{"id":59760,"date":"2024-09-30T11:14:50","date_gmt":"2024-09-30T11:14:50","guid":{"rendered":"https:\/\/biomedpharmajournal.org\/?p=59760"},"modified":"2024-10-10T11:24:36","modified_gmt":"2024-10-10T11:24:36","slug":"the-effect-of-keluwih-artocarpus-camansi-leaves-extract-on-pro-inflammatory-expression-growth-factors-and-bodies-in-zebrafish-larvae-danio-rerio-stunting-model","status":"publish","type":"post","link":"https:\/\/biomedpharmajournal.org\/staging\/vol17no3\/the-effect-of-keluwih-artocarpus-camansi-leaves-extract-on-pro-inflammatory-expression-growth-factors-and-bodies-in-zebrafish-larvae-danio-rerio-stunting-model\/","title":{"rendered":"The Effect of Keluwih (Artocarpus camansi) Leaves Extract On Pro-Inflammatory Expression, Growth Factors and Bodies in Zebrafish Larvae (Danio rerio) Stunting Model"},"content":{"rendered":"\n

Introduction<\/strong><\/p>\n\n\n\n

Stunting represents a significant\nnutritional challenge encountered by developing nations 1<\/sup>. Stunting\nis a persistent nutritional issue that manifests during the initial 1000 days\nof life, recognized as a critical window of opportunity 2<\/sup>. Indonesia\nhas one of the highest rates of stunting prevalence among middle-income\ncountries 3<\/sup>. <\/p>\n\n\n\n

Stunting can be caused by several\nfactors, including carrying out malnutrition care and assessing the\ninsufficient awareness of health and nutrition among mothers prior to pregnancy,\nduring pregnancy, and after the mother gives birth 3<\/sup>. Stunting\ncauses growth disorders, which are characterized by height that does not match\nage 4<\/sup>. Children who undergo stunting may exhibit suboptimal\nintelligence levels, rendering them more vulnerable to diseases and, in the\nlong term, exposing them to potential declines in productivity 5<\/sup>. In\naddition, stunting also has an impact on cognitive development disorders and\ndelays in motor development, most of which are irreversible 6<\/sup>.  <\/p>\n\n\n\n

The prevention of stunting is crucial to safeguard the quality of the next generation. By nurturing a superior\nnext generation, Indonesia can enhance its competitiveness on the global stage\nand effectively tackle future challenges 7<\/sup>. Family assistance teams have been\ndeployed across all regions of Indonesia with the aim of decreasing the\nstunting rate to 14% by 2024. According to data from the 2021 Indonesian\nToddler Nutrition Status Survey, the current prevalence of stunting stands at\n24.4% 8<\/sup>. <\/p>\n\n\n\n

Traditional medicine using herbal\nplants is still an option that can be used for treatment 9<\/sup>. When\ncompared to chemical drugs, traditional medicine has slower performance, but\nthe use of herbal plants as the main raw material makes traditional medicine\nhave milder side effects 10<\/sup>. Keluwih (Artocarpus camansi<\/em>) is a plant that is widely distributed in\ntropical and subtropical parts of Asia. Keluwih comes from Papua New Guinea,\nIndonesia, and the Philippines. One of the distribution areas of keluwih in\nIndonesia is Maluku, keluwih plants are also found in lowland areas 11<\/sup>.\nBased on the results of phytochemical screening, simplicia and ethanol extracts\nof keluwih leaves contain alkaloids, flavonoids, tannins, glycosides,\nanthraquinone glycosides, and steroids\/triterpenoids 10,11<\/sup>. The rich\ncontent of compounds in keluwih leaves is expected to provide benefits to the\nIndonesian people, especially in existing stunting conditions. It is known that\nthis keluwih has potential as an anti-inflammatory, antioxidant, antifungal,\nand antibacterial 12,13<\/sup>. Therefore, this study was undertaken to\nassess the potential of keluwih leaf extract against zebrafish stunting models\nagainst inflammatory markers, growth factors, and body size. Zebrafish induced\nby rotenone 12.5 ppb can be used as a stunting model in previous studies 14<\/sup>.<\/p>\n\n\n\n

