Activating α7nAChR suppresses systemic inflammation by mitigating neuroinflammation of the medullary visceral zone in sepsis in a rat model

2.1 Animals and experimental model of sepsis

A total of 64 adult, specific pathogen-free, male Sprague-Dawley rats (weighing 250–280 g, 8 weeks old) were purchased from the Experimental Animal Center of Three Gorges University, License Number: SYXK (Hubei) 2018-0104. About five rats were housed in each cage, and they were maintained under specific pathogen-free conditions in the Laboratory Animal Center of the First Hospital of Guiyang. The feeding condition was kept as follows: the temperature was controlled at 21 ± 0.5, a 12 h light–dark cycle (lights on during 07:00–19:00) was set, and standard chow and tap water were provided ad libitum. All rats adapted to the experimental settings for 7 days prior to the treatment.

Sepsis models were prepared by cecal ligation and puncture (CLP) operation [19] under isoflurane inhalation for anesthesia [20]. The CLP operation process is as follows: cut the abdominal cavity about 2 cm to find and isolate the cecum, then ligate about 1/3 of the cecum with a 5–0 suture, and pierce the ligated cecum with a 21-G needle twice. Gently squeeze a small amount of intestinal contents from the puncture hole; at last, return the processed cecum into the abdominal cavity and suture the abdominal cavity. After the CLP operation, the rats’ bodies were curled up and inactive, and they breathed fast and had little and slow response to the outside world. These appearances indicated that the model is successful. Afterward, they were accepted piperacillin (50 mg/kg, qd × 3d) for controlling infection and reducing the mortality and saline (1 mL/100 g, qd × 3d) for fluid resuscitation by intraperitoneal injection [21]. The operation was finished under anesthesia with isoflurane inhalation [22]. All experimental procedures were carried out in compliance with the guidelines of the Institutional Animal Care and Use Committee (IACUC).

2.2 Animal grouping and treatment

After a week of adaptive feeding, rats were assigned randomly to five groups, they were separately treated as follows: (1) Control group (eight rats): no interventions were took. (2) Sham group (eight rats): rats were underwent open and suture of the abdominal cavity without CLP. (3) Model group (16 rats): the standard CLP operation as above-mentioned was implemented for each rat. (4) GTS-21 group (16 rats), after CLP operation, rats were accepted intraperitoneal injection of GTS-21, a specific agonist of α7nAChR, which was used as an activator of CAP, referring to the related studies [23,24], the dose and duration are 4 mg/kg, qd × 3d. (5) Methyllycaconitine (MLA) group (16 rats): after CLP operation, rats were accepted intraperitoneal injection of MLA, the antagonist of α7nAChR, which was used as a blocker of CAP, referring to the related study [25], the dose and duration are 4.8 mg/kg, qd × 3d.

2.3 Rats’ mortality rate and MSS

Rats’ mortality rates among different groups were recorded and analyzed with Kaplan–Meier survival curve. Moreover, murine sepsis score (MSS) was assessed. MSS was a cumulative point from each rat’s points of appearance, consciousness, behavior, response to stimuli, reaction of opening eyes, breathing frequency, and quality [26]. MSS can be used to judge if or not the model rat develops into sepsis rat and evaluate its severity of sickness. Generally, when a rat’s MSS was more than 4 points, we can think that it is a sepsis rat. In addition, the higher the score, the more serious the rat [27]. Three experimentalists scored every rats separately, and the average points were taken as the MSS of the rat.

2.4 Enzyme-linked immunosorbent assay (ELISA)

After 3 days of treatment, there were, respectively, 9, 8, and 11 rats who died in the model group, the GTS-21 group, and the MLA group. All the survival rats were sacrificed to collect blood and medullary tissue for analysis under anesthesia with isoflurane inhalation. For ELISA analysis, 2 mL of blood was taken from the right atrium of the surviving rats under isoflurane inhaling for anesthetization and preserved at room temperature, 2 h later, blood samples were centrifuged at 3,000 rpm for 10 min and the serum was collected for detection. 100 μL of serum were taken to the wells of these kits such as TNF-α (batch number: E-EL-R0019c; Elabscience), IL-6 (batch number: E-EL-R0015c; Elabscience), IL-10 (lot number: E-EL-R0016c; Elabscience), HMGB1 (batch number: E-EL-R0505c; Elabscience), and sCD14 (batch number: CSB-E11178r; Cusabio), respectively, According to the manufacturer’s instructions, after such steps as incubation, washing, reaction with working solution and densitometry, serum contents of TNF-α, IL-6, IL-10, HMGB1, and sCD14 were determined with ELISA.

