miR-21 Regulates Immune Balance Mediated by Th17/Treg in Peripheral Blood of Septic Rats during the Early Phase through Apoptosis Pathway

Objective To study the mechanism by which miR-21 regulates the differentiation and function of Th17/Treg cells in sepsis. Methods A rat model with sepsis was made by cecal ligation and puncture (CLP). Then, some of the septic rats were transfected with miR-21 mimic or inhibitor by liposome. At 48 hours, lymphocytes and plasma from septic rats were isolated for further experimental detection. The expression of miR-21 in lymphocytes was detected by Polymerase Chain Reaction (PCR); the differentiation of Th17/Treg cells was counted by flow cytometry; lymphocyte apoptosis was observed by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) assay. The caspase-3/9 proteins were tested by Western blot; IL-10 and IL-17 were detected by enzyme-linked immunosorbent assay (ELISA). Results Compared with the sepsis group (SP group), the Th17 cells increased significantly, the Treg cells decreased significantly, the apoptosis rate of lymphocytes decreased significantly, the mRNA and proteins of caspase-3/9 decreased significantly, the IL-17 decreased, and the IL-10 increased in the sepsis group transfected with miR-21 (SP + miR-21 mimic group). After transfection of miR-21 inhibitor, the results were almost opposite to those of SP + miR-21 mimic group. Conclusions The differentiation and function of Th17/Treg cells were regulated by miR-21 in sepsis through caspase pathway.


Introduction
At present, sepsis is still one of the major diseases affecting the safety of human life in the world. In 2017, it was reported that 49 million cases of sepsis were recorded in the world, with 11 million deaths. is study pointed out that sepsis was one of the diseases with heavy medical burden in East Asia, Southeast Asia, South Asia, and South Africa [1]. Although the mortality rate of sepsis is very high, there is still no specific drugs that can effectively improve the prognosis [2]. It is widely known that "excessive inflammatory response" and "immune paralysis" are critical aspects in the pathophysiology of sepsis. Lymphocytes are very important in immune and inflammatory responses, especially 17/Treg cells. Studies in different fields have confirmed that 17/ Treg cells are closely related to inflammatory response. For example, Yan et al. have found that the imbalance between 17 and Treg cells was one of the causes of inflammatory enteritis.
17 promoted tissue inflammation, while Treg inhibited tissue immune response [3]. Moaaz et al. have also found that the imbalance of 17/Treg in autistic children led to the increase of proinflammatory factors and the decrease of anti-inflammatory factors [4]. Some scholars have proposed that the imbalance of 17/Treg cells led to the disorder of inflammatory response, which promoted the occurrence and development of chronic obstructive pulmonary disease [5]. Similarly, one study has shown that overexpression of TGF-β1 could affect the level of inflammation in the lung tissue of septic mice by regulating the imbalance of 17/Treg cells [6]. Another study also found that maresin-1 regulated the disorder of 17/Treg by increasing the number of Treg cells and reducing the number of 17 cells in the early stage of sepsis, so as to significantly inhibit the excessive inflammatory response in the process of acute lung injury and improve lung function [7]. With the development of the relevant research, the 17 and Treg cells have been gradually elaborated in sepsis [8,9]. e 17/ Treg ratio can change immune function and lead to the occurrence and development of sepsis [9,10]. erefore, regulating the 17/Treg ratio may be one of the effective therapeutic strategies of sepsis.
Many studies have pointed out that apoptosis of lymphocyte can lead to the impairment of immune cells in sepsis, such as T lymphocytes, B lymphocytes, and NK cells [11]. Meanwhile, other studies have shown that improving the apoptosis of immune cells can improve the survival rate in sepsis [12][13][14]. So, it is of great significance to regulate the 17/Treg cells ratio and promote the immune balance by preventing the apoptosis of lymphocytes in sepsis.
e researches have shown that microRNAs can regulate the differentiation and apoptosis of many cells [15,16] and are very important in controlling the development and function of immune cells [17]. miR-21 is one of the most studied members of microRNAs family. Early studies have shown that miR-21 has a positive regulatory effect on Treg cells in Kawasaki disease [18], thus affecting the 17/Treg ratio. Whether miR-21 can regulate the 17/Treg ratio and affect the apoptosis of lymphocytes in sepsis is still unclear. erefore, in our study, miR-21 was supposed to regulate the apoptosis of lymphocytes in sepsis, then changed the function and differentiation of 17/Treg cells, and ultimately improved the immune and inflammation status of sepsis.