Methods<\/strong><\/p>\n\n\n\n

Animal Care<\/strong><\/strong><\/p>\n\n\n\n

Adult male\nand female zebrafish sourced from the wild were identified at the reproduction\nlaboratory of the Faculty of Fisheries and Marine Sciences, Brawijaya\nUniversity. The zebrafish are housed in semi-static 60 L tanks, with\ntemperature in water maintained between 24-26.5\u00b0C and a light cycle of 14:10\n(dark:light) 15<\/sup>. The fish received three daily feedings using Tetra\nColor \u00ae Tropical Flakes from Blacksburg at Germany 16,17<\/sup>. <\/p>\n\n\n\n

Embryos from\nzebrafish are collected after male and female fertilization in a 2:1 ratio.\nThese embryos, aged 0-2 hours post-fertilization, are selected based on being\nround, clear, fertile, and free from mold. A total of 100 larvae are used in\nthe study. The research is approved by the University of Brawijaya’s Medical\nFaculty Ethics Committee (No. 149-KEP-UB-2023). The embryos are categorized\ninto five different groups, including a normal control, a negative control with\n12.5 ppb rotenone, and three experimental groups with varying levels of EEKL\nadded to 12.5 ppb rotenone.<\/p>\n\n\n\n

Embryo Media <\/strong><\/p>\n\n\n\n

The medium for\nembryo was prepared at a concentration of 10x, composed of 0.15 grams of CaCl,\n0.15 grams of KCl, 5 grams of NaCl, 0.815 grams of MgSO4, and 500 milliliters\nof distilled water 14<\/sup>.<\/p>\n\n\n\n

Extraction of Keluwih Leaves (Artocarpus camansi<\/em>)<\/strong><\/p>\n\n\n\n

Artocarpus camansi<\/em> has obtained\ncertification from UPT (Unit Pelaksana Teknis) Materia Medica, with number\n074\/124\/102.20-A\/2022. The extraction procedure followed the maceration method,\nutilizing 96% ethanol (at a ratio of 1:10) and conducted over a period of 3\ncycles, each lasting 24 hours. The extract obtained was evaporated until a\nconcentrated ethanol extract of Keluwih Leaves (EEKL) was obtained.<\/p>\n\n\n\n

Rotenone and EEKL Administration<\/strong><\/p>\n\n\n\n

Rotenone\n(R8875) purchased at sigma aldrich, with a purity of 95%, were dissolve in 1%\nDMSO to produce a stock solution. Rotenone was\nadministered at a concentration of 12.5 ppb 14<\/sup>, and the EEKL\nconcentration varied 2.5; 5; 10 ppm.<\/p>\n\n\n\n

Body Length Measurement<\/strong><\/p>\n\n\n\n

On the 9th\nday post-fertilization (dpf), measurements of zebrafish larvae’s body length\nwere conducted. The larvae were examined utilizing an Olympus SZ61\nstereomicroscope and subsequently quantified using calibrated Image J software.\nThe body’s length is determined by measuring from the nose’s tip (snout) to the\ntail fin’s base 18<\/sup>.  <\/p>\n\n\n\n

Growth factor (VEGF and TGF-\u03b2) and inflammation (IL-6 and TNF-\u03b1)\nmeasurements<\/strong><\/p>\n\n\n\n

Zebrafish\nlarvae aged 9 dpf were euthanized based on the NIH protocol. Whole zebrafish\nlarvae were placed in a microtube in ice water for at least 5 minutes and\nconfirmed that there was no movement. Then rinsed and fixed with cold methanol\n20 \u00b0C for 3-5 minutes, followed by inactivation using peroxide blocking\nsolution at 25 o<\/sup>C for 10 minutes and under running water for 5 minutes.\nWhereupon incubated in prediluent blocking solution for 10 minutes at room\ntemperature.<\/p>\n\n\n\n

