{"id":54339,"date":"2023-12-31T11:12:46","date_gmt":"2023-12-31T11:12:46","guid":{"rendered":"https:\/\/biomedpharmajournal.org\/?p=54339"},"modified":"2024-01-05T06:27:07","modified_gmt":"2024-01-05T06:27:07","slug":"green-tea-with-egcg-active-compound-decreases-nlrc3-expression-in-middle-cerebral-artery-occlusion-mcao-rats-model","status":"publish","type":"post","link":"https:\/\/biomedpharmajournal.org\/staging\/vol16no4\/green-tea-with-egcg-active-compound-decreases-nlrc3-expression-in-middle-cerebral-artery-occlusion-mcao-rats-model\/","title":{"rendered":"Green Tea with EGCG Active Compound Decreases NLRC3 Expression in Middle Cerebral Artery Occlusion (MCAO) Rats Model"},"content":{"rendered":"\n

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

Stroke is characterized by a sudden onset of neurological\ndysfunction and stands as one of the prevailing global culprits for fatalities\nand incapacitation. Stroke can be categorized into two types: ischemic stroke\n(~ 80%) and hemorrhagic stroke (~ 20%). As a result of hemiparesis or\nhemiplegia, both ischemic and hemorrhagic stroke patients require the usage of\nsupportive devices.\nIschemic stroke affects all aspects of quality of life,\nwith the exception of independence and the environment. The cerebral media\narteries are the most frequent places of occlusion when ischemia occurs.1,2<\/sup>\nThe preferred treatment is intravenous thrombolysis with r-TPA, but only 2-8.5%\nof patients receive it since many are contraindicated or have passed the\nthrombolysis phase.3,4<\/sup>\nNeuronal cell death plays an important role in ischemia-induced brain injury.\nHigh Mobility Group Box Protein 1 (HMGB1) and Phospholipase A2 (PLA2) are\nreleased from the injured and dead components of the neurovascular unit in\nischemic-induced brain injury. High Mobility Group Box Protein 1\n(HMGB1)binds to several Toll-Like Receptors (TLRs) on microglia cells to further\nupregulate NOD-like Receptor Protein-3 (NLRP3).5<\/sup>\nA recent study shows that the mammalian target of rapamycin (mTOR) plays a role\nin brain ischemia.6<\/sup>\nAnother study also explains that mTOR regulates NLRP3 activation via reactive\noxygen species (ROS) in murine lupus.7<\/sup><\/p>\n\n\n\n

Nucleotide-binding oligomerization domain containing 3 (NOD3) (as known as NLRC3) has an inhibitory\neffect on NLRP3, which inhibits the pyroptosis process in microglia\ncells. 8<\/sup>,<\/sup>9<\/sup> NOD3\nalso plays a role as an inhibitor of the mTOR pathway. The inhibition of mTOR\ncan induce the downregulation of the NOD3 protein.10<\/sup><\/p>\n\n\n\n

Green tea (Camellia sinensis) has a potential neuroprotection source\ndue to the catechins. 11<\/sup>\nGreen tea contains high levels of flavonoids: Epigallocatechin-3-gallate\n(EGCG), accounting for about 59% of the total content of catechins,\nepigallocatechin, and others.12<\/sup> <\/sup>Several studies have demonstrated the\nantioxidant effects associated with EGCG.13<\/sup>\nHowever, there are important variations in the processing of herbs, so the\ndifferences in EGCG concentrations in different types of tea may be responsible\nfor the different neuroprotective effects of some teas.14<\/sup>\nEGCG can inhibit mTOR.15<\/sup><\/p>\n\n\n\n

Based on the information above, it is known that green tea, containing the active compound EGCG, has the potential to prevent neuron cell death in ischemia conditions. This study analyzes the neuroprotective effect of green tea on NOD3 expression. This study might offer an alternate approach to solving ischemic stroke problems.<\/p>\n\n\n\n

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

The study procedure was reviewed and approved by the Animal Care and Use Committee (ACUC) of the Faculty of Veterinary Medicine at Universitas Airlangga No. 2.KE.161.09.2018 on 25 September 2018.<\/p>\n\n\n\n

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

Male Rattus norvegicus mice, aged four months, weighing 200-275\ngrams, were used for the study. They were acclimated to the animal housing\nenvironment for one week under specific conditions, which included a 12-hour\nlight-dark cycle and a temperature of \u00b0C. During this time, they had\nunrestricted access to water and standard rodent food. The criteria for animal\ncare and use, established by the Institutional Animal Ethics Committee at\nUniversitas Airlangga in Indonesia, were followed for all experimental methods.<\/p>\n\n\n\n

