Research Article

Litaf promotes atherosclerotic plaque formation by stimulating the NF-κB inflammatory pathway

https://doi.org/10.21203/rs.3.rs-2725698/v1

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Background

Litaf protein is a newly discovered inflammatory protein, whether it is involved in the formation of atherosclerosis remains unclear;

Methods

Ten C57BL/6J mice and ten C57BL/6J mice with knockout of Litaf gene (C57BL/6J^{− Litaf−}) were purchased from Animal Culture Center of Huazhong University of Science and Technology divided into two groups: control group and Litaf^−/− group. Animals were given a normal diet and euthanized after 16-week culture. Mice, heart and whole aorta were isolated, and the aortic root of the mouse was taken for frozen sections; used for oil red staining and immunohistochemical staining (α-actin, CD68, Masson), and then compared the oil red staining area and the positive proportion of immunohistochemical staining in each group; at the same time, a sufficient amount of mouse blood was collected for extraction Plasma protein and RNA, monitoring the expression of inflammation-related proteins by Western and RT-PCR.

Results

Oil red staining of the aortic root sections of the mice in each group showed that the area of atherosclerotic plaques in Litaf^−/− group was less than control group (P < 0.05). The aortic root sections of the mice in each group were immunohistochemical stained, and it can be found that the expression of α-actin and CD68 in Litaf^−/− group was higher than that in the control group, while the results were opposite after Masson stainning(P < 0.05). The expressions of P65, IκB, and caspase3 were monitored by Western and RT-PCR. As shown in Figs. 2 and 3, the expressions of P65, IκB, and caspase3 in Litaf^−/− group were lower than those in the control group (P < 0.05).

Conclusions

Litaf might participate in the formation of atherosclerosis through the NF-κB pathway and play a promoting role in the formation of atherosclerosis.

Litaf

NF-κB

inflammation

atherosclerosis

apoptosis

PIGs (p53 induced genes) play an important role as a bridge between p53 expression, activation, and apoptosis. Among them, PIG7 gene is closely related to inflammation and apoptosis, and has gradually become a research hotspot in related fields in recent years [1].

In 1997, Polyak et al. [2] used an adenovirus expression vector to transfer the exogenous p53 gene into the p53-deficient human colon cancer cell line DLD-1, and used serial analysis of gene expression (SAGE) technology to screen 14 genes whose expression was upregulated more than 10 times was named PIG 1–14, and the signal transduction model of p53-induced apoptosis was proposed, that is, "external damage-p53 expression-Pigs-induced expression monoxide free radical generation" -Mitochondrial oxidative damage-apoptosis initiates the chain signaling pathway, in which the No. 7 gene is named PIG7. There may be two different transcripts of this gene, and the difference between the two sequences is only 1 guanylate (G). Due to the frame shift, two completely different protein products at the C-terminus are, respectively named lipopoly saccharide-induced tumor necrosis factor α factor (LPS-induced TNF-α factor, Litaf and lysosomal late endosome small intrinsic membrane protein (small integral membrane protein in late endosome, Simple). Litaf plays a role in transcriptional activation during LPS-induced TNF-α expression, and simple may be involved in lysosome-mediated intracellular protein sorting and degradation [3]. Tang, et al [4] deeply studied the LPS-Litaf-TNF-α signaling pathway using gene knockout technology combined with kinase chip analysis. As an important PAMPs (Pathogen associated molecular patterns) member molecule, LPS can bind to TLR2/TLR4 [5]. TLR is assisted by the linker molecule MyD88 to recruit downstream signaling molecules to promote Litaf expression. They also found that Litaf knockout mice exhibited defective cytokine production but enhanced resistance to LPS-induced septic shock and had significantly higher survival rates than controls, confirming the critical role of Litaf in the inflammatory response [1].

Whether Litaf participates in the inflammatory immune response process of atherosclerosis through the NF-κB pathway is still unclear. This experiment intends to explore this problem through genetic engineering combined with drug intervention.

Mouse culture

Animal experiments were approved by the Experimental Animal Ethics Committee of Huazhong University of Science and Technology. Ten 7-week-old male C57BL/6J mice and ten 7-week-old male C57BL/6J^{− Litaf−} mice were purchased from Beijing Fuwai Animal Culture Center, and then divided into control group and Litaf^−/− group. These mice were cultured in the SPF Animal Culture Center of Wuhan Central Hospital for 16 weeks with 12/24 hours of light and unlimited feed and water supply during the culture period. And the animal feed was purchased from Animal Culture Center of Huazhong University of Science and Technology. The mice in the two groups were both given a normal diet, and the weight, heart beats, blood pressure, et al. of the mice were monitored weekly.

Oil red staining and immunehistochemical staining of mouse aortic root tissue sections After 21 weeks of culture with different diets, the aortas and hearts of mice in each group were separated from mice after the mice were euthanized, and the aortic roots of mice were taken for frozen sections (slice thickness 7um), and enough sections were obtained to carry out oil red staining and immunohistochemical staining (including α -actin, CD68, Masson staining), and compared the size of the positive staining area in each group to analyze the plaque area and the stability in each group. The software Pro-plus 6 software (Media Cybernetics, USA) and Graph Pad Prism version5.0 were used for image analysis. The specificity of each group was illustrated in Fig. 1[6][7].

