Xing-Yue Fang, Qi-Bing Liu, Xiao Yang, Guo-Hui Yi, Hao-Fei Fan, Jing Huang?

1. School of Basic and Life Science, Hainan Medical University，Haikou，571199

2. The first Affiliated Hospital of Hainan Medical University, Haikou，571199

3. Science Experiment Center of Hainan Medical College University，Haikou，571199

Keywords:THB OGD LPS Protective effect of cell stress

ABSTRACT Objective: To investigate the effects and mechanisms of tetrahydroberberine (THB) on vascular endothelial cell injury induced by oxygen glucose deprivation (OGD) and LPS-induced cell injury model. Methods: MTT assay was used to detect the effects of THB final concentration on the survival rate of EA.hy926 cells, which were 0 μg/mL, 2.5 μg/mL, 5 μg/mL, 10 μg/mL, 20 μg/mL and 40 μg/mL, respectively. Group I of the experiment: normal control group, OGD group, OGD+THB-10 μg/mL group, OGD+THB-20 μg/mL group, OGD+THB-40 μg/mL group; Experimental group II group: normal Control group, LPS group, OGD+ LPS group and OGD+ LPS+THB-20 μg/mL group. The relative expression of inflammation-related factors such as Caspase-1, ASC, IL-1β and IL-6 was detected by fluorescence quantitative PCR, and the expression of IL-1β and HO-1 was detected by Western Blot analysis. Results: The results of MTT assay showed that THB had no effect on the survival of EA.hy926 cells. The qPCR results of the experimental group I and II showed that the OGD model could play an antiinflammatory protective role by down-regulating the expression of Caspase-1, ASC, IL-1β and IL-6 genes. The anti-inflammatory effect of THB on the OGD model was general, but it was found that THB also exerted an anti-inflammatory effect by down-regulating the expression of inflammatory genes by OGD+ LPS group compared with OGD+ LPS+THB-20 μg/mL group. Western Blot analysis showed that the expression of IL-1β protein was consistent with the results of qPCR. The expression of HO-1 protein as a protective factor was contrary to that of inflammatory factor fluorescence quantitative analysis. Conclusion: OGD model can improve the protective effect of cell stress. THB plays an anti-inflammatory role by down-regulating the expression of Caspase-1, ASC, IL-1β and IL-6 genes, up-regulating the expression of HO-1 protein and inhibiting the activation of NLRP3 inflammatory bodies.

1. Introduction

Stroke is a common and frequently occurring disease in the world. In the world, stroke is the second death disease[1] and the third disability disease[2], which brings great burden to society and individuals. Among them, ischemic stroke is the most common type of stroke, accounting for 60-80% of all stroke. Vascular endothelial cell (VEC) injury due to lack of glucose and hypoxia is the first event of cerebral ischemia. In the process of ischemia-reperfusion injury, oxidative stress is closely related to a series of inflammatory reactions, which play a key role in vascular endothelial cell injury. At the same time, lipopolysaccharides (LPS) can induce dysfunction of vascular endothelial cells, promote inflammatory damage and oxidative damage of vascular endothelial cells [3-4].

Tetrahydroberberine (THB) is mainly found in corydalis and other Corydalis plants. It can also be reduced by berberine. Recently, it has been found that this kind of alkaloid has obvious cardiovascular effect, can block dopamine receptor in brain, and has central stabilizing effect. THB also has unique pharmacological properties on cardiovascular system, such as its ability to resist a variety of experimental arrhythmias [5], and its protective effects on myocardial [6], brain ischemia [7] and reperfusion injury. THB has also been reported to have certain antioxidant and anti-inflammatory activities, and is expected to be developed as an anti-inflammatory drug [8]. In this study, THB was used to induce vascular endothelial cell injury model induced by OGD and LPS, and to explore the intervention effect and mechanism of THB on human umbilical vein endothelial cell EA.hy926 injury.

