INTRODUCTION:

Gastric ulcer is a gastrointestinal mucosal damage disease that can occur in the esophagus, stomach or duodenum and can extend to the muscle mucosa¹. WHO data reports that deaths caused by gastric ulcers in Indonesia reached 0.99 percent, which was obtained from a mortality rate of 8.41 per 100,000 population. The high mortality rate is due to gastric perforation and H. pylori bacterial infection in the gastric mucosa².

Ethanol can cause gastric mucosal damage due to Reactive Oxygen Species (ROS)³. The ROS accumulation can cause damage to specific genes involved in cell growth or differentiation and can cause changes in levels of antioxidant enzymes resulting in oxidative stress which in turn can contribute to tissue damages⁴.

Oxidative stress can cause partial inactivation of COX-1 and reduce prostaglandin (PGE₂₎ in stomach⁵. Gastric PGE₂ and NO increase mucosal defense by increasing mucus and bicarbonate secretion, maintaining mucosal blood flow⁶. Ethanol can activate MAPK (Mitogen-Activated Protein Kinase). which plays an important role in signaling path^7,8 to activate CBP/p300 (Cyclic adenosine monophospate), while p38 dan JNK (c-Jun N-terminal kinase) MAPK activates AP-1 (Transcription factor activator protein I), which is a transcription factor needed for COX-2 expression. COX-2 is an enzyme expressed in inflamed tissue and plays a role in stimulation that occurs due to inflammatory processes such as pro-inflammatory cytokines and growth factors⁹. Increased expression of COX-1 can increase gastric mucosal defense, while inhibition of COX-2 expression can reduce inflammation which is expected to protect the stomach. The anti-secretory drugs such as omeprazole, lansoprazole, histamine, H₂- receptor blockers – ranitidine, famotidine are being used to control acid secretion. These drugs produce potential adverse effect on human health¹⁰.

The traditional medicine all over the world nowadays is revealed by an extensive activity of research on different plant species and their therapeutic principles¹¹. One of potential plants as a gastroprotector is green algae (Ulva lactuca L) which contains flavonoids and alkaloids that can overcome gastric ulcers¹². EEGH Ulva lactuca L. which originates from Drini beach, Gunungkidul, Yogyakarta contains alkaloids grade compounds, cardiac glycosides¹³ which has antioxidant effect. Antioxidant activity is effective in capturing hydroxyl radicals, peroxyls, alcohols and superoxide anions¹⁴ from ethanol. Previous studies of EEGH at a dose of 500 mg/kgBW had a gastric ulcer index of 0.5 and a protection ratio of 78.94% against the stomach of ethanol-induced rats¹⁵. Based on this background, it is necessary to conduct research on the mechanism of EEGH as a gatroprotector with parameters of histological features, ulcer index and COX-1, COX-2 expressions on the stomach of ethanol induced rats. The study results can explain the EEGH mechanism as a gastroprotector in gastric mucosal defense.

MATERIALS AND METHODS:

Materials:

The main ingredient used in this study is green algae (U. lactuca. L) originates from Drini beach Gunung Kidul Regency which were collected in January. The green algae were collected in the afternoon at low tide. The main chemicals used in this study were 96% ethanol as an extracting agent, reagent kit for IHC and Rabbit antibody COX-1 (FineTest) and also Monoclonal mouse antibody COX-2 (FineTest).

The main tools in this research are microtome, microscope (Olympus) along with optilab facilities, incubator, immunohistochemical device (ULTRATEK HRP anti-polyvalent (DAB) staining complete system PT. Medipath).

Methods:

The test animals used in this study were 2-3 months old Wistar rats from the Integrated Research and Testing Laboratory (LPPT) Gadjah Mada University with ethical approval from the UAD Research Ethics Commission No 011803034. The test animals were kept as a temperature of 24^o±2^o C, relative humidity of 30% -70% and 12 hours of light, 12 hours of darkness. The room was kept clean and given food according to laboratory standards¹⁶.

Green Algae Extracts:

The green algae powder was then extracted by maceration method with 96% ethanol. The ethanol extract of green algae is then evaporated by the solvent with a rotary evaporator at a temperature of 40ºC until a thick extracts were obtained¹⁵.

Treatment of Test Animals:

The test animals were divided into 6 groups of 6 animals each. Groups I and II were only given standard food and drink. Group III was given ranitidine 15.75 mg/kgBW. Groups IV, V and VI were given ethanol extract of green algae at doses of 125, 250 and 500 mg/kgBW. Treatments are given for 14 days. After 14 days, the rats were induced with ethanol at a dose of 2 ml/200g BW orally (except group I) and then fasted for 24 hours. Rats were sacrificed by evaporating carbon dioxide. Then operations were performed on the test animals¹⁵. The rats were operated and their stomachs were taken to make histological preparations for histopathological and immunohistochemical observations.