2.5 Flow cytometry (FCM) analysis

For FCM, 2 mL of fresh blood was collected from the right atrium and centrifuged at 400–500×g at room temperature for 30 min to collect leukocytes for testing. After repeated centrifugation and dilution, 1 × 10⁶ lymphocytes were collected into flow cytometry tubes and the relevant fluorescent-labeled antibodies were added and incubated, including CD25-PE monoclonal antibody (Invitrogen), lot number: 12-0390-82; CD4-FITC monoclonal antibody (Invitrogen), lot number: 11-0040-82; and IL-17A-PE monoclonal antibody (Invitrogen), lot number: 12-7177-81. At last, after repeated centrifugation and washing three times, the samples were resuspended in 0.2 mL of phosphate buffer solution (PBS) containing 0.5% bovine serum albumin and analyzed by flow cytometry.

2.6 Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL)

After 3 days, rats were sacrificed under anesthesia with inhalation of isoflurane, then they were perfused with heparinized saline transcardiac for 60 min, and medulla oblongata was collected for TUNEL and fluorescent immunolabeling analysis.

In the rostral to caudal direction, there were transitional changes in the size, shape, and number of nuclei in the MVZ. From the plane of area postrema (AP) to the obex, the nucleus tractus solitarius (NTS) and vagus dorsal motor nucleus (VDMN) become larger, and NTS becomes wider and divides into multiple subnuclei. To ensure the accuracy and comparability of the results, we specified the exact site where the sections were taken. Combined with the purpose of this study, the MVZ slices were derived from the plane of the AP to the obex. The medulla oblongata was embedded in paraffin and cut into thin slices (30 μm).

TUNEL was used to observe the neuronal apoptosis in MVZ. The sections were dewaxed, gently rinsed with PBS, and immersed in proteinase K solution. Afterwards, samples were infiltrated in 100 μL equilibration buffer, the TdT buffer was added to incubate for 10 min at room temperature, then the sections were washed three times with PBS prior to 4′,6-diamidino-2-phenylindole (DAPI) (produced by Beyotime Biotechnology, batch number: C1002) were added to stain the nuclei of cells, at last fluorescence quencher (produced by Southernbiotech, batch number: 0100-01) were used to seal the sections. Fluorescent microscope (Olympus, BX53, Japan) was used to observe and take photos of these sections. ImageJ software (National Institutes of Health, V1.8.0.112) was used to calculate the apoptosis index (the average ratio of the apoptotic cells to the total cells) [28].

2.7 Immunofluorescence

Sections were deparaffinized, antigen repaired and blocked with goat serum (1 h), afterward, the primary antibodies were added to incubate at room temperature, and the primary antibodies include anti-Caspase 3 (produced by Wuhan Sanyan Bio. Co., China. Lot: 66470-2-IG, dilution: 1:50), anti-tyrosine hydroxylase (TH, produced by Wuhan Boster Co., China. Lot: BM4568, dilution: 1:50), or anti-choline acetyltransferase (CHAT, produced by Wuhan Bioss Co., China; lot: bs-2423R, dilution: 1:50). Fifteen hours later, fluorescent-labeled second antibodies were added to incubate at 37°C for 1 h, and fluorescent-labeled second antibodies include FITC-labeled goat anti-rabbit IgG (produced by Wuhan Boster Co., China; lot: BA1105, dilution: 1:100) or Cy3 labeled goat anti-mice IgG (produced by Wuhan Boster Co., China; lot: BA1031, dilution: 1:100). Then, sections were washed four times with PBS for 3 min eACh time, and DAPI was added and incubated in the dark for 5 min to stain nuclei of all cells. Leica TCS SL laser scanning confocal spectral microscope (Leica Microsystems) was used to scan the images, and the latter were analyzed with ImageJ (NIH, USA). Nine MVZ images with 400 folds’ enlargement from three rats of every group were analyzed with Image Pro Plus 6.0 (ipp6.0, Mediacybernetics) software [29] and the average optic densities were acquired.

2.8 Statistics

All the survival rats were involved in the laboratory analysis. The data were expressed as mean ± standard deviation (X̄ ± SD) and were analyzed with the SPSS 19.0 software package (SPSS Inc, Chicago, IL, USA). The homogeneity test of variables by Levene’s test is performed first. Analysis of variance was used to compare the differences between any two groups, and the homogeneous data were determined by the Bonferroni test; otherwise, they were judged by the Tamhane test. P < 0.05 was considered statistically significant.

1. Ethical approval: The research related to animals’ use has complied with all the relevant national regulations and institutional policies for the care and use of animals. This research was approved by the Committee on Protection, Welfare and Ethics of Experimental Animals in the First Hospital of Guiyang, Guizhou Pro., China (no. 20190107).