Western blotting

Thirty-microgram of protein was loaded in each lane and separated by 10% SDS-PAGE. Mouse anti-NF-κB (P65/IκB) antibody (Cell Signal Technology, USA), anti-caspase-3 antibody (1:1,000), anti-β-actin antibody (Sigma Aldrich; 1:3,000), anti-mousesecondary antibody (Thermo Scientific) conjugated tohorseradish peroxidase (HRP) (1:3,000) were used in the Western blotting. The blots were developed with Bio-Rad (USA) and exposed to Clear Blue X-ray film (Pierce). Density of each band was quantified and normalized to β-actin by Image lab (Syngene, Cambridge, England). The bound antibodies were detected by ECL reagent (Gene Tex, USA) according to the manufacturer’s instructions. The expression of the protein P65/IκB/caspase-3and β-actin was detected at least three times by Western blot, and the mean protein expression of each group was figured out. The signification was shown in Fig. 2[8].

Quantitative reverse transcription polymerase chain reaction (RT-PCR)

Total RNA was extracted using Trizol reagent (Invitrogen, USA). RNA was transcribed into cDNA with the reverse transcription kit (TaKaRa, Dalian, China) as the template. The housekeeping gene glyceraldehydes-3-phosphate dehydrogenase (GAPDH) was used as an internal control. The following primers were designed according to the corresponding sequences obtained from GenBank: P65-3-F: 5'-CTGGTCCTGTGAAGCCTACA-3', P65-3-R: 5'-ATCCACCTCTTCACCAACGT − 3'; IκB -F: 5'-CCTGGAGATCCAGATCATGAGAAG-3', IκB-R: 5'-TCAAACTGGTTCAGGTACTTCCG-3'; caspase-3-F: 5'-GGTAT-CCATGGAGAACACT-3', caspase-3-R:5'-AAAAATAGAGTTCTTTTGTGAG-3'; GAPDH-F: 5'-ACC-ACAGTCCATGCCATCAC-3', GAPDH-R: 5'-TCCA-CCACCCTGTTGCTGTA-3'. The following reaction systems included 4 µL of 5× SYBR Green universal type qPCR Master Mix (TaKaRa, Dalian, China), 0.6 µL of primers, and 1 µL of cDNA [9]. The total reaction volume was 20 µL. The reaction mixture was centrifuged at 1,500 rpm/min, and PCR was conducted according to the following conditions: pre-degeneration at 95 ℃ for 30 s; degeneration at 95 ℃ for 3 min; annealing extension at 60 ℃ for 30 s. Melt curve was delineated. Finally, the data was read directly from the real-time PCR instrument (Bio-Rad, USA). The expression of the mRNA of P65/IκB/caspase-3 and GAPDH was checked more than three times by PCR, and then the mean expression result of each group was figured out. The specificity of each group was illustrated in Fig. 3 [10] [11].

Statistical analysis data are reported as mean ± standard deviation (SD). Comparisons were done with Student t-test for two groups and with one-way analysis of variance between groups. Statistical analysis was performed with Graph Pad Prism version 5.0. A value of P < 0.05 was considered statistically significant.

Oil red staining results

Oil red staining of the frozen section of the aortic root of mice, the red part is atherosclerotic plaque [12]; after 16 weeks of feeding with different diets, the oil red staining area of these two groups of mice was significantly different (P < 0.05). The area of oil red staining in the Litaf^−/− group was significantly less than that in the control group (P < 0.05), suggesting that there were significant differences in the average atherosclerotic plaque area among the two groups (P < 0.05).

Immunohistochemical staining results

The frozen sections of the aortic roots of each group were stained with α-actin, CD68, and Masson, respectively. As we known, α-actin is a surface marker of smooth muscle cells. After immunofluorescence staining, the cytoplasm of smooth muscle cells was red, and the more red staining, the more smooth muscle cells [13]. As is shown in Fig. 2, there were less smooth muscle cells in Litaf^−/− group in the control group (P < 0.05); CD68 was a macrophage-specific marker molecule, and the positive staining indicated the content of macrophages in the plaque [14]. As shown in the Fig. 2, the expression of CD68 in Litaf^−/− group was lower than that in control group (P < 0.05), which was consistent with the size of the atheromatous plaque. After Masson staining, muscle fibers were red and collagen fibers were blue [15], as shown in Fig. 2, the collagen fibers in Litaf^−/− group was more than that in the control group (P < 0.05).

Protein expression of NF-κB(P65)/IκB/caspase-3 in different groups

NF-κB pathway is one of the most common one related to inflammation, and P65/IκB is a key protein in this pathway [16]. Western blotting was employed to detect the expression of P65/IκB in the two groups. In addition, the expression of apoptosis related protein (caspase-3) was also detected. Compared with the control group, the expression of P65、IκB, and caspase-3 in Litaf^−/− group was much lower than in the control group (P < 0.05).

mRNA expression of P65/IκB/caspase-3 in two groups

Compared with the control group, the mRNA expression of P65/IκB and caspase-3 decreased significantly in Litaf^−/− group (P < 0.05). The result was almost the same with the result of Western blot.