Rat’s Gastric Histology Testing:

The stomach organs were taken and then washed with 0.9% NaCl, then kept in 10% formalin solution. Histopathological examination was based on work procedures established in the Pathology Laboratory, Faculty of Veterinary Medicine, Gadjah Mada University, including trimming, dehydration, cleaning, impregnation, embedding and cutting processes. Observations were made under a light microscope with a magnification of 100x. Then ulcer scoring was performed¹⁷. The changes observed were the presence, necrosis, degeneration and the presence of inflammatory cell infiltration that occurred in the sub-mucosa and mucosa in the rats stomach. Ulcer scores were performed as follows: no change (0), change in one place/focal (1), change in several places/multifocal (2) and change evenly/diffuse (3).

Immunohistochemical testing of COX-1 and COX-2 expressions:

Observation of glandular cell COX-1 protein expression and inflammatory cell COX-2 according to the standard methods of the Anatomical Pathology Laboratory of the Faculty of Medicine, Gadjah Mada University. After cutting the tissue preparations with a thickness of 3µm, they were dried at a temperature of 37°C by heating on a 60°C Warmer slide for 30-60 minutes. Deparafinisation was carried out in stages using Xylol I, Xylol II, and Xylol III, each was performed for 5 minutes. The rehydration process was carried out in stages using absolute alcohol, 96% alcohol and 70% alcohol for 4 minutes in each process and washed using running water for 5 minutes. Pre-treatment with Tris-EDTA pH 9.0 using a waterbath at a temperature of 95°C which was carried out for 10 minutes and then cooled at room temperature for ±30 minutes. It was followed by washing using PBS pH 7.4 twice for 2 minutes. Incubated with Peroxidase block for 10 minutes. Washed again with PBS pH 7.4 2 times each for 2 minutes. Reincubated with Super Block for 5-10 minutes. Washed with PBS pH 7.4 for 30 seconds. The next process was incubated with Primary Antibodies (anti-COX-1 or anti-COX-2) for 30-45 minutes and washed again with PBS pH 7.4 twice for 4 minutes. Incubation was performed with UltraTek Anti-Polyvalent secondary antibodies for 10 minutes and washed again with PBS pH 7.4 twice for 4 minutes. Incubated with UltraTek HRP (Horseradish peroxidase) for 10 minutes and washed again with PBS pH 7.4 twice for 3 minutes. The last incubation with DAB Solution which was always made fresh by mixing 1 mL of DAB Substrate High Contras and 50µL/1 drop of DAB Chromogen, this incubation was carried out for 1-5 minutes and after washing with running water for 5 minutes. Positive expression was shown in brown on the cytoplasm and cell membrane. The percentage of COX-1 and COX-2 expressions was calculated using Image J.

Data Analysis:

Gastroprotective activity performed in the stomach was observed from the results of gastric histology qualitatively and semi-quantitatively. Semiquantitative analysis was performed using ulcer score data. Gastroprotective activity was also analyzed from the COX-1 glandular COX-2 inflammatory cells expressions. Qualitative data was presented descriptively in the form of histological and quantitative images by calculating the percent value of protein expression using Image Analysis Software (Image J). The percent expression calculation was carried out in three different fields of view randomly for each preparation. Statistical analysis was performed on ulcer scoring, of COX-1 and COX-2 percent expressions with the SPSS program.

RESULT:

Rat’s Gastric Histology Test Results:

Gastric histological description of the various treatment groups are presented in Image 1. Gastric histology description was then performed the ulcer scoring according to¹⁷.

COX-1 Glandular Cell and COX-2 Inflammation Cell Expressions:

In immunohistochemical staining, cells expressing COX-1 and COX-2 proteins will be brown, while cells that did not express will be purplish blue¹⁸. The COX-1 microscopic results on glandular cells can be seen in Image 2, while the COX-2 microscopic results on inflammatory cells can be seen in Image 3.

Figure 1. Rat’s Gastric Histology Test

Figure 2. COX-1 microscopic results on gastric glandular cells of mice from various groups. Description: (a) Normal; (b) Negative; (c) Positive; (d) EEGH Ulva lactuca L. 125 mg/KgBB; (e) EEGH Ulva lactuca L. 250 mg/KgBB; (f) EEGH Ulva lactuca L. 500 mg/KgBB

Figure 3. COX-2 microscopic results on gastric inflammation cells of rats from various groups. Description: (a) Normal; (b) Negative; (c) Positive; (d) EEGH Ulva lactuca L. 125mg/KgBB; (e) EEGH Ulva lactuca L. 250mg/KgBB; (f) EEGH Ulva lactuca L. 500mg/KgBB

DISCUSSION:

Gastric histology testing was carried out to see the effect of ethanol administration on gastric histology and the EEGH effect to reduce gastric damage. The histopathological changes observed included necrosis, degeneration and the presence of inflammatory cell infiltration that occurred in the sub-mucosa and mucosa in the rats stomach. The average gastric ulcer score is presented in Table I.

