Folic Acid Can Reduce Hyperhomocysteinemia-induced Vascular Damage by Immune/Inammatory Response in Spontaneously Hypertensive Rats

Hypertension associated with hyperhomocysteinemia (HHcy) is correlated with a high risk of vascular diseases. However, the mechanisms of HHcy-associated hypertensive vascular damage and the ecacy of folic acid (FA) as a treatment have not been fully elucidated. The aim of the present study was to evaluate the role of immune/inammatory molecules and oxidizing factors in HHcy-associated hypertensive vascular damage, and to observe the intervention effect of FA on the two vascular injury factors. folic adhesion in Wistar-Kyoto spontaneously hypertensive rat (SHR), HHcy + SHR and HHcy + SHR + folic acid (FA) group. Immunostaining of rat carotid artery (a) showed that the expression of VAP-1 (arrowheads) was signicantly increased in HHcy + SHR group. And the average optical (AO) value of VAP-1 (b) was also signicantly increased in HHcy + SHR group (P <0.05). But the AO value of VAP-1(b) was signicantly reduced in HHcy + SHR + FA group (P = 0.001). Values represent means ± SD (*P<0.05 vs. WKY group, $P< 0.05 vs. SHR group, &P< 0.05 vs. HHcy + SHR group, n=3)


Background
Hyperhomocysteinemia (HHcy) is de ned as a homocysteine (Hcy) level above 10 µmol/L in human blood [1] . And hypertension associated with HHcy accounts for 75% of hypertension cases in China [2] . With the ageing of society and the decrease in the dietary intake of fresh vegetables, the incidence of hypertension associated with HHcy remains high. HHcy, known as the "new cholesterol of the 21st century", is an independent risk factor for cardiovascular and cerebrovascular diseases [3] .
Hypertension is known to be associated with increased vascular in ammation [4] . Patients of hypertension associated with HHcy have a higher incidence of vascular events and disability [5] than those of pure hypertension, suggesting that HHcy can aggravate vascular in ammation in hypertension. Previous studies indicate that HHcy can decrease endothelium-dependent and blood ow-mediated vasodilation [6] , increase oxidative stress [7][8][9] and in ammatory reactions [10,11] , and is closely related to the thickening of the arterial intima-media [12] and intravascular thrombosis [13] . HHcy is the abnormal aggregation of Hcy that is an intermediate product linking methionine metabolism and transsulfuration [14] in the body.
Therefore, HHcy can be regarded as the marker of abnormal methionine and / or abnormal trans-sulfur metabolism in the body. Additionally, Hcy unique structure can interfere with amino acid metabolism and form Hcy-proteins affecting protein structure and function [15][16][17] . Hcy is also involved in FA metabolism and affects methylation [18] . Therefore, we hypothesize that the pathological mechanism of HHcy is manifested by more Hcy-proteins triggering an overreaction of the immune system, which leads to acute arterial dysfunction and chronic structural change in hypertension associated with HHcy.
FA is generally used to lower HHcy and is a component of anti-hypertensive drug combinations for hypertension associated with HHcy [2] . FA plays a key role as a donor of single carbons in DNA synthesis and repair [19] . As an important methyl donor, FA also participates in the processes of Hcy remethylation to form methionine [14] , indirectly affecting gene expression, protease function, and disease status through methylation. FA is also a powerful antioxidant [20] and closely associated with immune cells that highly express FA receptors [21,22] . Studies have found that FA supplementation can reduce the risk of cardiovascular and cerebrovascular events [23] . Therefore, we hypothesize that the vasoprotective mechanism of FA, in addition to reducing the negative effects of HHcy, also involves antioxidant effects and inhibition of immune/in ammatory overreaction.
In this study, normal blood pressure Wistar-Kyoto rats (WKYs) and spontaneously hypertensive rats (SHRs) were administered DL-Hcy intraperitoneally to mimic human HHcy and hypertension associated with HHcy [24] . Vascular expression of immune/in ammatory molecules such as nuclear factor-κ-gene binding (NF-κB), tumour necrosis factor-alpha (TNF-a), interleukin-6 (IL-6), vascular adhesion protein-1 (VAP-1), monocyte chemotactic protein 1 (MCP-1), and CD68 and oxidative factors nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (Nox) such as Nox2 and Nox4 were measured and compared, in order to gain insights on the immune/in ammatory mechanism of severe vascular damage in hypertension associated with HHcy and the protective effects of FA on hypertension associated with HHcy through inhibiting oxidative stress and excessive immune responses. were provided from the Beijing Vital River Laboratory Animal Center (Beijing, China) and had free access to chow and water. The animal room was controlled at a constant temperature (22 ± 2 °C), humidity, and 12 h light/dark cycle.