2. Materials and Methods

2.1. Experimental Materials

EA.hy926 was purchased from Shanghai cell bank; LPS (Sigma, USA); DMEM medium (Gibco, USA); Fetal bovine serum (Gibco, USA); Trypsin (Sigma, USA); penicillin (Gibco, USA); Streptomycin (Gibco, USA); THB (China); RNA rapid Extraction Kit (Thermo, USA); Reverse transcription PCR Kit (Thermo, USA); Fluorescence quantitative Kit (Vazyme, China); DMSO (Sigma, USA); Antibody actin (Bi Yuntian, China); Anti-IL-1β (Santa Cruz biotechnology, USA), Anti-HO-1 (Cell signaling technology, USA); The primer sequence was synthesized by Biotechnology (Shanghai) Co., Ltd. see Table 1.

2.2 Instrument

CO2cell incubator (Thermo, USA); Confocal fluorescence microscope (LEICA, Germany); Enzyme-linked immunoassay (Thermo, USA);Cell culture bottle (Thermo, USA);Low speed centrifuge (Eppendorf, Germany);Programmed cell freezer (Thermo, USA).

2.3 Method

2.3.1 Cell culture

Cells were subcultured in DMEM medium containing 10% fetal bovine serum, penicillin and streptomycin 10 KU/L and cultured in 37 ℃ and 5% CO2saturated humidity incubator. Cells were subcultured within 20 generations and logarithmic growth stage cells were used for experiment.

2.3.2 MTT assay of tetrahydroberberine cytotoxicity

The cells were inoculated to a 96-well plate with a cell density of 105/mL, and then placed in a 37 ℃ incubator after the plate was laid. The cells were incubated until the bottom of the hole was full, the complete medium was absorbed, and the complete medium with final THB concentration of 0, 2.5, 5, 10, 20, and 40 μg/mL was added for 24 hours. Six rewells were set for each mass concentration gradient.

Add 20 μL MTT solution (5 mg/mL, 0.5% MTT) to each well and continue to culture for 4 h. Take out the cells and use all media in the pore carefully (contact with bottom deposits should be avoided).150 μL DMSO was added to each well, and the OD (absorbance) value of each well of the 96-well cell culture plate was determined at 490 nm wavelength of the enzyme-linked immunoassay (ELISA) after the 96-well cell culture plate was shaken on the shaking table for 10 min. Cell viability =OD experimental group/OD control group ×100%.

2.3.3 Establishment and grouping of OGD model

The OGD model was established [9-10], cells were collected from cell culture to logarithmic stage, cell density was adjusted to 105/mL, 2 mL/ well tiled in a 6-well plate, 5% CO2, incubated at 37℃, and cells were covered with 80% of the bottom of the 6-well plate. The final mass concentration of 0, 10, 20, 40 μg/mL THB predressing was added for 1 hour, and 3 rewells were set for each concentration gradient. Wash the cells with PBS for 3 times, then add Hanks buffer containing the corresponding final concentration above THB, transfer the 6-well plate to the saccharoxy-oxygen deprivation tank, adjust the pressure valve, and let in the mixed gas containing 95% N2+5% CO2at a flow rate of 20 L/min. After 5 min, remove the saccharoxy-oxygen deprivation tank to a 37 ℃ incubator for 6 hours. The medium was replaced with a complete medium containing the corresponding final concentration above THB, and the complex sugar and oxygen culture was continued for 24 hours (to simulate the reperfusion process).

Group I:

(1) normal control group: do not add THB, do not conduct OGD model, that is, blank control.

(2) OGD group: do not add THB, only conduct OGD model.

(3) OGD+ THB-10 μg/mL group: add 10 μg/mL THB and conduct OGD model.

(4) OGD+ THB -20 μg/mL group: add 20 μg/mL THB and conduct OGD model.

(5) OGD+ THB -40 μg/mL group: add 40 μg/mL THB and conduct OGD model.

2.3.4 Establishment of LPS damage model and grouping treatment

On the basis of Ma Ming-ming's LPS injury model [4], seed plate culture was carried out according to step 1.2.3 until the cells were monolayer covered with 80% of the bottom of the well, and the vascular endothelial cell injury model was established with the final concentration of 1 μg/mL of LPS injury for 30 h, namely the LPS treatment group.

Group II:

(1) Normal control group: no LPS, no OGD model, no THB, i.e. blank control.

(2) LPS group: add LPS (1 μg/mL), no OGD model, no THB.

(3) OGD + LPS group: add LPS (1 μg/mL) for OGD model without THB. In OGD treatment, LPS (1 μg/mL) was always contained in the culture medium.