Grouping of Septic Animal Models.
e animal experiment has been approved by the Ethics Committee of Bengbu Medical College (Ethics Statement Approval No.: 2018074). e model of cecal ligation and perforation was established in rats (CLP model); SD rats (male) weighing 200-250 g were selected; 5% Pentobarbital Sodium (5 mg/100 g) was injected intraperitoneally for anesthesia, and we found no signs of peritonitis, pain, or discomfort. After disinfection of the abdominal operation area, we cut the abdominal cavity about 2 cm along the abdominal white line to find out the cecum, ligated about one-third of the cecum with suture, punctured the ligated cecum twice with 21 g needle, and put the cecal ileum into the abdominal cavity and sutured. Finally, the rats were placed in the heating pad to maintain the anal temperature at 37 ± 0.5°C. e rats were divided into four groups by transfection in vivo, with eight rats in each group: control (NC rat) group, septic rat (SP rat) group, septic rats injected with miR-21 mimic (SP rat + miR-21 mimic) group, and septic rats injected with miR-21 inhibitor (SP rat + miR-21 inhibitor) group. e lymphocytes were also divided into four groups by transfection in vitro: NC group, SP group, SP + miR-21 mimic group, and SP + miR-21 inhibitor group. e rats were euthanized by cervical dislocation at the end of the study after the animals were anesthetized. We observed whether the breathing and heartbeat of the rats were completely stopped to confirm whether the rats died. e weight of the rats at the time of death was basically the same as that before the experiment (weighing 200-250 g). Based on the humane endpoints of the experimental animal center, we observed the animal performance every day during the experiment; if the rats were excessively listless or their body weight decreased >20%, we would terminate the experiment of the animals.

Isolation and Culture of Lymphocytes.
Lymphocytes in peripheral blood of rats were isolated by density gradient method. We used heart puncture blood collection method to collect the blood sample (2 ml) after the animals were anesthetized. After adding the lymphocyte isolation solution (USA, sigma) into the centrifuge tube, we carefully add the diluted peripheral blood (peripheral blood : dilution � 1 : 2), 1500 r/min; after centrifugation for 30 min, the lymphocytes above the isolation solution were obtained for subsequent culture. e lymphocytes were cultured in vitro with serum and RPMI-1640 medium.
e transfected liposome was Lipofectamine 3000 (lipo3000, Invitrogen L3000015). A suitable volume of serum-free medium and a suitable volume of transfection reagent were added to a transfection tube and cultured in incubator at 37°C for 48 h.

Western Blot Analysis.
e lymphocytes collected previously were lysed with lysis buffer, and the Bradford method was used to quantify the protein. e protein was electrophoretic by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE). e protein was transferred to the polyvinylidene fluoride membrane by electrospinning. Antibody was used to incubate overnight. We coupled antibody with horseradish peroxidase: Primary antibodies were caspase-3 (Abcam, ab184787, UK), dilution ratio: 1 : 2000, and caspase-9 (Abcam, ab184786, UK), dilution ratio: 1 : 1000. Secondary antibody was goat anti-rabbit IgG HRP (kgaa35, Jiangsu Kaiji Biotechnology Co., Ltd., China). Bio-Rad imaging was analyzed by quantity one software.

IL-17 and IL-10
Were Tested by ELISA. IL-17 and IL-10 in serum and cell culture medium were determined by using rat ELISA Kit (China Kaiji Biotechnology Co., Ltd, KGERC170-1, KGERC004-1), and the absorbance (OD value) of the above cytokines was measured by using microplate reader (MD SpectraMax M3, USA), and the corresponding concentrations were calculated.

Apoptosis of Lymphocytes Was Detected by TUNEL.
After the preparation of lymphocyte samples, they were immersed in paraformaldehyde solution overnight to improve the tissue permeability, and the samples were treated with methanol solution to inactivate enzymes. During TUNEL reaction, the samples were labeled with streptavidin TRITC working solution, and the lymphocyte samples were restained with DAPI working solution. After several times of washing with deionized water, the samples were observed with fluorescence microscope.

Detection of Caspase-3 and Caspase-9 Protein by
Immunofluorescence.
e separated lymphocyte was fixed with paraformaldehyde, inactivated with methanol solution, blocked with goat serum, incubated with primary antibody, and then incubated with FITC secondary antibody. DAPI staining solution was used to restain the cells, the caspase-3 and caspase-9 proteins were observed under fluorescence microscope, and photos were taken for preservation.

Statistical Analysis.
e data shown represent the mean ± standard; t-test or ANOVA was used in our research. Statistical significance was expressed by p value ( * p < 0.05 and * * p < 0.01).

Changes of CD4+T, 17, Treg Cells, and Inflammatory
Factors. We firstly found that the ratio of CD4+ T lymphocytes of septic rats was decreased when compared with the NC group [t value � 14.213, p < 0.01] (Figure 1(a)). Compared with the NC group, the ratio of 17 of septic rats increased, while the ratio of Treg decreased [t value � 5.687, p < 0.01; t value � 5.482, p < 0.01] (Figures 1(d) and 1(e)). Compared with the NC group, IL-17 in the peripheral blood of septic rats increased significantly, while the level of IL-10 decreased significantly [t value (IL-17) � 16.892, p < 0.01; t value (IL-10) � 16.312, p < 0.01] (Figures 1(b) and 1(c)).