Litaf protein is a newly discovered protein involved in the inflammatory response. At present, there are not many studies on whether Litaf is involved in the formation of atherosclerosis. In our study, Litaf knockout mice C57BL/6J^{− Litaf−} and C57BL/6J were cultured in the same conditions (normal diet, 12/24hours light and water unlimited) [17].After rearing for 16 weeks, the atherosclerotic plaque area and the proportion of related components in the two groups of mice were analyzed by oil red and immunohistochemical staining methods, and the role of Litaf in it was preliminarily analyzed; and the expression of NF-κB inflammatory pathway proteins was monitored by Western Blot and RT-PCR to further verify whether Litaf participates in and promotes the formation and progression of atherosclerosis through the NF-κB inflammatory pathway.

As can be seen in Fig. 1, after oil red staining, the red area, which also is the atherosclerotic plaques in the aortic root of mice was significantly different (P < 0.05), and in the Litaf^−/− group was significantly smaller than in the control group. We speculate that there may be the following situations: Litaf participates in the main inflammatory process of atherosclerosis, promoting arterial atherosclerotic plaque formation. As mentioned above, Litaf is an important protein of inflammatory response [5] [18], and should be involved in the inflammatory response process of atherosclerosis more or less. Litaf gene knockout inhibited the inflammatory response and inhibited plaque formation to a certain extent. Therefore, the atherosclerosis plaque areas in Litaf^−/− group should be smaller than in the control group. As we can see in Fig. 1, the plaque areas in the Litaf^−/− group are clearly smaller than in the control group, which is in consistent with our presume. CD68 is one of the molecular markers on the surface of macrophages. Macrophages after phagocytosis of lipids are the main components of atherosclerotic plaque [19]. α-actin is a specific molecular marker on the surface of smooth muscle cells. As shown in Fig. 1, the macrophages and smooth muscle cells in the Litaf^−/− group were more than those in the control group after staining by immunohistochemistry (P < 0.05). Masson staining makes collagen fibers appear blue and cells appear red, and collagen content is positively correlated with the stability of atherosclerotic plaques [20]. Our study detected that collagen staining in the Litaf^−/− group was higher than that in the control group (P < 0.05).

Then, what is the pathway through which Litaf participates in the inflammatory response and affects the formation of atherosclerotic plaques? Our previous study [10], has confirmed that NF-κB is the main inflammatory pathway involved in the formation of atherosclerosis, does Litaf regulate plaque formation through this pathway? Therefore, we extracted the plasma proteins and RNAs of the two groups of mice and performed Western and RT-PCR, respectively, to monitor the expression of NF-κB pathway proteins at different transcription levels. As can be seen in Fig. 2 and Fig. 3, the expressions of P65, IκB, and caspase3 are the same at the protein and RNA levels. And these results are consistent with the actual atherosclerosis formation and progression process, which is also giving us great encouragement.

Therefore, it is not difficult to conclude from the above analysis of the results that Litaf participates in the formation of atherosclerosis through the NF-κB pathway and promotes the progression of atherosclerosis. As for the role of Litaf in acute cardiovascular events, further research is needed to confirm.

Litaf may participate in the inflammatory immune response during the formation of atherosclerosis through the NF-κB pathway, and promote the formation of atherosclerosis. More research is needed to confirm whether Litaf is involved in the acute phase response of cardiovascular system lesions and the repair process after the acute phase.

PIGs: p53 induced genes

Litaf: LPS-induced TNF-α factor

Simple: Small integral membrane protein in late endosome

PAMPs: Pathogen associated molecular patterns

RT-PCR: Quantitative reverse transcription polymerase chain reaction

SD: standard deviation

Ethics approval and consent to participate:

Animal experiments were approved by the Experimental Animal Ethics Committee of Huazhong University of Science and Technology.

Consent for publication: Not application

Availability of data and materials: The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.Please contact email: [email protected] or [email protected]

Competing interests: The authors declare that they have no competing interests.

Funding: Not application

Authors^,contributions: LW and ZW analyzed data and performed Oil-red staining and immunohistochemical staining, and were major contributors in writing the manuscript. JX and LX raised the mice, isolated mouse aorta and heart, and performed frozen sections. DY and XH extracted plasma proteins and performed Western blot test. SJ and TG extracted plasma RNA and performed qt-PCR. TG, HL, and ZH prepared the antibodies and consumables required for the experiment, etc., reviewed and revised the manuscript. All authors read an approved the final manuscript.

Acknowledgements: Manhua Chen, Yumiao Wei

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Mice in the two groups were both treated with normal diet. Values are presented as the mean±SD, number: the number of mice in each group before investment. Number 16 weeks: the number of mice in each group after culturing for 16 weeks. Body Weight (g): the body weights of mice in each group before investment. Body weight 16weeks: the body weights of mice in each group after culturing for 16weeks. *P=0.056(Body weights), **P=0.053(Body weights after 16 weeks), ***P=0.055(Growth)

No competing interests reported.

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