The next\nstage was by incubating 100\u00b5L of commercial monoclonal primary antibody per\npreparation in the refrigerator for 24 hours, then washing it using PBS for 5\nminutes. Then added biotinylated universal secondary antibody for each IL-6\nobservation (100\u00b5L at 1:50 dilution, Sigma Aldrich, HPA035283); TNF-\u03b1\n(1-2\u00b5g\/mL, Sigma Aldrich, SAB1404480); VEGF (at 1:20 to 1:100 dilution, Sigma\nAldrich, AB1876-I); TGF-\u03b2 (with 1:100 dilution, Termofisher, MA5-16949) was\nthen incubated at 25 oC for 10 minutes. The incubated preparations were washed\nPBS (5 minutes) and again incubated using streptavidin\/peroxide complex reagent\n(10 minutes) and washed PBS (5 minutes).<\/p>\n\n\n\n

The next step\nis to detect the reaction by incubating with peroxide substrate solution (DAB)\n100\u00b5L per preparation for 2-10 minutes, washing with running water and adding\n100\u00b5L of Mayer’s hemoxylin (counterstrain) reagent per preparation and\nincubating for 1-3 minutes then washing under water flow. The final stage is\ndehydration and mouting as usual. Afterward, it was scrutinized using a Nikon\nEclipse type Ei light microscope, aided by an Optilab Microscope Camera that\nwas connected to a computer. <\/p>\n\n\n\n

Statistic analysis<\/strong><\/p>\n\n\n\n

Statistical evaluation was conducted through IBM ANOVA SPSS version 23.0, followed by the LSD post hoc test at a 95% confidence interval. Shapiro-Wilk was used for the normality assessment and the Levene test was used for checking homogeneity.<\/p>\n\n\n\n

Results and Discussion  <\/strong><\/p>\n\n\n\n\n\n
\"\"<\/td>\n\n

Table 1: Visualization of positive cells containing observed proteins<\/strong><\/p>\n

<\/p>\n

Click here to view Table<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

The use of pesticides as an environmental factor\ncan trigger stunting. Rotenone is a pesticide with a concentration of 12.5 ppb,\nwhich can induce stunting 19<\/sup>. Rotenone works to inhibit mitochondrial\ncomplex I and inhibit the process of ATP synthesis so that the amount of ATP\ndecreases and induces an elevation in ROS (reactive oxygen species), leading to\npotential cell death, cellular damage, and other oxidative risks 20<\/sup>.<\/p>\n\n\n\n

Giving rotenone with a concentration of 12.5 ppb to zebrafish larvae affected growth factors and inflammatory cytokines, which manifested in the body length of the larvae. Rotenone administration can increase levels of inflammatory cytokines (IL6 and TNFa) and decrease growth factors (TGF-\u03b2 and VEGF). This can be seen from the many brown images in Figure 1, which show a positive immunohistochemical reactivity. Rotenone administration also showed a shorter body length in zebrafish larvae compared to the normal group (Table 2) 14<\/sup>. <\/p>\n\n\n\n\n\n
\"\"<\/td>\n\n

Table 2: Body length measurements of zebrafish larvae<\/strong><\/p>\n

<\/p>\n

Click here to view Table<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

The images obtained were measured quantitatively to determine the persentage positive area in immunohistochemical (immunoreactive) observations. The results of the quantification values obtained can be seen in Figure 1.<\/p>\n\n\n\n\n\n
\"\"<\/td>\n\n

Figure 1: Quantification of % immunoreactive positive areas<\/strong><\/p>\n

<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

Giving EEKL can improve the condition\nof the stunting model in zebrafish. It has been proven to reduce inflammatory\ncytokine levels, increase growth factor levels, and improve body length in\nzebrafish stunting larvae. The optimal dose of EEKL is 2.5 ppm, because this is\nthe lowest dose that has an effect on improving stunting conditions in\nzebrafish larvae.<\/p>\n\n\n\n