Changes of Apoptosis of Lymphocytes.
As reported, apoptosis is related to the differentiation and function of lymphocytes [9,10]. So we detected apoptosis of lymphocytes by flow cytometry. We found that the apoptosis rate of lymphocytes of septic rats increased [t value � 16.387, p < 0.01] (Figure 2(a)). It is well known that the caspase family is responsible for selectively cleaving certain proteins, resulting in apoptosis. We detected the levels of caspase-3/9 mRNA by PCR. Compared with the NC group, the expression of caspase-3/9 mRNA in lymphocytes of septic rats increased significantly [t value (caspase-3) � 26.246, p < 0.01; t value (caspase-9) � 18.489, p < 0.01] (Figures 2(b) and 2(c)).

Changes of miR-21 in Lymphocytes.
As reported, miR-21 is closely related to apoptosis in medical fields [19][20][21][22]. We detected the expression of miR-21 by PCR. We found that the expression of miR-21 in lymphocytes of septic rats was significantly decreased [t value � 23.037, p < 0.01] (Figure 3).

Observation of Lymphocytes Transfected with miR-21.
To observe whether miR-21 can regulate the differentiation and function of lymphocytes, we firstly need to establish a successful model of miR-21 transfection into lymphocytes in sepsis. In this study, we used miR-21 mimic and inhibitor for transfection into lymphocytes in sepsis. Compared with NC group and sepsis group, the expression of miR-21 in sepsis + miR-21 mimic group was increased, while the relative expression of miR-21 in sepsis + miR-21 inhibitor group was significantly reduced [t value (NC versus SP) � 5.436, p < 0.01; t value (miR-21 mimic versus SP) � 23.988, p < 0.01; t value (miR-21 inhibitor versus SP) � 18.393, p < 0.01] (Figure 4).

miR-21 Regulated the Differentiation and Function of 17 and Treg Cells.
To observe the effect of miR-21 on lymphocytes in sepsis, we firstly enhanced or decreased the expression of miR-21 by lentivirus transfection. Next, we counted the 17 and Treg cells ratio by flow cytometry again. e proportion of 17 cells was significantly reduced, while the proportion of Treg cells was significantly raised in sepsis + miR-21 mimic group. ere was no difference in the ratio of 17 cells, while the ratio of Treg cells was significantly reduced in sepsis + miR- 21 (Figures 6(a)-6(c)).