Table 1. Picture of Gastric Histology and Average Scores of Gastric Ulcers in Ethanol-Induced Rats

Group	Rats	Histopathological Analysis Results Rat’s Gastric	Average Scores of Ulcers (±Sd)
Normal (Healthy)	1	No changes	0 ± 0.00
	2	No changes
	3	No changes
Negative	1	Necrosis/erosion and hemorrhage	2 ± 0.00*
	2	Necrosis/erosion and hemorrhage
	3	Necrosis/erosion and hemorrhage
Positive (Ranitidine) Doses of 15.75mg/ kgBB	1	Hemorrhage	1.66 ± 0.58
	2	Edema
	3	Edema
EEGH Doses 125mg/kgBB	1	No changes	1.66 ± 1.53
	2	Edema and necrosis / erosion
	3	Edema and necrosis /erosion
EEGH Doses 250 mg / kgBB	1	Hemorrhage and edema	1.33 ± 1.15
	2	Hemorrhage and edema
	3	No changes
EEGH Doses 500 mg / kgBB	1	No changes	0 ± 0.00#
	2	No changes
	3	No changes

Ethanol 2ml/100gBW administration caused necrotic lesions by direct necrotizing action which in turn reduces defensive factors, the secretion of bicarbonate and production of mucus¹⁹, also haemorrhage with an average score of 2.00±0.00 (Table 1). The statistical analysis results of the Mann-Whitney test, the ulcer score of normal healthy control and the negative group showed a significant difference with the p value of 0.025 (<0.05), this indicates that there was damage caused by ethanol. Ethanol irritates the stomach by increasing free radicals production in the form of Reactive Oxygen Species (ROS) thereby reducing the ability of cellular antioxidants to maintain a balance between aggressive and defensive factors²⁰. The Reactive Oxygen Species (ROS) generated by the metabolism of arachidonic acid, platelets, macrophages and smooth muscle cells may contribute to gastric mucosal damage²¹. Ethanol rapidly penetrates the gastric mucosa apparently causing cell and plasma membrane damage leading to increased intra cellular membrane permeability to sodium and water. The massive intracellular accumulation of calcium represents a major step in the pathogenesis of gastric mucosal injury. This leads to cell death and exfoliation in the surface epithelium²².

Based on Image 1, necrosis existed in groups II and IV. Necrosis is irreversible cell or tissue death in living organisms and necrosis is a pathological process that is often caused by external factors (eg infection, toxins and trauma) to cells or tissues²³. So that necrosis can occur even in normal cells. Then in groups II, III and V, edema was seen, which is a reaction to the defense of organisms and tissues against damage such as bacteria or foreign objects²⁴.

Ranitidine administration as a positive control had a p value> 0.05, namely 0.114, which means that there was no significant difference with the negative group, meaning that the administration of ranitidine suspension had no protective effect on the stomach. According to research Tarnawski 2005, that ranitidine is not effective in gastric mucosal wounds or gastric necrosis caused by ethanol induction²⁵.

Administration of EEGH at a dose of 125mg/kgBW still showed edema and necrosis with an average ulcer score of 1.66±1.53, while the EEGH dose of 250mg/kgBW showed hemorrhage and edema with an average ulcer score of 1.33±1.15. EEGH administration at a dose of 500 mg/kgBW showed protection against the stomach with an average ulcer score of 0±0.00 and no visible tissue damage, both hemorrhage, necrosis and edema. Different test results showed that administration of EEGH Ulva lactuca, L. at doses of 125mg/kgBW dan 250mg/kgBW had value of p>0,05 namely 1 and 0,317 so that it could be concluded that there was no significant difference, which means EEGH Ulva lactuca, L. at doses of 125mg/kgBW dan 250mg/kgBW had not been able to protect the gastric mucosa, while EEGH Ulva lactuca, L. administration at a dose of 500 mg/kgBW, showed p value < 0.05 that is 0.025, which means EEGH Ulva lactuca, L. at a dose of 500 mg/kgBW gave gastroprotective effect based on a significant reduction in ulcer scores compared to negative controls.

Based on Image 2, the ethanol induction presence causes a reduction in COX-1 protein so that the bonds between the substrate and the chromogen formed are also small which tends to be purplish blue. In the ranitidine group, the brown color intensity was dominant, indicating that the COX-1 protein was expressed quite a lot. In the treatment group, EEGH Ulva lactuca L. administration in various doses of brown color intensity were formed more than the negative control but less than the positive control. The microscopic results are in line with the calculation of COX-1 expression which is presented in Table 2.