Within several steps, the rats were conditioned to be middle\ncerebral artery occlusion (MCAO). Ketamine 80 mg\/kg Body Weight and xylazine 10\nmg\/kg Body Weight were used intraperitoneally to anesthetize Rattus norvegicus.\nExcision was performed in the right neck of Rattus norvegicus until carotid\ncommunis could be seen, and then the carotid artery was separated from the\ninner carotid and locked for 180 minutes using a small bulldog clamp. At the\n180-minute mark, the bulldog clamp was removed, and the neck incision was\nsealed. The rat consciousness was observed while observing whether or not the\nstroke model had emerged.16<\/sup>\nAlthough the bulldog clamp technique was simpler than the other technique, it made\nthe MCAO model in Rattus norvegicus well. <\/p>\n\n\n\n

Eleven male rats were randomly\nassigned to each of the six groups. The groups are sham\n= healthy rats\u2019 group, P0 = control group, P1 = EGCG 10mg\/kg Body Weight, P2 =\nEGCG 20mg\/kg Body Weight, P3 = EGCG 30mg\/kg Body Weight, and P4 = green tea\nextract ‘Meditea’ 30mg\/kg Body Weight. The extract of EGCG or green tea was\ndiluted with aquades, a concentration of 1 mg\/ml, then delivered to the samples\nbefore they had a meal using rat sonde every morning. The intervention was\ngiven once a day for seven days.<\/p>\n\n\n\n

After the seventh day, the samples were anesthetized using propofol 0.1\nmg\/100 g intravenous rodent. A coronal section of the brain tissue from the\ninfarcted hemisphere was removed. The rat brains were then preserved in 4%\nformalin and subsequently encased in a paraffin block for immunohistochemical\n(IHC) analysis.17<\/sup><\/p>\n\n\n\n

Chemicals<\/strong><\/p>\n\n\n\n

Anti NOD3 pAb Lsbio ABIN625967, EGCG (from Xi\u2019an Rongsheng\nBiotechnology CO., LTD, Keji 3rd Road, Xi\u2019an, China) or green tea extract (obtained\nfrom PT. Dharma Putra Airlangga, Tegalsari, Surabaya, Indonesia) extract dose\n10, 20, and 30 mg\/kg Body Weight; standardized green tea extract label\n‘Meditea’; aquadest; ketamine 80 mg\/kg Body Weight; xylazine 10 mg\/kg Body Weight;\npropofol 0,1 mg\/100 gr IV; anti-NOD3 antibodies; and BSA 1 %.<\/p>\n\n\n\n

Immunohistochemical Examination<\/strong><\/p>\n\n\n\n

Samples of brain tissue were preserved in a solution containing 4%\nbuffered neutral formalin; then, they were encased in paraffin, sectioned, and\nsubjected to immunohistochemical staining to assess the NOD3 expression under a\nlight microscope.<\/p>\n\n\n\n

Anti-NOD3 pAb Lsbio ABIN625967 was used for immunohistochemical\ndetection of NOD3 expression. Immunohistochemical (IHC) staining was carried\nout with anti-NOD3 antibodies. The slides were added with enzyme conjugate,\ndiluted with BSA 1%, and incubated for an hour at ambient temperature; a\nchromogen was applied for a duration of 10 minutes, and the slides were rinsed\nwith flowing water. If appropriate, counterstain was also given. <\/p>\n\n\n\n

The result of the IHC staining was examined using a microscope with\na magnification of 400x microscope. Scoring guidelines according to DC Allred,\nMD’s proportion score is as follows: 0 indicates no marker expression in any\nspecimens; 1 indicates > 0\u20131% marker expression; 2 indicates > 1\u201310%\nmarker expression; 3 indicates > 10\u201333.3 % marker expression; 4 indicates\n> 33.3-66.6 % marker expression; 5 indicates > 66.6\u2013100% marker\nexpression; and strength score is as follows: 0 indicates negative marker\nexpression; 1 denotes low expression, 2 represents moderate expression, and 3\nsignifies high expression. The ratio plus the strength score adds up to the\ntotal scores.18<\/sup><\/p>\n\n\n\n

