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Research Article
The effect of 1.3 bis(p-Hydroxyphenyl)urea compound on IL-6, IL-1β, TNF-α and COX-2 protein expression on λ-Carrageenan-induced rats
expand article infoDenny Satria‡, Syukur Berkat Waruwu‡, Yuandani Yuandani‡, Hari Purnomo§, Urip Harahap‡
‡ Universitas Sumatera Utara, Medan, Indonesia
§ Universitas Gadjah Mada, Yogyakarta, Indonesia

Corresponding author: Urip Harahap ( uriphrhp@gmail.com )

Academic editor: Rumiana Simeonova
© 2022 Denny Satria, Syukur Berkat Waruwu, Yuandani Yuandani, Hari Purnomo, Urip Harahap.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation: Satria D, Waruwu SB, Yuandani, Purnomo H, Harahap U (2022) The effect of 1.3 bis(p-Hydroxyphenyl)urea compound on IL-6, IL-1β, TNF-α and COX-2 protein expression on λ-Carrageenan-induced rats. Pharmacia 69(4): 927-934. https://doi.org/10.3897/pharmacia.69.e89217
Open Access

Abstract

Objectives: This study aims to determine the protein expression of IL-6, IL-1β, TNF-α, and COX-2 using an immunohistochemistry method based on the anti-inflammatory activity of intraplantar carrageenan solution in male rat paws.

Materials and methods: Animals were provided with 1.3 bis(p-Hydroxyphenyl)urea at 12.5, 25, 50, 100, and 200 mg/kg BW. This was administered approximately one hour before the 1% carrageenan induction. As a control measure, diclofenac sodium and CMC sodium 0.5% was similarly administered. After 360 mins, all of the rats were sacrificed, and the inflammatory tissue in the rat’s paw was acquired. The immunohistochemistry preparations were carried out to observe the expression of IL-6, IL-1β, TNF-α, and COX-2.

Results: The result showed a decrease in the expression of IL-6, IL-1β, TNF-α, and COX-2, all groups given 1.3 bis(p-Hydroxyphenyl)urea and diclofenac sodium compared to the control group CMC sodium 0.5% CMC sodium 0.5%. From the results of the mean difference test, it can be seen that the IL-1β expression at doses of 50, 100, and 200 mg/Kg BW was not significantly different from that of diclofenac sodium (p > 0.05). Meanwhile, the expression of COX-2, TNF-α, and IL-6 at doses of 100 and 200 mg/Kg BW was not significantly different from that of diclofenac sodium (p > 0.05).

Conclusion: The 1.3 bis(p-Hydroxyphenyl)urea has anti-inflammatory activity by decreasing the percentage of IL-6, IL-1β, TNF-α, and COX-2 protein expression in rat’s paw inflammation tissue.

Keywords

anti-inflammatory, IL-6, IL-1β, TNF-α, Cox-2, 1.3 bis (p-Hydroxyphenyl)urea

Introduction

Inflammation is the immune system’s response to pathogens, damaged cells, toxic compounds, or irradiation. By producing cytokines and mediators of inflammation, the inflammatory process serves as the primary defense mechanism for health (Medzhitov 2010; Chen et al. 2018). Following infection, cells, particularly macrophages, dendritic cells, and mast cells, secrete various cytokines, chemokines, lipid mediators, and bioactive amines (Lawrence 2010). The inflammatory response in the body is characterized by the presence of numerous mediators, including pro-inflammatory cytokines such as Interleukin-1 (IL-1), Tumor Necrosis Factor (TNF), interferon (INF)-c, Interleukin-6 (IL-6), Interleukin-12 (IL-12), and Interleukin-1 (IL-1), in addition to Nitric Oxidase and COX-2 (Setia and Tjitaresmi 2014). Almost all inflammatory processes result in the activation and infiltration of tissue macrophages and blood monocytes. This activation results in numerous changes in cells, including the production of TNF, IL-1, and IL-6, which are pro-inflammatory cytokines with various effects on the host. These include the induction of fever, an acute phase response in the liver characterized by leukocytosis and the production of acute-phase proteins such as C-Reactive Protein (CRP), and the differentiation or activation of T cells, B cells, and macrophages (Ishartadiati 2009; Warsinah 2017).