Table 2. Comparison of the average percent expression of COX-1 glandular cells and COX-2 in rat’s gastric inflammatory cells from various groups

Group	Average percent expression	Average percent expression	Results
	COX-1	COX-2	COX-1/ COX-2
I.	266.411 ± 9.694	0	ꝏ
II	98.907 ± 27.409	154.717 ± 37.327	0.6393
III	223.028 ±16.137*	62.339 ± 15.831**	3.5777
IV	132.386 ± 4.882	182.265 ± 27.544**	0.7263
V	150.803 ±23.242*	132.238 ± 4.785**	1.1404
VI	171.755 ±25.003*	124.047 ± 3.771**	1.3846

In the groups which were given ethanol, there was a decrease in COX-1 expression from 266,411±9,694 to 98,907±27.409. While in the groups which were given ranitidine, EEGH doses of 125mg/kgBW, 250mg/kg BW and 500mg/kg BW, respectively 132.386±4.882; 150,803±23,242 and 171,755±25,003 can increase COX-1 expression. EEGH administration at doses of 250 and 500mg/kgBW significantly increased COX-1 expression with p<0.10.

Based on Image 3, the normal group did not show any COX-2 protein expressions. The negative control group that showed a lot more brown color intensity compared to the normal and positive control group, which means that the presence of ethanol causes a lot of expressed COX-2 protein which will bind to chromogen. In the groups which were given EEGH Ulva lactuca L. at doses of 125mg/KgBW, 250mg/KgBW and at a dose of 500mg/KgBW, it can be seen from the brown color that was formed successively, the intensity of the brown color decreases. The microscopic results are in line with the calculation of COX-2 expression which is presented in Table 2. In the groups which were given COX-2 expression ethanol as of 154,717±37.327. While in the groups which were given ranitidine, EEGH doses of 125 mg/kgBW, 250mg/kgBW and 500mg/kgBW, can decrease COX-2 expression consecutively 62.339±15.831; 182.265±27.544; 132.238±4.785 and 124.047±3.771.

To see the trend of the effect of EEGH U Ulva lactuca L. on the stomach of ethanol-induced rats, a comparison was made between the percent expression of COX-1 and the percentage of COX-2 expression contained in Table II. EEGH Ulva lactuca L. administration was able to increase COX-1 expression significantly and was not able to significantly reduce COX-2 expression when compared to negative controls. The comparison results of percent expression of COX-1/COX-2 in glandular cells and rats gastric inflammatory cells were directly proportional to the increase in the dose of Ulva lactuca L. where consecutively the results obtained were 0.7263; 1,1404 and 1.3846.

The overall results showed that EEGH administration at a dose of 500mg/kgBW had a protective ability in the stomach of ethanol-induced rats with the parameters of decreased ulcer score, decreased edema, hemorrhage, necrosis, increased percent expression of COX-1 and decreased COX-2. This protective ability is alleged to be due to EEGH content which functions as an antioxidant. EEGH contains a melatonine compound which is an alkaloid in gastric mucosal defense which increases gastric microcirculation and inhibits neutrophil activation, an effect that is partially mediated by the NOS/NO and PG/COX systems. Melatonine inhibits the inflammatory cascade via down regulation of transcription facto NF‐κB²⁶. Also, EEGH Ulva lactuca L. contains flavonoids²⁷. Flavonoids can increase prostaglandins, inhibit histamine secretion and secrete prostaglandins-like compounds²⁸. Flavonoids can bind free radicals as well as antioxidants and act as anti-secretory and increase mucus²⁹. The antioxidant activity of flavonoids is efficient in trapping superoxide anion, hydroxyl, peroxyl and alcohoxyl radicals²¹. EEGH Ulva lactuca L. also contains tocopherol which is included in the vitamin E group which is believed to be an antioxidant that can prevent lipid peroxidation from unsaturated fatty acids in cell membranes^30,31. Humans need tocopherol because it has a balance in stabilizing cells from free radicals and inhibiting the oxidation process³¹.

CONCLUSION:

The results showed that EEGH at doses of 500 mg/kgBW had a protective effect as seen from a decrease in the ulcer index and a reduction in necrosis, edema, and haemorrhage. EEGH Ulva lactuca L. were given at a dose of 500mg/KgBW was able to significantly increase the percentage of COX-1 expression in glandular cells but was not able to significantly reduce COX-2 expression.

CONFLICT OF INTEREST:

There are no conflict of interest.

ACKNOWLEDGMENTS:

We would like to thank the UAD Institute for Research and Community Service (Lembaga Penelitian dan Pengabdian Masyarakat / LPPM) who have funded this research with a research agreement letter Number: PJB-001/SP3/LPPM-UAD/VI/2019.

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Received on 16.11.2021 Modified on 09.04.2022

Research J. Pharm. and Tech 2023; 16(2):727-732.

DOI: 10.52711/0974-360X.2023.00124