Experimental grouping
WKYs were randomly distributed into two experimental groups: WKY group and HHcy group, 8 rats in each group; SHRs were randomly distributed into three experimental groups: SHR group, HHcy + SHR group and HHcy + SHR + FA group, 8 rats in each group. WKY group and SHR group were administered physiological saline (PS, 5 ml/kg, twice a day) intraperitoneally for 12 weeks; HHcy group, HHcy + SHR group and HHcy + SHR + FA group were injected intraperitoneally with 2% DL-Hcy (5 ml/kg, twice a day, H4628, Sigma-Aldrich, St. Louis, USA) for 12 weeks. At the same time during the last 8 weeks of the experiment, HHcy + SHR + FA group was given FA (0.4 mg/kg/d, F7876, Sigma) by gavage; and the other four groups were given gavage of the same amount of PS. FA was freshly dissolved in 0.5 ml PS immediately before the experiment.

Blood pressure measurement
Blood pressure was measured by a tail-cuff method using non-invasive rat tail artery manometer (Beijing Ruolong Biotechnology Company, BP-2010A). At least three measurements were taken for each animal, each measurement was more than 5 minutes, and the mean of three measurements was calculated as the ultimate blood pressure.

Specimen collection
The rats were anesthetized by intraperitoneal injection of 10% chloral hydrate (0.3 ml/100 g) to collect blood from vena cava for measuring Hcy, SOD and MDA, then to harvest the thoracic aorta and bilateral carotid artery. Part of the arterial tissue was prepared by standard methods for morphometric and immunostaining analyses. And the remaining arterial tissue was immediately frozen in liquid nitrogen and stored at − 80 °C for measuring protein level by Western blotting and semiquanti cation of mRNA expression by quantitative real-time polymerase chain reaction (qRT-PCR).

Measurement of Hcy, MDA and SOD
The blood was drawn from vena cava of rats and sent to the laboratory of Qianfoshan Hospital. The concentration of plasma Hcy was measured by using a Cobas8000 automatic biochemistry analyzer (Roche, Switzerland). The activity of serum SOD and the level of serum MDA were determined using commercial kits (Jiancheng Institute of Biological Technology, Nanjing, Jiangsu, China) according to the manufacturer's instruction.

Histopathology and immunohistochemistry
Five-micron sections of formalin xed, para n-embedded aorta and carotid were stained with hematoxylin and masson to assess vascular pathology, vessel wall thickness and collagen deposition. Each sample slice was observed under the microscope (Olympus, Tokyo, Japan) at a magni cation of 200 ×.
Immunohistochemistry analysis for CD68 and VAP-1 was performed according to the manufacturer's instructions. Antigen retrieval was performed on serial artery sections and incubated with antibodies against CD68 (1:100, Wuhan Three Eagles, GB11067) and VAP-1 (1:100, Proteintech, ab181168), then the artery sections were incubated with a uorescent secondary antibody. The protein expression of CD68 and VAP-1 were assessed on a Nikon Eclipse Ti-E inverted epi-uorescent microscope (Nikon Instruments, Tokyo Japan). Brown areas were considered positive. And immunohistochemistry analysis for CD68 and VAP-1 was performed by Image-pro plus 6.0 (Media Cybernetics, Inc, Rockville, MD, USA).

