Potensi Inhibitor HMGB1 terhadap IL-6, TNF-alfa, dan IL-18 pada Interaksi HMGB1 dan RNA SARS-CoV-2 dengan TLR4, RAGE, dan TLR7 sebagai Kandidat Target Terapi COVID-19

R. AJ. SRI WULANDARI, Prof. dr. Tri Wibawa, Ph.D, Sp.MK (K); Prof. Dr. dr. Hartono, M.Si

2025 | Disertasi | S3 Kedokteran Umum

Latar Belakang. Interaksi HMGB1 dengan RNA SARS-CoV-2 menginduksi pelepasan sitokin proinflamasi pada COVID-19 lewat aktivasi TLR4, RAGE, dan  TLR7. Glycyrrhizin dan haptoglobin dapat mengikat HMGB1 ekstraseluler untuk mencegah interaksinya dengan reseptor. Penelitian ini bertujuan untuk menganalisis kadar HMGB1 dan IL-18 pada COVID-19 dibandingkan non-COVID-19, mengeksplorasi mekanisme HMGB1 dan RNA SARS-CoV-2 dalam memicu produksi sitokin proinflamasi serta potensi penghambatannya dengan glycyrrhizin dan haptoglobin.
Metode. Penelitian tahap pertama secara observasional dilakukan pada sampel serum yang dikirim ke Lab COVID-19 FK-KMK UGM. Berdasarkan konfirmasi RT-PCR swab nasofaring, serum dikelompokkan menjadi kelompok COVID-19 dan non-COVID-19. Kadar HMGB1 dan IL-18 dideteksi pada kedua kelompok menggunakan metode ELISA. Penelitian tahap berikutnya secara eksperimental pada kultur sel THP1 (monocyte cell line) yang dipaparkan dengan rekombinan HMGB1 manusia dan RNA SARS-CoV-2 sintetik. Potensi penghambatan jalur ini diobservasi melalui penambahan glycyrrhizin dan haptoglobin. Ekspresi TLR4, RAGE, dan TLR7 dideteksi dengan qPCR. Kadar IL-6, TNF-alfa, dan IL-18 dideteksi pada supernatan kultur dengan metode ELISA. Data yang diperoleh dianalisis menggunakan uji t berpasangan/uji Wilcoxon.
Hasil. Kadar serum HMGB1 meningkat pada pasien non-COVID-19 (p = 0,0199) dan COVID-19 rawat inap (p = 0,0316). Kadar serum HMGB1 berkorelasi positif dengan kadar IL-18 pada pasien COVID-19 rawat inap (r = 0,5500; p = 0,0337). Ekspresi mRNA TLR4, RAGE, dan TLR7 meningkat pada paparan  HMGB1 dan RNA SARS-CoV-2 (p=0,0408; p=0,0083; dan p=0,0014). Peningkatan kadar IL-6, IL-18 dan TNF-alfa terjadi pada konsentrasi tinggi HMGB1 dan RNA SARS-CoV-2 (p=0,0078; p=0,0287; dan (p=0,0350). Glycyrrhizin 20 ?M menurunkan ekspresi TLR4 dan TLR7, sementara haptoglobin 10 ?g/ml menurunkan ekspresi TLR4, RAGE, dan TLR7 akibat paparan HMGB1 dan RNA SARS-CoV-2.
Kesimpulan. Kadar serum HMGB1 – IL-18 berkorelasi positif pada COVID-19 rawat inap. Peningkatan kadar IL-6, IL-18 dan TNF-alfa terjadi pada paparan HMGB1 dan RNA SARS-CoV-2 konsentrasi tinggi. Glycyrrhizin dan haptoglobin menurunkan ekspresi TLR4, RAGE, dan TLR7 pada paparan HMGB1 dan RNA SARS-CoV-2.

Background. HMGB1- SARS-CoV-2 RNA interaction induces the release of proinflammatory cytokines in COVID-19 via activation of TLR4, RAGE, and TLR7. Glycyrrhizin and Haptoglobin bind extracellular HMGB1 to prevent its interaction with the receptor. This study aims to analyze HMGB1 and IL-18 levels in COVID-19 compared to non-COVID-19,exploring the mechanism of HMGB1 and SARSCoV-2 RNA in triggering the production of pro-inflammatory cytokines and their potential inhibition by glycyrrhizin and haptoglobin.
Methods. In the observational study, research was carried out on serum samples sent to the UGM FK-KMK COVID-19 Lab. Based on RT-PCR confirmation of nasopharyngeal swabs, sera were grouped into COVID-19 and non-COVID-19 groups. HMGB1 and IL-18 levels were estimated in both groups using the ELISA method. In the experimental study, THP1 (monocyte cell line) cell cultures were exposed to recombinant human HMGB1 and synthetic SARS-CoV-2 RNA. This pathway is potentially inhibited by adding glycyrrhizin and haptoglobin to the THP1 culture. Expression of TLR4, RAGE, and TLR7 was detected by qPCR. The ELISA method detected IL-6, TNF-alpha, and IL-18 levels in culture supernatants. The data obtained was analyzed using pair t-test/Wilcoxon test.
Results. Serum HMGB1 levels were increased in inpatient non-COVID-19 (p = 0.0199) and inpatient COVID-19 (p = 0.0316). Serum levels of HMGB1 were positively correlated with IL-18 levels in COVID-19 patients inpatients (r = 0.5500; p = 0.0337). TLR4, RAGE, and TLR7 mRNA expression increased upon exposure to HMGB1 and SARS-CoV-2 RNA (p=0.0408; p=0.0083; and p=0.0014). Increased levels of IL-6, IL-18, and TNF-alpha occurred at high concentrations of HMGB1 and SARS-CoV-2 RNA (p=0.0078; p=0.0287; and (p=0.0350). Glycyrrhizin 20 ?M decreased TLR4 and TLR7 expression, and haptoglobin 10 ?g/ml decreased TLR4, RAGE, and TLR7 expression due to exposure of HMGB1 and SARS-CoV-2 RNA.
Conclusion. HMGB1 – IL-18 serum levels are positively correlated in COVID-19 inpatients. Increased levels of IL-6, IL-18, and TNF-alpha occurred upon exposure to high HMGB1 and SARS-CoV-2 RNA concentrations. Glycyrrhizin and haptoglobin decreased the expression of TLR4, RAGE, and TLR7 upon exposure to HMGB1 and SARS-CoV-2 RNA.

Kata Kunci : HMGB1, RNA SARS-CoV-2, PRR, glycyrrhizin, haptoglobin