 and PD-L1 plays an important role in the development of sepsis. Secretome produced by mesenchymal stem cells (MSC) improves sepsis due to their anti-inflammatory and immunomodulatory properties, thereby inproving the prognosis of sepsis. This study aims to analyze the effect of MSC secretome administration on NF-κB expression (p65 and p50), levels of TNF-α, IL-6, IL-10, IL-4 and expression of PD-1, PD-L1, and survival rate in rat model of sepsis. Method: Forty-eight male Rattus norvegicus rats were divided into four groups:K (-), rats without sepsis induction and were given 300 μL physiological saline; K (+), rats with sepsis induction and were given 300 μL saline + antibiotics; P1, rats with sepsis induction and were given 150 μL secretome + antibiotics; P2, rats with sepsis induction and were given 300 μL secretome + antibiotics. Measurement of the expression of NF-κB (p65 and p50), PD-1, PD-L1, and levels of TNF-α, IL-6, IL-10, and IL-4 in rat was carried out by the Polymerase Chain Reaction (PCR) method and Enzyme-linked Immunosorbent Assay (ELISA) against whole blood and rat serum. Survival rate was observed by assessing the number of rat that died and lived in each group every 6 hours for 48 hours. Data analysis was performed with a statistical program to observe the relationship between the effect of secretome administration and the mean relative expression of NF-κB (p65 and p50), PD-1, PD-L1, and the levels of TNF-α, IL-6, IL-10, IL-4, as well as the survival rate in rat model of sepsis. One way Anova, Kruskal-Wallis, post-hoc LSD and Mann-Whitney tests were used to analyze the mean of biomarkers. Survival rate data were plotted using the Kaplan-Meier method. If p < 0.05, the result is significant. Results: This study found a significant relationship between MSC secretome administration and the decreased expression of NF-κB (p65 and p50) (p = 0.000 and p=0.001), levels of TNF-α and IL-6 (p = 0.004 and p=0.028), and increased levels of IL-10 and IL-4 (p = 0.024 and p = 0.015). Administering 300 μL of secretome significantly reduced NF-κB expression (p65 and p50), TNF-α and IL-6 levels, and increased IL-10 and IL-4 levels compared to the K (+) group. Administering 150 μL of secretome significantly reduced TNF-α and IL-6 levels compared to the K (+) group. There was also a decrease in relative expression of PD-L1 expression in the septic rat group, but not in the relative expression of PD-1 (p=0.002 and p=0.379). The Kaplan-Meier Log Rank test showed a significance (p=0.016) in the distribution of survival. Conclusion: Administration of MSC secretome significantly reduced NF-κB (p65 and p50) expression, TNF-α and IL-6 levels, and increased IL-10 and IL-4 levels. There was a significant decrease in the relative expression level of PD-L1, but not PD-1. This study also found that MSC secretome administration increased the survival rate in the septic rat group.