qRT-PCR
Total RNA was extracted from the homogenate of fresh-frozen thoracic aorta without adipose tissue using TRIzol reagents (Invitrogen, 15596026). The RNA was quanti ed spectrophotometrically (Spectrophotometer, Merinton, SMA4000), and two micrograms of RNA was reverse transcribed into cDNA with RT reagent kit with gDNA Eraser (Takara, RR047A) for qRT-PCR. The cDNAs of MCP-1, VAP-1, TNF-α, IL-6, NF-κB p65/Rela, NF-κB2, Nox2, Nox4, transforming growth factor (TGF)-β1 and TGF-β3 were used to determine gene expression using TB Green Premix Ex Taq (Takara, RR820A) in a real-time PCR machine (ABI ViiA 7, Applied Biosystems, Foster City, CA). And the program was run with reaction cycling of initial denaturing (95℃, 30 s), followed by 40 cycles of denaturing (95℃, 5 s) and extension (60℃, 30 s). A melting curve was run to con rm speci city of PCR products. Gene expression levels were normalized to GAPDH expression levels. The relative quantity of mRNA expression was calculated according to the cycle threshold (2 −△△Ct ) method. Target genes for ampli cation are listed in Table 1.
The band intensities were determined using Image Lab software and expressed relative to GAPDH.

Statistical analysis
Statistical analysis was conducted using one-way ANOVA followed by LSD test. Results were expressed as means ± SE. All statistical analyses were performed using the SPSS software version 13.0. P < 0.05 was considered statistically signi cant.

Comparison of immune in ammation and oxidative stress indicators between groups
Levels of the mRNA relative expression of immune in ammatory molecules such as TNF-α, IL-6, NF-κB p65 / Rela, and NF-κB 2 , especially IL-6 and NF-κB p65 / Rela were signi cantly increased in arterial tissue homogenate of HHcy-associated hypertensive rats (P < 0.05). But FA treatment group showed signi cant reductions in the mRNA levels of immune in ammatory molecules, especially the decline of IL-6 and NF-κB p65 / Rela (P < 0.05). Levels of the mRNA relative expression of oxidative stress indicators (Nox2 and Nox4) were only an increasing trend in arterial tissue homogenate of HHcy-associated hypertensive rats, and FA treatment group did not show signi cant change in the mRNA levels of oxidative stress indicators (P > 0.05). Levels of the mRNA relative expression of TGF-β1 and TGF-β3 also showed an increasing trend in arterial tissue homogenate of HHcy-associated hypertensive rats, and the change of mRNA was also not obvious in FA treatment group (P > 0.05). Levels of the mRNA relative expression of adhesion factors (MCP-1 and VAP-1) also showed an increasing trend in arterial tissue homogenate of HHcy-associated hypertensive rats, and the change of mRNA was also not obvious in FA treatment group (P > 0.05). (Fig. 3) The protein relative expression levels of immune in ammation indicators (TNF-α and IL-6) were signi cantly increased in arterial tissue homogenate of HHcy-associated hypertensive rats (P < 0.05). But the protein relative expression of IL-6 showed signi cant reductions in FA treatment group (P < 0.05). The protein relative expression level of oxidative stress indicator Nox2 was also signi cantly increased in arterial tissue homogenate of HHcy-associated hypertensive rats (P < 0.05). And the protein relative expression of Nox2 also showed signi cant reductions in FA treatment group (P < 0.05). (Fig. 4)

