The effect of Helicobacter pylori on the gastric expression of LOX-1 and CD-36: the role of CRP and ox-LDL

Gholamreza Ghanbariha Semnan University of Medical Sciences and Health Services Reza Dabiri Semnan University of Medical Sciences and Health Services Majid MirmohammadKhani Semnan University of Medical Sciences and Health Services Mojdeh Habibzadeh Semnan University of Medical Sciences and Health Services Vahid Semnani Semnan University of Medical Sciences and Health Services Abbas Pakdel (  pakdel@semums.ac.ir ) Semnan University of Medical Sciences and Health Services https://orcid.org/0000-0002-7173-367X


Introduction
Helicobacter pylori (H. pylori) is a gram-negative, spiral, microaerophilic, and agellate bacterium localized in the human stomach. Approximately half of the world's population is infected with this bacterium. H. pylori can colonize gastric tissue and attach it to epithelial cells with its agella and adhesion molecules. By releasing toxins, it can cause tissue damage and in ammation [1]. Chronic gastritis is usually an asymptomatic disease that is highly important, since it causes stomach cancer in some patients. There are two types of gastritis. In most cases, gastritis is caused by H. pylori infection. The autoimmune type of gastritis is scarce [2].
Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) is a type II membrane protein that structurally belongs to the type C lectin family [3]. In addition to being an oxidized low-density lipoprotein (ox-LDL) receptor, this protein can bind to C-reactive protein (CRP) in endothelial cells with a lower a nity than that of ox-LDL. Despite its lower CRP a nity for binding to this receptor, it induces LOX-1 expression and can play a role in carcinogenesis [4-6, 3, 7-9]. H. pylori-induced acute and chronic gastritis can cause many changes in gene expression. It may lead to gastric cancer. Elevated serum levels before CRP treatment (> 10 mg / L) are signi cantly associated with poor prognosis in patients with gastric cancer, both in early and advanced stages [10,11]. Activation of LOX-1 causes the transformation and growth of several cancer cells, while decreased LOX-1 expression can prevent such processes [12]. Some studies have demonstrated that overexpression of LOX-1 is involved in tumor progression in prostate, colorectal, gastric, pancreatic, and breast cancers [13,6,5,4,14].
Cluster of differentiation 36 (CD-36) is an 88 kDa membrane glycoprotein belonging to the scavenger   receptor B family. CD-36 is present on the surface of several cells, such as fat cells, monocytes,  macrophages, platelets, endothelial, cardiac, skeletal, and smooth muscle cells, dendritic cells, retinal pigment epithelium, and red blood cell hematopoietic precursors. CD-36 plays a minor role in the onset of primary tumors. CD-36 is a glycoprotein that functions as a transporter of long-chain fatty acids and ox-LDL, and is involved in angiogenesis, immune responses, adhesion, and metastasis. This protein is expressed in stromal, immune, and cancer cells. Different patterns of expression have been observed from the onset to the spread of cancer in different malignancies. Today, this protein is considered one of the biomarkers and therapeutic targets of cancer. The expression is also generally reduced in tumor microvessels supporting tumor progression and invasion. In Tumor Stromal cells, CD-36 expression is extremely low. The lower the CD-36 level in Cancer-Associated Fibroblasts (CAFs) is, the more aggressive the tumor is [15][16][17][18].
In recent years, it has been indicated that fat metabolism has been involved in processes related to cell transformation and tumorigenesis [19,20]. The results of various studies showed that serum levels of ox-LDL and hs-CRP increased the risk of cancers [21][22][23][24]. The relationship between LOX-1 ligands and CD-36 and LOX-1 expression in patients with H. pylori infection is unclear. In this study, we investigated the relationship between serum CRP and ox-LDL levels and CD-36 and LOX-1 gene expression.