Statistical Analysis<\/strong><\/p>\n\n\n\n

All variables were analyzed with the descriptive test. The data underwent\nanalysis to check for normal distribution using the Kolmogorov-Smirnoff and\nLevene tests for variance homogeneity. The independent sample T-test was\nemployed to examine the variance in EGCG effects among groups, while the\nPearson test was utilized to assess the correlation between variables. Data was\nanalyzed using the Statistic Package for Social Science Software, Version 21.Results<\/p>\n\n\n\n

\nThe result of the IHC staining for NOD3 expression examination\nunder the microscope with a magnification of 400 x (figure 1).\n\n<\/p>\n\n\n\n\n\n
\"\"<\/td>\nFigure 1: Morphology of the microglial cell given NOD3 marker. Healthy-rats group (Sham), control group (P0), EGCG 10mg\/kg Body Weight (P1), EGCG 20mg\/kg Body Weight (P2), <\/strong>

<\/p>\n

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

The mean score of NOD3 expression in sham = 2,636, P0 = 5,636, P1 =\n4,636, P2 = 4,000, P3 = 2,636, P4 = 2,909 (figure 2).<\/p>\n\n\n\n\n\n
\"\"<\/td>\nFigure 2: The mean score of NOD3 expression in all treatment groups and the comparison result of each group with P0a<\/sup> and shamb<\/sup>, a<\/sup>Significantly less than 0.05 compared to the P0 (control group).<\/strong>

<\/p>\n

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

The research data was subjected to analysis using the Independent\nSample T-test. To use this test, a few requirements must be met: each group’s\ndata must have a normal distribution, The sample should be derived from\nseparate data sources, and there should be uniform variance among the groups.\nThis study’s homogeneity test, which used the Levene test, produced a sig value\ngreater than 0.05 (p = 0.01), signifying that the data exhibited homogeneity;\nthe normality test, which used the Kolmogorov-Smirnov test, also produced\na sig value. > 0.05 (p = 0.01) indicates normally distributed data for this study.\nWe compared every group with P0 and Sham.<\/p>\n\n\n\n

The Independent sample T-Test result compared to sham in the NOD3 expression\nin the P0 = 0.00; P1 = 0.001; P2 = 0.001; P3 = 1.000 and P4 = 0.235. While if\ncompared to P0, the results are Sham = 0.001; P1 = 0.001; P2 = 0.001; P3 = 0.001\nand P4 = 0.001.<\/p>\n\n\n\n

The relationship between these two variables was assessed through\nthe Pearson correlation test. The significance is 0.007, with a value of r = –\n0.330.<\/p>\n\n\n\n

Discussion <\/strong><\/p>\n\n\n\n

This study divided Rattus norvegicus into six groups, each with 11\nmale rats. All groups except Sham were conditioned as stroke rats. We carried\nout middle cerebral artery (MCA) occlusion. The stroke condition was created using the MCAO model by locking the\ninternal carotid artery with a bulldog clamp for 180 minutes. Due to its\nsupply of a significant portion of the basal ganglia, internal capsule, and\nlateral brain surface, the MCA is the most often affected in stroke cases.19<\/sup>\nVarious studies have shown that other cells besides neurons have a role in the\npathophysiology of ischemia. A functional “neurovascular unit” is\nalso formed by combining the vascular, glial, and neuronal components.<\/p>\n\n\n\n

We observed NOD3 expression in microglia cells, which play an\nessential role in pyroptosis, which leads to ischemic stroke.20,21<\/sup>\nAs a result, P0 demonstrated the highest mean expression of NOD3 in comparison\nto the other groups, while P3 had the smallest mean, followed by P4.<\/p>\n\n\n\n

We also analyze the data using a descriptive test. Figure 2 shows\nthe data on the mean total score of NOD3 marker expression in six groups of\nmice. The graph presents that the intervention of EGCG and green tea extract\ncan reduce the NOD3 expression in microglia cells. Decreased expression of NOD3\nindicates the inhibition of mTOR by EGCG. Xie et al. (2014) reported that\ninhibition of mTOR signalling modulates macrophages\/microglia after focal\nischemia. Inhibition of mTOR through the administration of rapamycin\nsignificantly reduced IL-1\u03b2 expression in microglia, inhibiting the pyroptosis\nprocess, which plays an important role in ischemic stroke.22<\/sup><\/p>\n\n\n\n