1.3 bis(p-Hydroxyphenyl)urea is one of the modified p-aminophenol compounds that have analgesic and anti-inflammatory activity (Purnomo 2016; Waruwu et al. 2021). A previous study demonstrated that the compound 1.3 bis(p-Hydroxyphenyl)urea inhibited inflammation in carrageenan-induced rat paws while having fewer toxic side effects (Waruwu et al. 2022). Based on the inhibition of the cyclooxygenase (COX-2) enzyme, this compound may have analgesic and anti-inflammatory properties (Purnomo 2016). In comparison, the in silico test for COX-1 and TNF-α demonstrated that 1.3 bis(p-Hydroxyphenyl)urea had a greater affinity for COX-1 (1CQE) and TNF-α (2AZ5) than the control compounds dexamethasone and diclofenac. This compound may be developed into an anti-inflammatory agent (Harahap et al. 2021). This study aims to determine the expression of COX-2, TNF- α, IL-1, and IL-6 using an immunohistochemical method based on the anti-inflammatory activity of intraplantar carrageenan solution in male rat paws.

Materials and methods

The performance of this study was conducted based on the guidelines and approval of the Ministry of Health and the Ethics Committee. In addition, all operations were approved by the Animal Research Ethics Committees (AREC) of the University of Sumatera Utara’s Faculty of Mathematics and Natural Science, Biological Department, with the approval number 0423/KEPH-FMIPA/2021, 14 July 2021. The animals were provided with 1.3 bis(p-Hydroxyphenyl)urea compound at 12.5, 25, 50, 100, and 200 mg/Kg BW. This was administered at approximately one hour before the 1% carrageenan induction, which was injected through the intraplantar route into the right hind leg of the rat at 0.1 ml. As a control measure, diclofenac sodium and CMC sodium 0.5% was similarly administered. After 360 mins, all rats were sacrificed, and the inflammatory tissue in the rat’s paw was acquired. The principle of euthanasia in test animals before being sacrificed was for them to be anaesthetized first. Animals were handled with care without causing any fear, then animals were sacrificed with one of the techniques in a separate place from other animals, and no living animals were kept in the vicinity. Euthanasia was carried out by competent personnel, and was accompanied by a confirmation process to confirm death (BPOM RI 2014).

Reagents and chemical

λ-Carrageenan 1% (Sigma Aldrich), Formaldehid 10% (Smart Lab), Na2HPO4 (Smart Lab), CMC sodium 0.5% (Sodium-Carboxy Methyl Cellulose), diclofenac sodium (Novell), Hematoxylin and Eosin, IL-6 Antibody (Abclonal), IL-1β Antibody (Abclonal), TNF-α Antibody (Abclonal), and COX-2 Antibody (Abclonal), Triton (E. Merck), Aquades (Smart Lab), and Ethanol (Smart Lab).

Analysis of IL-6, IL-1β, TNF-α, and COX-2 expression procedures by immunohistochemistry

The immunohistochemistry preparations were carried out to observe the expression of IL-6, IL-1β, TNF-α, and COX-2. Immunohistochemical staining using peroxidase blocking with 0.3% H2O2 followed by washing with PBS 3 times. The surgically removed tissue was fixed in a 10% formalin buffer solution. Dehydration is done gradually. First, dehydration is carried out in 50%, 70%, 80%, 95%, and 100% alcohol solutions with the same length of time for each alcohol content of 90 minutes 2 times. Clearing, the tissue is put into a solution containing xylol for 90 minutes. Infiltration was carried out by inserting the tissue into a 90-minute paraffin solution and carried out 2 times. Infiltration was carried out in an oven at 60 °C. Embedding, tissue, and paraffin solution were put into a paraffin block mold and left for ± 3 hours or until the paraffin solidified. Sectioning, the formed paraffin blocks were cut with a rotary microtome. The tissue was cut with a thickness of 3–4 m and placed on a glass poly-L-lysine object, then left in an incubator overnight at 40 °C. Deparaffinization, sequentially put into a solution of xylol, 100% alcohol, 95% alcohol, 80% alcohol, 70% alcohol, 50% alcohol, for 90 minutes each 2 times. Peroxidase blocking with 0.3% H2O2 in methanol for 20 minutes. The preparations were washed with 10% Phosphate Buffer Saline (PBS) for 3× 5 minutes. Then non-specific blocking was performed with 10% normal serum for 30 minutes followed by incubation with primary antibodies at 4 °C for 18–22 hours. The preparations were washed with 10% Phosphate Buffer Saline (PBS) 3 times for 5 minutes, dropped with secondary antibody (universal antibody) for 30 minutes. Then the preparations were washed with 10% Phosphate Buffer Saline (PBS) 3 times for 5 minutes, dropped with Chromogen 3,3-diaminobenzidine for 5–10 seconds, washed with distilled water, counterstained with Hematoxylin Mayer for 5–10 seconds followed by washing with running tap water for 10–15 minutes. Dehydration was carried out by putting in 80% alcohol, 95% alcohol, and xylol 2 times. Mounting using the E. Z mount (Lab Vision, Cat#MS-1378-PO). Increased expression of COX-2, TNF-, IL-1β, and IL-6 which is characterized by dark brown, medium brown, and purplish light brown, while those that do not express it are purple. These were calculated and analyzed using a light microscope with 400 times magnification. In addition, each preparation was observed in 6 view fields, with the average being calculated (Kim et al. 2016; Sugihartini et al. 2017; Permata and Ahmad 2019).