3.1 Mortality and MSS of rats among different groups

Despite strict disinfection and anti-infection measures being adopted, rats in the septic group still had a high mortality rate. Three days after the operation, there were, respectively, 9, 8, and 11 rats died in the model group, the GTS-21 group, and the MLA group, and the mortality rates were, respectively, 56.3, 50, and 68.8% in these groups. There was no rat died in the control group and the sham group (Figure 2a). The MSS scores of the model group (19.48 ± 2.815), the GTS-21 group (17.64 ± 2.468), and the MLA group (21.10 ± 3.194) were significantly higher than that of the control group (0) and the sham group (0.8750 ± 1.424), all the P values <0.0001, GTS-21 obviously reduced the MSS score of sepsis (P < 0.05), whereas MLA significantly increased the MSS of the GTS-21 group (P < 0.001). In addition, the scores of the same group on different days were not significantly different (Figure 2b).

Figure 2

Kaplan–Meier survival curve and MSS among different groups. (a) The Kaplan–Meier survival curve suggests that there was a significant difference in survival percentage among five groups (log rank test: χ ² = 15.43, P < 0.0001); however, there was no significant difference among the model group, the GTS-21 group, and the MLA group (χ ² = 1.21, P > 0.05). (b) The scatter plots of MSS show that the average MSS scores of the model group, the GTS-21 group, and the MLA group were much higher than those of rats in the control group. *P < 0.05; ***P ＜ 0.001.

3.2 Inflammatory cytokines levels and α7nAChR intervention

Inflammatory cytokines serum concentration (including TNF-α, IL-6, IL-10, sCD14, and HMGB1) in the model group, the GTS-21 group, and the MLA group were significantly higher than those in the control group and the sham group; for TNF-α, they are, respectively (pg/mL), 127.41 ± 13.23, 91.48 ± 13.18, 146.81 ± 18.61 vs 47.40 ± 8.31; for IL-6, they are, respectively (pg/mL), 99.41 ± 12.70, 81.56 ± 9.49, 108.75 ± 12.57 vs 39.16 ± 7.04; for IL-10, they are, respectively (pg/mL), 90.17 ± 18.77, 66.36 ± 18.55, 114.92 ± 18.61 vs 32.39 ± 7.78; for HMGB1, they are, respectively (pg/mL), 1098.46 ± 143.84, 859.65 ± 127.21, 1250.11 ± 215.91 vs 570.54 ± 101.67; for sCD14, they are, respectively (ng/mL), 367.86 ± 66.21, 290.62 ± 57.43, 481.65 ± 80.72 vs 184.70 ± 45.08, all the P values <0.01. All these cytokines show no difference between the control group and the sham operation group.

Besides IL-10 and sCD14, serum concentrations of TNF-α, IL-6, and HMGB1 in the GTS-21 group were significantly lower than those in the model group; they are, respectively: 174.86 ± 15.66 vs 210.56 ± 18.76; 91.48 ± 13.18 vs 127.41 ± 13.23; and 859.65 ± 127.21 vs 1098.46 ± 143.84, all the P values <0.01; all inflammatory cytokines levels reached the highest in the MLA group, and there were significant differences when compared to the GST-21 groups (P < 0.01 or P < 0.05) (Figure 3).

Figure 3

Concentration of serum cytokines among different groups of rats. TNF-α: tumor necrosis factor alpha; IL-6: interleukin 6; IL-10: interleukin 10; sCD14: soluble CD14, precepsin; HMGB-1: high mobility group box-1. (a–c) Serum concentration (pg/mL) of TNF-α, IL-6, and IL-10 among different groups; (d) serum sCD14 concentration (ng/mL) among different groups, usually as an inflammatory marker in early stage of sepsis; (e) serum concentration of HMGB1 (pg/mL), usually as an inflammatory marker in late stage of sepsis; sepsis causes serum level of inflammatory cytokines, including TNF-α, IL-6, IL-10, sCD14, and HMGB1 significantly increased; these cytokines levels in the model group, the GTS-21 group, and the MLA group were significantly higher than those in the control group and the sham group, except IL-10 and sCD14, GTS-21 significantly decreased the other cytokines’ level; all inflammatory cytokines levels reached the highest in the MLA group, and there were significant differences when compared to the GST-21 groups. *P < 0.05; **P < 0.01.