Discussion
Studies have found that HHcy has a close relation with a variety of diseases [25] , such as cardiovascular disease, kidney disease, brain parenchymal disease, metabolic disease, and even tumors. The development of disease in HHcy involves endothelial dysfunction, thrombosis, thickening of blood vessel walls, oxidative stress, in ammatory responses, metabolic syndrome, and changes in disease-associated small mRNA [26] . Hcy is the intermediate product of methionine metabolism and transsulfuration [14] . An abnormal upstream methionine cycle affects the essential methylation process, leading to the high expression of disease-associated small mRNA, while aberrant downstream transsulfuration affects sul de metabolism, causing abnormalities in gas signal molecules and the redox state. In this study, WKYs and SHRs were administered DL-Hcy intraperitoneally to establish HHcy and hypertension associated with HHcy models. Based on the Hcy-protein antigen theory proposed by Fergusond [27] and the conclusion of Jakubowskie [28,29] that antigen-antibody interactions persist on the intima, this study aims to evaluate the hypothesis that HHcy aggravates vascular damage in hypertension associated with HHcy mainly through immune/in ammatory mechanisms.
Based on the premise that HHcy induces the abnormal activation of Hcy-protein antigen, which subsequently triggers an immune/in ammatory response, this study investigates, at the molecular level, the role of immunoin ammation and oxidative stress in the pathogenic mechanism of HHcy. We have found that vascular in ammatory factors such as TNF-a and IL-6 and classical immune/in ammatory pathway indicators such as NF-κB p65/Rela and NF-κB 2 were much higher at the mRNA levels in HHcyassociated hypertensive rats compared to the WKYs (P<0.05); The mRNA relative expression of IL-6 and NF-κB in HHcy-associated hypertensive rats was also signi cantly higher than that in SHRs (P<0.05). Additionally, vascular protein relative expression of TNF-a and IL-6 in HHcy-associated hypertensive rats was notably increased compared with that in normotensive rats (P<0.05); and IL-6 protein expression in HHcy-associated hypertensive rats was also higher than that in SHRs (P<0.05). We also studied the oxidative stress indicators of the Nox family [30] , which are highly expressed in blood vessels. The mRNA relative expression of Nox2 and Nox4 in HHcy-associated hypertensive rats trended towards an increase compared to that in normotensive rats but was not signi cantly higher than that in SHRs (P 0.05). Similarly, vascular Nox2 protein relative expression in HHcy-associated hypertensive rats was higher than that in normotensive rats but was not signi cantly different from that in SHRs (P 0.05). Therefore, HHcy signi cantly stimulates the intramural immune/in ammatory response, especially the NF-κB pathway, thereby aggravating arterial vascular injury, rather than directly increasing the expression of oxidative stress pathway molecules.
We also analyzed and selectively measured indicators for oxidative stress and in ammation in the blood circulation of HHcy-associated hypertensive rats and found that compared with SHRs, HHcy-associated hypertensive rats manifested higher levels of MDA, a serum oxidative product (P = 0.004), and lower levels of SOD, a serum antioxidant molecule (P = 0.000), suggesting that HHcy is associated with increased oxidative stress, which is in line with the pathogenic phenomena that Guo G [7] and Chan S [8] observed in HHcy. Previous studies also demonstrated that in ammatory indicators such as C-recreative protein [11] and the neutrophil/lymphocyte ratio [10] were signi cantly higher in patients with HHcy, which suggested that immunoin ammation did participate in the pathogenesis of hypertension associated with HHcy. The above ndings are pathological phenomena seen in HHcy or hypertension associated with HHcy, rather than su cient explanations or evidence regarding whether HHcy directly stimulates oxidative stress or induces in ammation; they also do not provide clues about which plays the dominant role, in ammation or oxidative stress? Thus, more convincing exploration at the molecular level is needed.
The persistent antigen-antibody interaction on the vascular intima inferred from the abnormal Hcy-protein antigen theory in the literature [31][32][33] and the stronger expression of immune/in ammatory molecules than oxidative stress pathway molecules observed in our study both support the HHcy-induced immune/in ammatory response playing a dominant role in vascular damage of hypertension associated with HHcy. Therefore, we performed immunohistochemical tests for CD 68 (an immune cell positive marker) and VAP-1 in the blood vessels in each group of rats and found that the arterial wall of HHcyassociated hypertensive rats had slightly more CD 68 -positive cells (P 0.05) and much higher VAP-1 expression (P<0.05) than did other groups, including the SHR group (P = 0.001). These results suggest that the expression of immune cells and immune cell adhesion factors in the arterial wall increases during hypertension associated with HHcy, which supports our hypothesis.