Poor nutrition and ongoing inflammation\ndriven by pro-inflammatory cytokines play a role in causing growth delays. At\nthe initial stage, the levels of several highly inflammation mediators such as\nTNF-\u03b1, IL-6, and IL-12 were observed to be reduced in stunted children when\ncompared to those in the normal control group 21<\/sup>. Inflammatory\ncytokines observed in this study were IL6 and TNF-\u03b1. TNF consists of two\nrelated proteins mainly produced by mononuclear lymphocytes (TNF-\u03b2) and phagocytes\n(TNF-\u03b1). Assessing TNF-\u03b1 levels in malnourished kids is crucial as low TNF-\u03b1\ncan weaken the immune system, while high levels can worsen nutrition by causing\nanorexia and cachexia. IL-6 is key in triggering acute phase protein synthesis\nin liver cells. Excessive IL-6 in children can cause chronic inflammation and\ncontribute to growth issues, including stunting. It negatively affects liver\nIGF-I gene activity and facilitates the reduction of IGFBP-3 (insulin-like\ngrowth factor-binding protein-3).<\/p>\n\n\n\n

Sederquist and colleagues discovered\nthat multiple inflammatory cytokines like TNF-\u03b1 and IL-6 can singly or jointly\nimpact child growth. These cytokines can operate through overall systemic\npathways or specifically target the growth speed of long bones 22,23<\/sup>.\n<\/p>\n\n\n\n

The cytokine Transforming Growth Factor\n\u03b2 (TGF-\u03b2) holds a pivotal role in regulating cell growth and differentiation\nacross diverse tissues. Additionally, it is involved in processes such as\ninflammation, autoimmunity, and tumor development 24<\/sup>. Under normal\ncircumstances, local sources maintain tissue homeostasis by preserving baseline\nlevels of TGF-\u03b2 signaling. Following tissue damage, TGF-\u03b2 is extensively secreted\nby blood platelets and other stromal elements to aid in tissue repair, wound\nhealing, and reducing inflammation. The interaction between TGF-\u03b2 signaling and\nreactive oxygen species (ROS) metabolites is crucial 25<\/sup>.<\/p>\n\n\n\n

Reduced ROS levels play crucial roles\nin determining cell fate and cellular responses affecting cell proliferation,\ndifferentiation, and death 26<\/sup>, similar to TGF-\u03b2 signaling. If ROS\nlevels surpass the body’s antioxidant defenses, this imbalance leads to\noxidative stress, harming proteins, nucleic acids, and lipids either directly\nor indirectly 27<\/sup>. TGF-\u03b2 is abundant in bones and cartilage. It promotes\nthe growth, differentiation, and formation of osteoblasts from osteoprogenitor\ncells 28<\/sup>.  <\/p>\n\n\n\n

Under normal circumstances, increased\nROS also causes hypoxia, which stimulates VEGF expression. Hypoxia boosts VEGF\nthrough enhanced mRNA transcription and stability, triggering blood vessel\nformation to sustain oxygen levels. Excessive ROS from oxidative stress damages\ncells by mutating VEGF via intricate signaling routes. Chronic hypoxia lowers\nVEGF expression, impairs tissue vascularization, and causes endothelial\ndysfunction. Such dysfunction disrupts angiogenesis, regulated by VEGF’s\ninteraction with VEGFR-2, affecting cell proliferation and growth 29<\/sup>.<\/p>\n\n\n\n

Conclusion<\/strong><\/p>\n\n\n\n

Artocarpus camansi<\/em> leaves ethanol extract (EEKL) can improve stunting conditions through increasing growth factor expression, decreasing pro-inflammatory cytokines and increasing body length in zebrafish larvae induced by rotenone of 12.5 \u03bcg\/mL significantly, optimal concentration of Artocarpus camansi<\/em> leaves ethanol extract in overcoming stunting conditions it’s 2.5 ppb.<\/p>\n\n\n\n

Acknowledgment<\/strong> <\/p>\n\n\n\n

Thank you to all the teaching staff, especially laboratory staff, who helped with this research.<\/p>\n\n\n\n

Conflict of Interest<\/strong><\/p>\n\n\n\n

The authors declare no conflict of interest.<\/p>\n\n\n\n

Funding Sources<\/strong><\/p>\n\n\n\n

No external funding for this study.<\/p>\n\n\n\n

References <\/strong><\/p>\n\n\n\n

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     <\/h1>\n","protected":false},"excerpt":{"rendered":"

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