Immunohystochemical procedures

Statistical analysis

The results were presented as means ± SD. The statistical analysis was carried out by using SPSS edition 22.

Results and discussion

Immunohistochemistry was performed to reveal the total expression of IL-6, IL-1β, TNF-α, and COX- 2 on the connective tissue area of the wound edge using a light microscope 400 times. The expression of IL-6, IL-1β, TNF-α, and COX-2 was indicated by brown granules in the plasma and nuclear membrane. Each IHC preparation was observed in 6 fields of view, and the average was calculated and then analyzed by Oneway Anova. The results observed a decrease in the amount of expression of COX- 2, TNF-α, IL-1β, and IL-6 in all groups given the 1.3 bis(p-Hydroxyphenyl)urea and diclofenac sodium compared to the control group CMC sodium 0.5%. The graph of the average amount of expression of COX-2, TNF-α, IL-1β, and IL-6 in mouse paws is given in Fig. 1.

Figure 1. 

Graph of observations of the average amount of COX-2, TNF-α, IL-1β, and IL-6.

After analyzing the average number of IL-6, IL-1β, TNF-α, and COX-2 proteins expression using the Oneway Anova method, it showed differences in the amount of COX-2, TNF-α, IL-1β, and IL- 6 that were found. A significant difference between the test group and the negative control group was found. From the Tukey HSD mean difference test results, it can be seen that the IL-1β expression at doses of 50, 100, and 200 mg/Kg BW was not significantly different from that of diclofenac sodium (p > 0.05). Meanwhile, the expression of COX-2, TNF-α, and IL-6 at doses of 100 and 200 mg/Kg BW was not significantly different from that of diclofenac sodium (p > 0.05).

COX-2

The results showed a decrease in COX-2 expression in all groups given the 1.3 bis(p-Hydroxyphenyl)urea and sodium diclofenac compared to the control group CMC sodium 0.5%. The least COX-2 expression occurred in the diclofenac sodium group and the 1.3 bis(p-Hydroxyphenyl)urea at doses of 100 and 200 mg/Kg BW. This indicates that the 1.3 bis(p-Hydroxyphenyl)urea can inhibit inflammation by reducing the amount of COX-2 expression. Cyclooxygenase-2 (COX-2) is an enzyme whose presence is influenced by tissue stimulation. These stimuli can be cytokines, bacterial lipopolysaccharides, inflammation, or other pathological conditions (Gilroy et al. 1999). NSAIDs work primarily by blocking the COX pathway. In this pathway, most NSAIDS work reversibly by preventing the arachidonic acid from meeting with the active site of the COX enzyme so that prostaglandin biosynthesis reduces inflammation (Flood et al. 2015). The microscopic picture of COX-2 expression can be seen in Fig. 2. In injured tissue, a wound healing process begins with the formation of blood clots and is followed by an inflammatory phase and remodeling after the injury (Kumar et al. 2010). The wound healing process will not occur if there is no inflammation and will be a source of pain. Inflammation causes many substances to be released endogenously, known as inflammatory mediators. Arachidonic acid is one of the essential inflammatory mediators; arachidonic acid plays a role in the biosynthesis of prostaglandins through the cyclooxygenase pathway (Robbins and Kumar 2015).

Figure 2. 