Discussion
Sepsis is a common disease in the ICU, with the characteristics of high mortality [23]. " e early inflammatory storm" theory cannot fully explain the whole pathophysiological process of sepsis, and various anti-inflammatory treatments alone can not improve its prognosis, so "immune paralysis" theory has gradually been widely valued by scholars [24]. Some scholars have proposed that apoptosis of immune cells induced by sepsis is one of the pathophysiological mechanisms of immunosuppression [2]. A study showed that T cell response was significantly reduced in patients with posttraumatic sepsis, in which Treg was significantly increased and 17 was significantly reduced [10]. is result showed that the imbalance of 17/Treg cells led to the disorder of inflammatory response and finally led to the occurrence of immunosuppressive state in sepsis. One basic experimental study also found that the level of Treg cells in the serum of septic mice increased, and the corresponding inflammatory factors secreted were significantly excessive. It was further found that knockout of TLR4 could In in vitro culture, the protein levels of caspase-3 and caspase-9 in eight-paired NC rat group, SP rat group, SP rat + miR21 mimic group, and SP rat + miR21 inhibitor group were determined by Western blot. NC group: normal control group; SP group: sepsis group; SP rat + miR21 mimic group: sepsis group with miR-21 activator transfected into lymphocytes; SP rat + miR21 inhibitor group: sepsis group with miR-21 inhibitor transfected into lymphocytes. * * p < 0.01.
reduce Treg activity and restore the balance of proinflammatory cytokines to improve immune paralysis [25]. erefore, one of the recent studies about the treatment of sepsis mainly focuses on how to regulate the immune balance, that is, the so-called "Immunomodulatory erapy" [26]. For example, Zou found that miR-126 could control the inflammatory response in peripheral blood by regulating the balance of 17/Treg cells. In our study, in early stage of sepsis, 17 cells increased significantly in the peripheral blood of rats, while Treg cells decreased significantly. e imbalance of 17/Treg ratio is consistent with Zou's research [27]. We also found that IL-17 increased, while IL-10 decreased, indicating that this study is in line with "excessive inflammatory response" in early stage of sepsis.
One of the early characteristics of sepsis is that a large amount of lymphocyte apoptosis leads to decline in the number of lymphocytes [12,[28][29][30][31]. is study confirmed the same conclusion that the count of CD4+ T lymphocytes of septic rats reduced significantly. Similarly, clinical studies have found that the apoptosis of lymphocytes with sepsis patients increased significantly, while the lymphocyte count decreased significantly [32]. In order to find out the cause of the decline in the number of T lymphocytes, we found that the apoptosis rate of T lymphocytes in septic rats increased significantly. It is well known that the most important pathway related to apoptosis is caspase signaling pathway. Caspase family belongs to cysteine protease, which is responsible for selectively cleaving some proteins, resulting in apoptosis. Caspase-3 can directly degrade the structural and functional proteins in cells, thus causing apoptosis. After stimulation, caspase-9 can be activated by self-cutting and then cause caspase cascade reaction. In this study, we found that numerous apoptotic lymphocytes appeared, and caspase-3 and caspase-9 significantly increased, indicating that caspase pathway is involved in apoptosis of lymphocytes. Most scholars believe that apoptosis of lymphocyte is one of the causes of "immune paralysis" in sepsis, but some scholars have questioned this view [33][34][35][36][37]. In the early stage of sepsis, we found that inflammatory factors in peripheral blood were released in large quantities and apoptosis of lymphocytes appeared. Combined with the pathophysiological process of sepsis, the body was in the stage of "excessive inflammatory response" in the early stage. We also confirmed that the release level of IL-17 increased significantly but that of IL-10 decreased significantly, which was related to the imbalance of 17/Treg ratio. is shows that the proportion of 17/Treg cells is changed by apoptosis of lymphocyte in early sepsis, which makes the body in the stage of "excessive inflammatory response." Similarly, in late sepsis, we guess that the proportion of 17/Treg was affected by apoptosis of lymphocyte, which makes the body in the stage of "immune paralysis." is may be the reason why simple anti-inflammatory treatment in the early stage of sepsis has not achieved good curative effect. rough our study, we speculate that the proportion of 17/ Treg should be balanced by immunotherapy in the early or late stage of sepsis, so as to make the body in the stage of "controllable inflammatory response" in the early stage and avoid "immune paralysis" in the late stage.
Many research works have explored miR-21 involvement in many aspects of sepsis, such as inflammation and MODS (liver, kidney, and lung) [38][39][40]. Our study proved that the 13   expression of miR-21 in lymphocytes of sepsis also changed significantly, so miR-21 may regulate the immune cells, thereby affecting the immune state. Clinical studies found that miR-21 was negatively correlated with inflammation and disease severity in sepsis [41]. Animal model validation also obtained the same results; that is, miR-21 reduces the production of proinflammatory factors through its downstream pathway and reduces the level of inflammatory response in sepsis, thereby reducing tissue damage and organ dysfunction [40]. In this study, we also found that the increase of miR-21 can lead to the change of inflammatory factors in peripheral blood of septic rats, such as IL-17 and IL-10. is study further found that miR-21 moderates the inflammatory response through affecting the differentiation of 17/Treg; that is, the increase of miR-21 can reduce the count of 17 cells but increase the count of Treg cells, resulting in different proportions of inflammatory factors secreted by them. Some articles have demonstrated that miR-21 participates in the process of apoptosis; for example, knockout of miR-21 gene can increase the apoptosis of renal tubular endothelial cells induced by ischemia-reperfusion [42]. With the increase of miR-21, the apoptosis of lymphocytes was significantly reduced, and caspase-3 and caspase-9 proteins were significantly decreased, and vice versa. ese results indicate that miR-21 in lymphocytes of sepsis affects the differentiation of 17/Treg cells through apoptosis pathway, thereby affecting inflammatory response and immune function.
In conclusion, in the early stage of sepsis, the body is in the stage of excessive inflammatory response, which may be caused by the imbalance of lymphocyte subsets ( 17/Treg cell ratio). e apoptosis process involved in caspase pathway changes the whole process of differentiation and function of 17/Treg cells. Regulating apoptosis of lymphocyte to improve immune balance may be the key to improve the inflammatory response in the early stage of sepsis and may also be one of the means to prevent "immune paralysis" in the late stage of sepsis. Our study found that miR-21 exhibited a certain effect on differentiation and function of lymphocyte subsets in sepsis and can affect the inflammation and immune balance of sepsis. It can be used as one of the markers of sepsis and a potential target for sepsis treatment, However, in order to achieve the balance of 17/Treg cells in sepsis, the opportunity of miR-21 activation or inhibition is unclear and needs to be confirmed by further research.

Data Availability
e data used to support the findings of this study are included within the manuscript , and if needed more, can be asked to submit more by corresponding author.

Conflicts of Interest
e authors declare that there are no conflicts of interest.