In this study, the independent sample T-Test result in the\nexpression of NOD3 in the P0-P2 groups showed a significant difference with\nSham with a significance value of 0.001. However, P3 and P4 did not show a\nsignificant difference to Sham. These results indicate a significant repair of\nmicroglia cells in each treatment group until it is even close to normal in the\nP3 and P4 groups, as evidenced by the significance value of P3 and P4 with a\nsham> 0.005. P3 had the biggest significance value compared to Sham, meaning\nthat P3 is the best treatment, followed by P4 because the IHC score is closest\nto the normal group.<\/p>\n\n\n\n

Independent sample T-Test results on the expression of NOD3 in the P1,\nP2, P3, and P4 group showed a significant difference with P0 with a\nsignificance value of 0.001. This implies that every dosage of EGCG and tea\nextract exhibited a noteworthy impact compared to the control group. These\nfindings align with Zhou’s (2014) previous studies, which revealed the\ndownregulation of NOD3 protein after being induced by mTOR inhibition.10<\/sup><\/p>\n\n\n\n

The Pearson correlation test showed that the correlation was\nsignificant with a negative correlation. The negative correlation indicates\nthat taking more green tea active ingredient EGCG or green tea extract will\ndecrease the NOD3 expression markers in the brain of the Rattus norvegicus MCAO\nmodel.<\/p>\n\n\n\n

Ischemic stroke is generally caused by thromboembolism, where the\ninjured and dead neurovascular unit releases HMGB1 and Phospholipase A2 (PLA2),\nwhich are variations of DAMPs, HMGB1 binds to TLR receptors (TLR2, TLR4, and\nRAGE) in microglial cells inducing further upregulation of NLRP3.5<\/sup> NLRP3\nis recognized as a key mediator of the innate immune response to danger signals\nand increases inflammation. It induced IL-1B and IL18 activation, leading to\npyroptosis. Pyroptosis plays a crucial role in ischemic stroke pathogenesis.23,24<\/sup><\/p>\n\n\n\n

A recent study by Perez-Alvarez et al. shows that mTOR plays a role\nin brain ischemia6<\/sup>,\nLi et al. explain that mTOR regulates NLRP3 activation via ROS in murine lupus7<\/sup>.\nRecently, it was discovered that NOD3 is an inhibitor of NLRP3. NOD3 competes\nwith the ASC adapter to bind to the CARD domain (Caspase Recruitment Domain),\nwhich has previously bound to pro-caspase 1 so that pro-caspase 1 cannot be\nactivated to caspase 1 and the pyroptosis process is inhibited.8<\/sup> <\/sup>Moreover, research conducted by\nHawley et al. revealed that NOD3 acts as a nutrient sensor for the mTOR signaling\npathway.25<\/sup><\/p>\n\n\n\n

Green tea contains the active ingredient EGCG, which acts as an\nantioxidant. EGCG has been shown to inhibit mTOR in the NF-kB pathway. Research\non the effect of EGCG on PI3K\/mTOR signaling and protein translation inhibition\nshows that inhibition of PI3K\/mTOR can be accomplished at EGCG doses ranging\nfrom 320-380 nM EGCG.23,26<\/sup><\/p>\n\n\n\n

Using the NOD3 marker is one of the strong points in this research\nsince it is one of the NLR families that has not been studied much before\ncompared to other NLR families. Our research can also be applied to humans\nbecause it uses markers found in the brain where MCAO occurs. Another strong\npoint is the design of this research. It is true experimental research, so the\nresearcher can control the confusing factors affecting the research result. <\/p>\n\n\n\n

Apart from strong points, this research also has some limitations. We performed IHC as our method, which is a semi-quantitative method, so it is less valid when compared to quantitative methods. The marker used is only a NOD3 marker, so the neuroprotective effect of green tea (EGCG) on other markers and pathways is unknown. And also, the cells studied were only on microglia cells, so green tea\u2019s neuroprotective effect on other cells in the brain was unknown. <\/p>\n\n\n\n

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

The intervention of EGCG and green tea extract results in a downregulation of NOD3 expression. There was a noticeable change in NOD3 expression after the 10 mg\/kg Body Weight treatment.  EGCG (the active component of green tea) and NOD3, are correlated.  We can conclude that Green tea, which contains the active ingredient EGCG, could be used to treat ischemic stroke.<\/p>\n\n\n\n

Acknowledgement  <\/strong><\/p>\n\n\n\n

This work was supported by all the staff of the Faculty of Medicine, Airlangga University – Dr. Soetomo General Academic Hospital.<\/p>\n\n\n\n

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

All authors in this article declared no potential conflict of interest.<\/p>\n\n\n\n

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

No funding<\/p>\n\n\n\n

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

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