Overview of COX-2 expression in rat paw inflammation tissue. Notes: (yellow) Ù = Express, (red) Ù = Not Expressing, A. CMC sodium 0.5%, B. Diclofenac Sodium, C. Dose 200 mg Kg/BW, D. Dose 100 mg/Kg BW, E. Dose 50 mg/Kg BW, F. Dosage 25 mg/Kg BW, G. Dose 12.5 mg/Kg BW.

TNF-α

The statistical test results showed that the amount of TNF-α at doses of 100 and 200 mg/Kg BW was not significantly different from that of diclofenac sodium (p > 0.05). Based on Fig. 3, it can be seen that there was a decrease in the amount of TNF-α expression in all groups given the 1.3 bis(p-Hydroxyphenyl)urea and sodium diclofenac compared to the control group CMC sodium 0.5%. This indicates that the 1.3 bis(p-Hydroxyphenyl)urea can inhibit inflammation by decreasing the amount of TNF-α expression. TNF-α is an essential mediator in the inflammatory response. The pro- inflammatory cytokine TNF-α is mainly produced by immune cells such as macrophages and lymphocytes. TNF-α acts as an immune response to inflammation, infection, and tissue damage. Excessive production of TNF-α triggers a decrease in nitric oxide (NO), thereby inducing an increase in endothelin1 (ET-1), causing the proliferation of vascular smooth muscle (Barale and Russo 2020). TNF-α levels represent the severity of inflammation; the higher the TNF-α levels, the higher the severity of inflammation and vice versa; the lower the TNF-α levels, the lower the severity of inflammation and the faster healing process (Souto 2014).

Figure 3. 

Overview of TNF-α expression in rat paw inflammation tissue. Notes: (yellow) Ù = Express, (red) Ù = Not Expressing, A. CMC sodium 0.5%, B. Diclofenac Sodium, C. Dose 200 mg Kg/BW, D. Dose 100 mg/Kg BW, E. Dose 50 mg/Kg BW, F. Dosage 25 mg/Kg BW, G. Dose 12.5 mg/Kg BW.

IL-1β

Based on observations, it can be seen that there is a decrease in the amount of IL-1β expression in all groups given the compound 1.3 bis(p-Hydroxyphenyl)urea and diclofenac sodium. Statistical test results show that the amount of IL-1β doses of 50, 100, and 200 mg/Kg BW was not significantly different from diclofenac sodium (p > 0.05). This shows that the compound 1.3 bis(p-Hydroxyphenyl)urea can inhibit inflammation by reducing the amount of IL-1β expression. The microscopic picture of IL-1β expression can be seen in Fig. 4. The inflammatory cytokine IL-1β, through its receptor (IL-1R) on cells and other chemotactic factors, serves as a beacon to attract neutrophils and other immune cells, thereby eliciting an inflammatory response to tissue injury (Shi 2010). Il-1β is a critical inflammatory cytokine that is a significant mediator of the inflammatory response and upregulates the proinflammatory chemokine IL-18 (Cullen et al. 2015). Tissue injury can damage cell membranes without recognizing a cell receptor which causes the accumulation of inflammatory cytokines (Wen et al. 2012). The expression of Il-1β and IL-18 plays an essential role by acting as early mediators of inflammation (Li et al. 2019).

Figure 4. 

Overview of IL-1β expression in rat paw inflammation tissue. Notes: (yellow) Ù = Express, (red) Ù = Not Expressing, A. CMC sodium 0.5%, B. Diclofenac Sodium, C. Dose 200 mg Kg/BW, D. Dose 100 mg/Kg BW, E. Dose 50 mg/Kg BW, F. Dosage 25 mg/Kg BW, G. Dose 12.5 mg/Kg BW.

IL-6

Based on observations, it can be seen that there is a decrease in the amount of IL-6 expression in all groups given the 1.3 bis(p-Hydroxyphenyl)urea and diclofenac sodium. The statistical test results showed that the amount of IL-6 at doses of 100 and 200 mg/Kg BW was not significantly different from that of diclofenac sodium (p > 0.05). This indicates that the 1.3 bis(p-Hydroxyphenyl)urea can inhibit inflammation by reducing the amount of IL-16 expression. IL-6 functions as a pro-inflammatory and anti-inflammatory cytokine secreted by T cells and macrophages to stimulate the body’s immune response during infection. In the acute inflammatory stage, pro- inflammatory cytokines are released, including IL-6 as a marker of inflammation (Kovacs et al. 2014; Gulati et al. 2016). Macleod and Mansbridge (2015) explained that IL-6 is essential in delaying wound healing, where IL-6 helps stimulate the formation of keratinocytes that play a role in wound healing. IL-6 is very beneficial in the wound healing process at the beginning of injury that stimulates epithelial proliferation; however, inhibition of IL-6 has proven successful in the treatment of cases of systemic inflammation (Kuhn et al. 2014). The microscopic picture of IL-6 expression can be seen in Fig. 5.