(4) OGD + LPS + THB -20 μg/mL group: Add 20 μg/mL THB, add LPS (1 μg/mL) to make OGD model. When the final concentration of THB was 20 μg/mL and applied for 1 h in advance, LPS (1 μg/mL) and THB (20 μg/mL) were always contained in the culture medium when OGD was applied.

2.3.5 Fluorescence quantitative PCR detection

The RNA of each treatment group was extracted by RNA rapid extraction kit (thermo). The RNA was reversed into cDNA by reverse transcription PCR kit. Caspase-1 (cysteinyl aspartate specific protein-1), ASC (apoptosis associated specific like protein) IL-1β（Interleukin-1β）,IL-6（Interleukin-6）were detected The expression levels of these inflammation related genes were detected by fluorescence quantitative Kit (Vazyme) according to the primers in Table 1.

For the specific operation of RT-PCR, please refer to the instructions. The reaction conditions are: 42 ℃, 60 min; 70℃, 15 min, 4 ℃ storage. QPCR system refers to the specification, and the reaction procedure is as follows: 94 ℃, 3 min; 94 ℃, 15 s, 58 ℃, 30 s, 72 ℃, 30 s, 40 cycles; 72 ℃, 10 min. In the annealing step, fluorescence signals were collected, and each template repeated 3 parallel amplification.

Table 1 Primer sequence of quantitative detection of inflammation related factors by fluorescence

2.3.6 Western Blot analysis

The total protein of each group was extracted by RIPA Kit (Bi Yuntian), and the total protein concentration was determined by BCA protein quantitative kit. After 30 g protein on each swimming lane, SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was applied to the membrane on PVDF membrane, then sealed with 1 h at room temperature of 5% skim milk, 3 times TBST, 5 min each time, then anti- IL-1 beta (1: 500) and min were added respectively. Anti actin (1:1000) and anti actin (1:10000) were incubated overnight at 4 ℃. TBST was washed for 3 times, 5 min each time, and the corresponding secondary antibody (1:1000) was added. After incubation for 2 h at room temperature, TBST was washed for 3 times again, 5 min each time. ECL chemiluminescence kit was used to develop color. Super sensitive multi-functional imager was used to collect protein bands, and quantity one software was used to analyze the protein bands.

2.4 Statistical analysis

SPSS software was used to analyze and process the original data, and graphpad prism 8.0.2 was used to map and analyze the data. The statistical data was expressed as mean ± standard deviation. The method of paired t test was used for experimental comparison, and the difference was statistically significant with P ＜ 0.05.

3. Results

3.1 Cytotoxicity of THB on EA.hy926 cells

MTT colorimetry was used to detect the cell activity of each group. The experimental results showed that the cell survival rate of 2.5 μg/mL THB group was 96.1 ± 4.5% slightly lower than that of control group. The activity of 5, 10, 20 and 40 μg/mL THB cells of other groups were 98.1 ± 1.2%, 98.8 ± 0.6%, 101.1 ± 0.9%, 103.2% ± 3.2% similar to that of control group (Figure 1). Therefore, within the error range of data, THB of different concentrations has no effect on cell survival and no cytotoxicity.

Fig. 1 Effect of THB at different concentrations on the activity of EA.hy926 cells (Each column represents the mean ± SD，n=6.)

3.2 Expression results of inflammation related genes

3.2.1 Caspase-1 and ASC gene expression

Paired t test was used to compare the QPCR results with the Control group. In the results of group I (figure 2, 3- I), the expression levels of Caspase-1（OGD+THB-0 μg/mL t=16.0, P＜0.01）and ASC（OGD+THB-0 μg/mL t=24.3, P＜0.01）were significantly reduced by OGD treatment. At the same time, the relative mRNA expression levels of Caspase-1（OGD+THB-40 μg/mL t=13.5, P＜0.01 and ASC（OGD+THB-40 μg/mL t=32.8, P＜0.01）showed an overall trend of decreasing with the increase of THB concentration. It is speculated that OGD treatment can stimulate the stress protection mechanism of cells, THB has a certain protective effect on cells, OGD treatment can slightly weaken the stress response, but the protective effect increases with the increase of concentration. In the results of group II (Fig. 2, 3- II), there was no significant change in the expression of Caspase-1 (t = 0.28, P ＞ 0.05) and ASC (t = 1.1, P ＞ 0.05) in LPS group. The relative expression of Caspase-1 (t = 8.7, P ＜ 0.05), ASC (t = 9.9, P ＜ 0.05) in OGD + LPS group was higher than that in LPS + OGD + THB-40 μg/mL group (t = 11.2, P ＜ 0.01) and ASC (t = 12.9), P ＜ 0.01), indicating that THB has anti-inflammatory effect.