Subjects
In this cross-sectional study, we investigated 74 outpatients referred to the endoscopy department based on gastritis status in gastric biopsy and H. pylori infection in three groups: 1-H. pylori/gastritis positive 2-H. pylori-negative / gastritis positive 3 H. pylori-negative/gastritis negative. Gastritis was determined by a pathologist based on the Sydney criterion. Gastric tissue biopsy and serum specimens were used for molecular and biochemical experiments. People who, at the time of the study, had cancer, diabetes, infectious diseases, cardiovascular disease, high blood pressure and hyperlipidemia, pregnancy, anemia, kidney disease, autoimmune and in ammatory diseases, and gastrointestinal disorders, and thyroid problems, were omitted. Likewise, smokers and alcoholics and people taking anti-in ammatory, steroids, antacids, proton pump inhibitors, bismuth, and antibiotics drugs were excluded from the study.

Sampling
Blood samples were taken from the participants, and after centrifugation, its serum was isolated and kept at -70°C until we conducted experiments. Gastric antrum biopsy specimens were immediately stored in 10% formalin. After xation, the samples were molded, cut, and stained by hematoxylin and eosin (H&E).
We used the rest of the tissue samples for RNA extraction and gene expression analysis. To detect H. pylori in the gastric mucosa, we used a urease test and direct observation of bacteria in stained tissue sections.

Rna Extraction And Real-time Rt-pcr
Total RNA was extracted from antrum biopsies according to the method of the kit manufacturer (Gene All). For cDNA synthesis, according to the instructions of the cDNA synthesis kit (YTA.Co), in the initial stage, 2µg of the total RNA with 2µl of primer (each of the oligo (dT) and random hexamer 1µl) and volume was raised to 13.4 µl by DEPC-H20. It was located at 70°C for 5 minutes. In the second step, 4µl of buffer (5X), 1µl of dNTPs, 0.5µl of RNase Inhibitor, and 1µl of M-MLV enzyme were added and incubated at 37°C for 60 min and then at 70 ° C for 5 min. AllelID 7.5 software was used to design the primers ( Table 1). For the real-time RT-PCR, 10µl of 2x SYBR Green (YTA Cat No: YT2551) was mixed with 0.4µl of each forward and reverse primer, and 1.5µl of cDNA with 1µl of passive reference dye (50X) and DEPC H2O. The volume was brought to 20µl and placed in a thermocycler. After an initial denaturation phase at 95°C for 3 minutes, the denaturation phase at 95°C for 5 seconds, and then the annealing/extension phase at 60 ° C for 20 seconds were performed in 40 cycles. BACT gene was an internal control, and after obtaining CT of each sample for genes, the mean was taken from CT. Afterward, the results were analyzed using the ∆CT method. Statistical analysis SPSS 24 and GraphPad software programs were used for data analysis and graphs drawing, respectively. Shapiro-Wilk test was used to check the normality of the data. To compare data to normal distribution, a one-way ANOVA parametric test, and then the Tukey test was used to examine the differences between groups. Data with abnormal distributed nonparametric Kruskal-Wallis tests, and then groups were compared to the pairwise comparisons of the group test. Spearman (nonparametric) correlation was used to investigate the correlation between serum analyte concentration and gene expression. The value of P ≤0.05 was considered a signi cant level in this study.

Results
Of the 74 participants in the study, 43 were female, and 31 were male. Pathological ndings and the RUT test revealed gastritis and H. pylori infection in the groups ( Table 2). The results indicated that 40 of these patients were H. pylori-negative, and 34 were H. pylori-positive. In addition, 11 patients had not gastritis, and 63 patients had gastritis. Comparison of BMI between the groups by the one-way ANOVA test showed no signi cant difference in the participants' BMI (P = 0.496). Comparison of sex and age variables in the groups using the Chi-Square test showed no signi cant difference between age and sex traits (P = 0.835).