Figure 5. 

Overview of IL-16 expression in rat paw inflammation tissue. Notes: (yellow) Ù = Express, (red) Ù = Not Expressing, A. CMC sodium 0.5%, B. Diclofenac Sodium, C. Dose 200 mg Kg/BW, D. Dose 100 mg/Kg BW, E. Dose 50 mg/Kg BW, F. Dosage 25 mg/Kg BW, G. Dose 12.5 mg/Kg BW.

Conclusion

The 1.3 bis(p-Hydroxyphenyl)urea has anti-inflammatory activity by decreasing the percentage of COX-2, TNF-α, IL-1β, and IL-6 expression in rat paw inflammation tissue.

Conflict of interest

The authors declare no conflict of interest in conducting this study.

Acknowledgements

This research is supported by funds from Ministry of Education, Culture, Research and Technology Indonesia through Basic Research Grant (2020–2022).

References

  • Barale C, Russo I (2020) Influence of cardiometabolic risk factors on platelet function. International Journal of Molecular Sciences [2020 Jan] 21(2): e623. https://doi.org/10.3390/ijms21020623
  • BPOM RI (2014) Peraturan Kepala Badan Pengawas Obat Dan Makanan Republik Indonesia Nomor 7 Tahun 2014 Tentang Pedoman Uji Toksisitas Nonklinik Secara In Vivo. Jakarta: Badan Pengawasan Obat dan Makanan RI; 2014. Hal. 3–4, 9, 11–12, 28–32.
  • Chen L, Deng H, Hengmin C, Fang J, Zuo Z, Deng J, Li Y, Wang X, Zhao L (2018) Inflammatory Responses and Inflammation-associated Diseases in Organs. Oncotarget 9: 7204–7218 [online] https://doi.org/10.18632/oncotarget.23208
  • Cullen SP, Kearney CJ, Clancy DM, Martin SJ (2015) Diverse activators of the NLRP3 inflammasome promote IL-1beta secretion by triggering necrosis. Cell Reports 11(10): 1535–1548. https://doi.org/10.1016/j.celrep.2015.05.003
  • Flood P, Rathmell JP, dan Shafer S (2015) Stoelting’s Pharmacology & Physiology in Anesthetic Practice. Edisi kelima. Wolters Kluwer Health, Philadelphia.
  • Gilroy DW, Colville–Nash PR, Willis D, Chivers J (1999) Inducible cyclooxygenase may have anti-inflammatory properties. Nature medicine 5: 698–701. https://doi.org/10.1038/9550
  • Gulati M, Nobile CJ (2016) Candida albicans biofilms: development, regulation, and molecular mechanism. Microbes and Infection 18(5): 310–321. https://doi.org/10.1038/9550
  • Harahap U, Purnomo H, Satria D (2021) In-silico analysis Of 1,3-BIS (p-Hydroxyphenyl)urea as anti- inflammatory through inhibition of COX-1 and TNF-α. Rasayan Chemical Journal 14(3): 1489–1492 [ISSN: 0974–1496|e-ISSN: 0976–0083|CODEN:RJCABP] https://doi.org/10.31788/RJC.2021.1436163
  • Ishartadiati K (2009) Peranan TNF, IL-1, dan IL-6 pada respon imun terhadap protozoa. FK Universitas Wijaya Kusuma, Surabaya.
  • Kim So-W, Jin R, Park C-S (2016) Immunohistochemistry for pathologists: protocols, pitfalls, and tips. Journal of Pathology and Translational Medicine 50(6): 411–418. [Published online 2016 Oct 13] https://doi.org/10.4132/jptm.2016.08.08
  • Kovacs R, Czudar A, Horvath L, Szakács L, Majoros L, Kónya J (2014) Serum interleukin-6 levels in murine models of Candida albicans infection. Acra Microbiologica et Immunologica Hungarica 61(1): 61–69. https://doi.org/10.1556/amicr.61.2014.1.6
  • Kumar V, Abbas AK, Fausto N, Mitchell R (2010) Basic Pathology 8th China: Saunders, Elsevier. h. 45–8.