LPS did not play an inflammatory role by activating the inflammatory body of NLRP3 (NLR family pyrin domain containing 3), so there was no significant change in the expression of caspase-1 and ASC under LPS treatment. As caspase-1 and ASC are important components of NLRP3 inflammatory body, they all show a consistent downward trend under the action of THB, so we can speculate that the anti-inflammatory mechanism of THB may be related to the activation and assembly of NLRP3, and the specific mechanism of action remains to be explored.

Fig. 2 Effect of THB on Caspase-1 gene expression in EA.hy926 cells (Each column represents the mean ± SD，n=3，compare with control group：*P＜0.05,**P＜0.01）

Fig. 3 Effect of THB on ASC gene expression in EA.hy926 cells (Each column represents the mean ± SD，n=3，compare with control group：*P＜0.05,**P＜0.01）

3.2.2 IL-1 β and IL-6 gene expression

IL-1 β and IL-6 are common inflammatory cytokines, their expression is generally closely related to inflammation. By detecting the relative expression of IL-1 β and IL-6, we can infer the severity of cell inflammation.

The QPCR results of IL-1 β and IL-6 were compared with Control group in two ways respectively. The data were processed by paired t test. In the results of experiment group I (figure 4, 5- I), the expression levels of IL-1β in OGD treatment (OGD+ THB-0 μg/mL t=14.6, P ＜ 0.01) and IL-6 (OGD+THB-0 μg/mL t=25.1, P ＜ 0.01) were significantly reduced. At the same time, the expression levels of IL-1 β(t=19.5, P ＜ 0.01), IL-6 (t=25.3, P ＜ 0.01) in OGD+ THB-20 group and IL-1β（t=14.6, P＜0.01), IL-6(t=14.6, P＜0.01) were lower than those in OGD group, and the anti-inflammatory effect of 20 μg/mL THB was the best.

In the results of experiment group II (figure 4, 5- II), the expression levels of IL-1 β (t=70.5, P ＜ 0.001), IL-6 (t=16.4, P ＜ 0.01) were significantly increased in LPS group. The relative expression levels of IL-1β (t=27.2, P ＜ 0.01) and IL-6 (t=9.9, P ＜ 0.01) in OGD+LPS group were significantly higher than that of IL-1β (t=11.2, P ＜ 0.01) and IL-6 (t=11.8, P ＜ 0.01) in LPS+OGD+THB-20μg/Ml group. THB has anti-inflammatory effect.

Fig. 4 Effect of THB on IL-1β gene expression in EA.hy926 cells (Each column represents the mean ± SD，n=3，compare with control group：*P＜0.05,**P＜0.01 ,***P＜0.001）

Fig. 5 Effect of THB on IL-6 gene expression in EA.hy926 cells (Each column represents the mean ± SD，n=3，compare with control group：*P＜0.05,**P＜0.01）

3.3 Western blot results

The protein expression of HO-1 (heme oxygenase-1) and IL-1β showed that OGD could alleviate the inflammatory injury induced by LPS by up regulating HO-1 and down regulating the expression of IL-1β. THB also plays an anti-inflammatory role by up regulating HO-1 and down regulating the expression of IL-1 β protein.

Fig. 6 Effect of THB on the expression of HO-1 and IL-1β in EA.hy926 cells

4. Discussion

At present, the classic in vitro OGD model simulating human stroke is used in many researches to simulate human cerebral infarction by depriving cells of nutrition supply and oxygen [9-10]. Ischemiareperfusion injury is an important cause of aggravation in patients with cerebral infarction, which is accompanied by activation of NLRP3 inflammatory body and further aggravation of cell damage [11]. The regulation of NLRP3 inflammatory corpuscles includes transcription and posttranscriptional level. The transcription is induced by Toll like receptor / nuclear factor - κ B (TLR / NF - κ b) pathway, which activates NLRP3 inflammatory corpuscles by up regulating the expression of NLRP3. The posttranscriptional level is transduced by PAMP and damp, which activates NLRP3 inflammatory corpuscles by initiating the assembly of NLRP3 multi protein complex.