Viel EC [4] proposed that the degree of in ltration of immune cells is associated with the degree of blood pressure elevation. So, we compared the blood pressure between SHR group and HHcy + SHR group of more immune cell in ltration and in ammation. The SBP and DBP of HHcy-associated hypertensive rats were slightly higher than those of SHRs, but the difference was not statistically signi cant. We also compared the blood pressure in HHcy and WKY group and obtained similar results. We reviewed the relevant literature about the relation between HHcy and blood pressure. Some authors [34][35][36][37][38] stated that HHcy elevated blood pressure and was a pathogenic or risk factor for hypertension, while others [39] argued that HHcy could not raise blood pressure. Our ndings show that HHcy seems to in uence blood pressure but does not signi cantly increase it. We analyzed the possible reasons: the experimental period of our animal models was only 4 months, which is not enough time to mimic the end-stage vascular events of hypertension associated with HHcy; because of the sample size, our study does not have the same statistical impact that large clinical trials provide; thus, signi cantly elevated blood pressure caused by HHcy was not observed. The pathological analysis of blood vessels with HE and Masson stains also demonstrated that there was no noticeable difference between HHcy-associated hypertensive rats and SHRs. The arterial vascular pathology results for each group also support the above analysis.
We also studied the mechanism of FA's protective effects on the cardiovascular and cerebrovascular systems in hypertension associated with HHcy. Our ndings demonstrate that FA does reduce serum Hcy; however, it cannot completely counteract HHcy from external sources; that is, the Hcy level in HHcy + SHR + FA group was still higher than that in the SHR group (P<0.05), suggesting that the FA-dependent transfer of methyl groups, which promotes Hcy's conversion into methionine, can only partially reduce the negative effects of HHcy. As it is known that FA has antioxidant effects, we explored changes in oxidative stress levels and found that FA intervention in HHcy-associated hypertensive rats signi cantly reduced serum MDA levels (P = 0.000) and signi cantly increased serum SOD levels (P = 0.000); SOD was elevated so remarkably that it exceeded the baseline levels in the SHR group (P = 0.000). We also tested the effects of FA on oxidative stress and in ammatory responses at the molecular level and found that FA reduced the mRNA relative expression of IL-6 and NF-κB p65/Rela in HHcy-associated hypertensive rats (P<0.05), but did not change the mRNA relative expression of Nox2 and Nox4 (P 0.05). Experiments showed that the protein level of IL-6 and Nox2 was signi cantly reduced in HHcy + SHR + FA group (P<0.05). The above ndings suggest that the strong antioxidant effects of FA may not be entirely dependent on inhibiting molecules in the main oxidative stress pathway; there may be another independent antioxidative stress pathway that plays a role, such as the nuclear factor erythroid2-related factor2 (Nrf-2)/heme oxygenase-1(HO-1) pathway. Our team is currently studying how FA helps antioxidant stress via the Nrf2/HO-1 pathway. Meanwhile, we have also found that FA can regulate immune/in ammatory response, but the mechanism remains unknown and needs to be explored by further experiments.
In short, FA has a unique effect as a compound preparation for the treatment of hypertension associated with HHcy. FA can both ght HHcy to reduce its negative effects, and also protect tissue cells from damage through independent antioxidant capacity. In addition, FA may also regulate immune cells and inhibit the immune/in ammatory response that might play a leading role in the vascular damage of hypertension associated with HHcy.
The limitations of this study are that the experimental period for the HHcy model did not provide enough time to mimic the end-stage vascular damage in hypertension associated with HHcy; therefore, signi cant difference in the vascular pathology of HHcy-associated hypertensive rats and SHRs were not detected.
There may also be sampling bias regarding vascular pathology, PCR, and western blot (n = 3), which affects the interpretation of the ndings. The independent antioxidant effects of FA require further exploration at the molecular level. The potential effects of FA on immune/in ammatory response is a new research direction that we have discovered and requires veri cation by further research.

Conclusion
Immune/in ammatory response plays a leading role in arterial vascular pathology of hypertension associated with HHcy. The classic in ammatory pathway molecule NF-κB p65 / Rela can still be used as an intervention target for immune regulation. IL-6 can be a reliable observation target for immune/in ammatory response in hypertension associated with HHcy. FA has unique therapeutic value for HHcy, showing strong antioxidant capacity and potential immunomodulatory effects. Table 1 Primer sequences for quantitative real-time polymerase chain reaction (qRT-PCR) analysis from Takara.