  • Lawrence T, Fong C (2010) The resolution of inflammation: anti‐inflammatory roles for NF‐ kappa B. The International Journal of Biochem-istry & Cell Biology 42(4): 519–523. https://doi.org/10.1016/j.biocel.2009.12.016
  • Li H, Qian F, Liu H, Zhang Z (2019) Elevated uric acid levels promote Vascular Smooth Muscle Cells (VSMC) proliferation via an nod-like receptor protein 3 (nlrp3)-inflammasome- dependent mechanism. Medical Science Monitor 25: 8457–8464. https://doi.org/10.12659/MSM.916667
  • Medzhitov R (2010) Inflammation 2010: New Adventures of an Old Flame. Department of Immunobiology and Howard Hughes Medical Institute, Yale University School of Medicine, New Haven. Cell 140(6): 771–776. https://doi.org/10.1016/j.cell.2010.03.006
  • Shodiq PF, Ahmad Fe (2019) The ointment of peel dragon fruit (Hylicereus costaricensis) extract decreased the Interleukin-2 (IL-2) expression and the number of mononuclear inflammation cells toward open wound in skin of rats wistar. Veterinary Biomedical and Clinical Journal 1(2): 24–34
  • Purnomo Ha, Jenie UA, Nugroho AE, Pranowo HD (2016) Synthesis and structure elucidation of 1,3 bis(p-Hydroxyphenyl)urea. Jurnal Ilmu Kefarmasian Indonesia 14(1): 33–37. [ISSN 1693–1831]
  • Purnomo Ha, Jenie UA, Nugroho AE, Pranowo HD (2016) In silico and in vivo qualitative relationships of para-aminophenol analogues. International Journal of Pharmaceutical and Clinical Research 8(5)Suppl: 367–371.
  • Robbins SL, Kumar V, Cotran RS (2015) Robbins and Cotran Pathologic Basis Of Disease. 8th Edn. Elsavier Saunders, Philadelphia.
  • Setia AID, Tjitaresmi A (2014) Aktivitas Antiinflamasi Dari Berbagai Tanaman : Sebuah Review. Fakultas Farmasi Universitas Padjajaran. Farmaka 14(3): 77–86.
  • Shi Y (2010) Caught red-handed: Uric acid is an agent of inflammation. Journal of Clinical Investigation 120(6): 1809–1811. https://doi.org/10.1172/JCI43132
  • Souto GR, Queiroz-Junior CM, de Abreu MHNG, Costa FO, Mesquita RA (2014) Pro-inflammatory, Th1, Th2, Th17 cytokines and dendritic cells: A Cross-sectional study in chronic periodontitis. PLoS ONE 9(3): e91636. https://doi.org/10.1371/journal.pone.0091636
  • Sugihartini N, Saridewi R, Ramdhani U, Rahmawanti F, Yuliani S, Sophia V (2017) Anti-inflammatory activity of Camellia sinensis, l. extract cream combined with vitamin C as antioxidant on croton oil-induced inflamation in male mice strain BALB/C. Traditional Medicine Journal 22(2): 73–79 https://doi.org/10.22146/tradmedj.27915
  • Naji DH, Badran M (2017) A review on the cell biology and toxicology within the correlation to a new achievement. IOSR Journal Of Pharmacy 7(4): 15–17. https://doi.org/10.9790/3013-0704011517
  • Warsinah, Baroroh HN, dan Sunarto (2017) Terapi Inflamasi Non Infeksi Berbasis Herbal Daun Jarak Pagar Dengan Pendekatan Biomolekuler. Jurusan Farmasi Fakultas Ilmu Kesehatan Universitas Jenderal Soedirman. Prosiding Seminar Nasional dan Call for Papers “Pengembangan Sumber Daya Perdesaan dan Kearifan Lokal Berkelanjutan VII” 17–18 November 2017, Purwokerto, 655–664.
  • Wen H, Ting JP, O’Neill LA (2012) A role for the NLRP3 inflammasome in metabolic diseases – did Warburg miss inflammation? Nature Immunology 13(4): 352–357. https://doi.org/10.1038/ni.2228
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