LOX-1 and CD-36 gene expression
According to the statistical analysis of LOX-1 expression data using the Shapiro-Wilk test (P ≤0.05), the data distribution was not normal. Comparison of the gene expression in the three groups using the Kruskal-Wallis test showed a signi cant difference (p = 0.033). Comparison of the results between the groups using the pairwise comparisons of the group test demonstrated a signi cant difference between H. pylori/gastritis positive and H. pylori /gastritis negative (p = 0.025) and between H. pylori-negative / gastritis positive and H. pylori/gastritis negative groups (P = 0.010). However, no signi cant difference was observed between H. pylori/gastritis positive and H. pylori-negative/gastritis positive (p = 0.597) groups (Fig. 1).
Comparison of CD-36 expression using the Independent Samples Median Test showed a signi cant difference between the groups (p = 0.013). The comparison between the groups using the pairwise comparisons of the group test showed a signi cant difference between H. pylori/gastritis positive and H. pylori/gastritis negative (p = 0.046). There was no signi cant difference between H. pylori-negative / gastritis positive and H. pylori / gastritis negative (p = 0.288), and between H. pylori / gastritis positive and H. pylori-negative / gastritis positive (p = 0.712) (Fig. 2).

Hs-crp And Ox-ldl Results
Comparison of the results of the hs-CRP concentration measurement in the three groups performed using the Kruskal-Wallis test showed a signi cant difference in the hs-CRP variable (p<0.001). The groups were compared by the pairwise comparisons of the group between H. pylori/gastritis positive and H. pylori/gastritis negative (p = 0.013), and between H. pylori-negative / gastritis positive and H. pylori/gastritis negative (p<.001). There was a signi cant difference between H. pylori/gastritis positive and H. pylori-negative / gastritis positive groups (p = 0.016).

Analysis of the ox-LDL concentration between the groups by the pairwise comparisons of the group test
showed a signi cant difference between H. pylori-negative / gastritis positive and H. pylori and gastritis negative groups (p = 0.032). Furthermore, no signi cant difference was observed between H. pylori/gastritis positive and H. pylori/gastritis negative groups (p = 0.440), and between H. pylori / gastritis positive and H. pylori-negative / gastritis positive (p = 0.051).
Correlation between LOX-1, CD-36 expression level, and serum hs-CRP and ox-LDL concentration Due to the abnormality of the data, the Spearman test was used to measure the relationship. There was a weak correlation between serum hs-CRP concentration and LOX-1 expression (r = 0.219, P = 0.058).
Owing to the abnormality of the data, the Spearman test was used to measure the relationship between them. There was no correlation between serum ox-LDL concentration and LOX-1 and CD-36 expression.