3.3 Immunity and α7nAChR intervention

In the model group, the GTS-21 group, and the MLA group, blood lymphocytes percentages of CD4⁺CD25⁺Treg and CD4⁺IL17⁺TH17 were significantly higher than those in the control group and the sham group, they were, respectively (%), 14.62 ± 0.78, 13.50 ± 0.58 and 16.57 ± 0.73 vs 9.30 ± 0.82; 2.92 ± 0.18, 2.11 ± 0.38 and 4.11 ± 0.34 vs 0.95 ± 0.12, all the P values <0.01. The ratios of Treg/TH17 in the model group, the GTS-21 group, and the MLA group were significantly lower than that in the control group; they were, respectively, 5.02 ± 0.57, 6.51 ± 0.86, and 4.06 ± 0.50 vs 9.82 ± 0.41, P < 0.05 or 0.01. GTS-21 evidently reduced the percentage of TH17 lymphocyte (2.11 ± 0.38 vs 2.92 ± 0.18, P = 0.02), but it neither reduced Treg lymphocyte percentage nor increased the ratio of Treg/TH17 significantly in sepsis, (13.50 ± 0.58 vs 14.62 ± 0.78, P = 0.62; 5.02 ± 0.57 vs 6.51 ± 0.86, P = 0.51); the percentages of Treg and TH17 lymphocytes in the MLA group were significantly higher than those in the GTS-21 group (P < 0.05 or 0.01) (Figure 4).

Figure 4

Blood lymphocyte percentages of Treg and TH17 among different groups. (a) Representative scatter plot images of CD4⁺CD25⁺Treg and CD4⁺IL-17⁺TH17 lymphocyte of different groups. (b) Histogram of double positive pencentage of CD4⁺CD25⁺Treg lymphocytes among different groups. (c) Histogram of double positive pencentage of CD4⁺IL-17⁺TH17 lymphocytes among different groups. (d) Histogram of the ratio of Treg/TH17 among different groups. Sepsis causes a significantly increase of Treg and TH17 lymphocytes and signifies activation of innate and acquired immunity. GTS-21 obviously decreases these two kinds of lymphocytes, especially TH17 lymphocyte percent, suggesting activation of CAP curbs the immunity, whereas MLA’s effect is the opposite, denoting that blocking CAP exaggerates the immunity. It can also be seen from the ratio of Treg/TH17. *P < 0.05; **P < 0.01.

3.4 The pathological features of MVZ

TNUNEL analysis shows that compared to the control and sham groups, there were much more apoptotic neurons in the septic groups, especially in the MLA group and the model group. GTS-21 had the tendency to reduce apoptosis of MVZ neurons in septic rats.

From fluorescence double-labeling analysis, the expression of TH obviously decreased in other four groups compared to the control group, and GTS-21 had the tendency to up-regulate it; on the contrary, MLA furtherly reduced it remarkably, whereas the expression of CHAT did not decrease significantly in sepsis except in the MLA group. The expression of Caspase 3 was evidently up-regulated in three septic groups when compared to the control group and the sham group, GTS-21 had the tendency to curb its expression, and MLA boosted its expression (Figure 5).

Figure 5

Pathological study of MVZ in sepsis and intervened by CAP (TUNEL and double immunofluorescence-labeling experiments). Figure 4 shows that sepsis induces MVZ neurons (including cholinergic neurons and catecholaminergic neurons) apoptosis and inactiveness. (a) TUNEL and double immunofluorescence labeling images of MVZ. The red fluorescence in the TUNEL column indicates apoptotic cells. In the TH/Caspase 3 column, the green fluorescence indicates the expression of TH, and the red fluorescence indicates the expression of Caspase 3 in the catecholaminergic neurons. In the CHAT/Caspase 3 column, the green fluorescence indicates the expression of CHAT, and the red fluorescence shows the expression of Caspase 3 in the cholinergic neurons. From these images, we can see that sepsis not only causes significant apoptosis of catecholaminergic and cholinergic neurons in MVZ but also affects their activeness. Intervention with CAP can affect the activity and apoptosis of functional neurons in the MVZ area: α7nAChR agonists GTS-21 have the tendency to prevent the cholinergic and catecholaminergic neurons from apoptosis and inactiveness in MVZ, while α7nAChR antagonist MLA significantly accelerates apoptosis and inactiveness of these two kinds of neurons. (b) The histogram of TUNEL analysis, which confirms that sepsis-induced significant apoptosis in MVZ, activating CAP had the tendency to reverse it; on the contrary, blocking CAP exacerbated the apoptosis. (c) The histogram of TH/Caspase 3 expressions in MVZ, which suggests that sepsis causes significant apoptosis of catecholaminergic neurons in MVZ; GTS-21 can reverse the situation, whereas MLA is the opposite; in addition, both sepsis and even sham operation cause significantly low expression of TH, especially when rats were treated by MLA. (d) The histogram of CHAT/Caspase 3 expressions in MVZ, which shows that even though sepsis caused significant apoptosis of cholinergic neurons in MVZ; it did not induce the significantly reduced expression of CHAT except in the MLA group.