NLRP3 is a member of the pattern recognition receptor nod like receptor family in the cytoplasm. When the cells are stimulated by infection, injury, hypoxia, etc., the activated NLRP3 can recruit pro-caspase-1 through the binding protein ASC to form NLRP3 inflammatory complex. Under the action of inflammatory complex bodies, pro-caspase-1 is activated as caspase-1, and the precursor of IL-18 and IL-1 β is cleaved into important bioactive inflammatory factors, participating in the inflammatory response [12]. The core protein of NLRP3 inflammatory body is NLRP3 protein, which has the function of exogenous and endogenous signal receptors. It can recognize a variety of stimuli, such as ATP [13], β - amyloid protein [14], influenza virus [15]and collect ASC and caspase-1 to assemble and activate NLRP3 inflammatory body, and then activate caspase-1 to form NLRP3 / ASC /Caspase-1 cascade reaction pathway, release IL-1β, IL-6 and other inflammatory factors, mediate tissue inflammatory damage [16]. After LPS stimulation, the secretion of TNF - α, IL-6, IL-1 β and other inflammatory factors can be increased by activating TLR4 / NF - κ B signaling pathway, resulting in the inflammatory damage of vascular endothelial cells [17]. In addition, it has been reported that berberine can inhibit the expression of IL-1, IL-6 and the activation of NF - κ B, at the same time, it can reduce LPS induced injury by increasing SOD activity [18-20]. The mechanism of THB improving the injury of vascular endothelial cells provides theoretical basis.

Studies have shown that inhibition of HO-1 will lead to upregulation of the expression of inflammatory factor NF-κB, which will further aggravate the inflammatory injury. Therefore, the presence of high levels of HO-1 in the serum can alleviate the inflammatory stress injury caused by cerebral hemorrhage [21]. According to Li Minzhang et al[22], the primary cortical neurons pretreated with sevoflurane had a very significant neuroprotective effect when they were exposed to OGD model, and the inflammatory damage was significantly lower than that of the control group. This effect is related to the reduction of oxidative stress by Erk1/2-HO-1 signaling pathway.

The results showed that the decrease of the increase of HO-1 by the inflammatory factors Caspase-1, ASC, IL-1 β and IL-6 after OGD model was probably due to the stress protection mechanism of endothelial cells. When OGD is damaged, cells can reduce their damage in many ways, that is, to enter the stress state to protect cells. X. Wu found that by releasing H2, cells could induce the expression of PINK1 protein (pten-induced putative kinase protein 1) and Parkin protein, and reduce ROS damage and cell apoptosis in the OGD model [23]. In addition, Yu Diao and other researchers also found that when neurons suffer from OGD damage, they can also reduce the production of no by increasing the expression of KCNQ / M channel opener, so as to reduce the damage of OGD on cells. Their study also found that KCNQ / M channel will be activated after OGD injury, and the activation of this channel significantly reduces the damage to neurons, but the detailed mechanism remains to be studied [24]. In addition, Tan Ting and others have found that when neurons are injured by cerebral ischemia-reperfusion (OGD), the Ca2+in cells will become unbalanced, which will lead to endoplasmic reticulum stress. At this time, cells can spontaneously carry out unfolded protein response to endoplasmic reticulum stress. In addition, it can also regulate the imbalance of intracellular Ca2+through TRPC ion channel, so as to reduce the damage of OGD and protect neurons [25]. In conclusion, the experimental results of the decrease of inflammatory factors caspase-1, ASC, IL-1 β, IL-6 and the increase of inflammatory protective factor HO-1 in OGD model are probably due to the stress protective mechanism of endothelial cells when they are injured by OGD. THB may play an anti-inflammatory role by up regulating the expression of inflammatory protective factors such as HO-1, down regulating the expression of inflammatory factors such as IL-1 β, IL-6, down regulating the expression of genes related to NLRP3 inflammatory bodies such as caspase-1 and ASC, inhibiting the activation and assembly of NLRP3 inflammatory bodies, but its specific regulatory mechanism needs further study.