Discussion
In this study, the expression levels of LOX-1 and CD-36 genes, and serum levels of hs-CRP and ox-LDL in our target groups were compared based on gastritis and H. pylori. The serum levels of hs-CRP and ox-LDL were higher in H. pylori-positive patients. Our results indicated that H. pylori increased LOX-1 and decreased CD-36 gene expression. Moreover, a weak correlation was observed between the serum level of CRP and the expression of LOX-1, while there was no correlation between the serum level of ox-LDL and the expression of genes. In summary, our results demonstrated that there were higher serum levels of CRP and ox-LDL and expression of the LOX-1 gene in the group with negative H. pylori-gastritis positive than in the control group. Although in the H. pylori and gastritis-positive group, the serum level of CRP and LOX-1expression was higher, the expression of CD-36 decreased despite the high level of ox-LDL (Fig. 5).
In this work, the effects of smoking, alcohol consumption, use of any drugs, use of antacids, use of antiin ammatory medications, antibiotics, anti-H. Pylori drugs were excluded according to the inclusion and exclusion criteria. It furthermore balanced the body mass index in the three groups. In addition, none of the people in their rst-degree relatives had a history of gastric cancer. Therefore, the effects seen were due to infection with this bacterium and gastritis.
The role of H. pylori, in ammation and hyperlipidemia in the evolution of distal gastric adenocarcinoma has been demonstrated [25][26][27] ; however, its molecular mechanism is unclear. According to the results of the study conducted by Huang et al., plasma levels of hs-CRP and ox-LDL were higher in atherosclerotic CHD patients with Cag + H. pylori infection [28]. Studies by Jafarzadeh et al. showed that although serum hs-CRP levels increased in H. pylori infection, this increase in hs-CRP was not affected by the bacterial CagA status [10]. In our study, it appears in the H. pylori/ gastritis positive group, H. pylori were the leading cause of the increase in serum hs-CRP levels.
Studies show that in gastric cancer, ox-LDL interaction with the LOX-1 receptor causes NF-κB pathway activation and increases vascular endothelial growth factor C (VEGF-c) expression [26]. Furthermore, the use of ox-LDL binding inhibitor inhibits VEGEF-c expression. Pascual et al. in their study found that CD-36 + metastasis initiating cells in highly invasive melanoma, lung carcinoma, bladder, and breast cancer patients were associated with a poor prognosis. Moreover, Inhibition of CD-36 in animal models of breast cancer and melanoma prevented metastasis [17].
In study conducted by the Kashihara et al. Investigating noncancerous gastric tissue obtained from 39 gastrectomy patients, the level of glycoprotein thrombospondin 1 (TSP-1), which is a CD-36 ligand, was measured, and patients in both groups with high and low TSP-1 were compared. Noncancerous gastric tissue of patients with high TSP-1 levels had higher carcinogenesis and more active CD-36 signaling. Although in these studies several patients had H. pylori infection, the effect of H. pylori on CD-36 gene expression was not investigated [29].
Some studies have investigated the effect of palmitic acid fatty acids as CD-36 ligand on signal transduction pathways in gastric cancer metastasis [30,15]. Accordingly, CD-36 levels show a signi cant upregulation in malignant tissue. In these studies, they introduced CD-36 as an oncogenic factor involved in cancer progression. However, another study reported downregulation of CD-36 in Cancer-Associated Fibroblasts (CAFs), in more aggressive tumors [18]. Although low levels of CD-36 are associated with low TNM (tumor, node, metastasis) classi cation in pancreatic ductal adenocarcinoma (PDAC) cancer, they are associated with a larger tumor size and a weaker disease prognosis [31].
Analysis of the mRNA extracted from gastric tissue samples of 10 patients with H. pylori showed that out of 13817 transcripts, 98 transcripts increased after H. pylori treatment and bacterial eradication (P<0.02 fold change threshold) [32]. CD-36 was one gene whose expression increased signi cantly (approximately 2.95 times). In agreement with the investigation conducted by Resnick et al., the expression analysis in our study exhibited a decrease in CD-36 expression in the H. pylori group.
According to the results, the primary reason for high LOX-1 expression in the H. pylori group is the activation of the in ammatory pathway. It appears that CRP acts as the dominant ligand for the LOX-1 receptor. There is evidence that CRP is suggested as a ligand for LOX-1 and induced genes involved in tumorigenesis [33,34]. In addition to the ligands for LOX-1 and CD-36, direct bacterial interaction with these receptors may have caused the molecular changes observed in gastric cells. Kattoor et al. in their study revealed that infectious agents, such as Chlamydia pneumoniae and H. pylori, could increase their expression by direct interaction with the LOX-1 receptor [35]. In this study, they showed that Chlamydia pneumoniae bound to LOX-1 in human umbilical vein endothelial cells (HUVECs) and allowed ox-LDL to enter the cells. They also observed this mechanism in the lung and adductor tissues of mice. However, this study showed that Chlamydia pneumoniae was only useful on LOX-1 and had no effect on the expression and signaling of the CD-36 gene [36]. One limitation of this study was the small amount of biopsy and serum samples of patients; therefore, molecular studies at the protein level were impossible to conduct.

Conclusion
Based on the investigation results, two factors, H. pylori, and gastritis, increase LOX-1 expression and decrease CD-36 expression. The increase in plasma hs-CRP levels may be associated with an increase in LOX-1 expression. In future studies, the effect of H. pylori and gastric cells coculture on the expression of LOX-1 and CD-36 genes and the impact of bacteria on the internalization of ox-LDL should be investigated.
Declarations Figure 2 Comparison of CD-36 relative expression levels in the three groups. Data were expressed as median