Test objects and methods
Test subjects
Experimental case group
From January 2012 to October 2013, 36 patients with glioma who underwent neurosurgery in our hospital were included in the experimental group, aged from 12 to 65 years old, with an average of 39.6 years old, including 20 males and 16 females. Infectious diseases, neurological diseases, tumors, and autoimmune diseases were excluded, and their liver and kidney function were normal and they had no history of trauma or other chronic diseases. All cases were diagnosed as glioma patients by the pathology department of our hospital, and all glioma patients were divided into low-grade group (grade I-II) and high-grade group (grade III-IV) according to WHO brain tumor classification. In the experimental group, cerebrospinal fluid was collected 24 hours before, 24 hours after, 72 hours after, and 120 hours after surgery, respectively, in strict accordance with the lumbar puncture protocol. After the collection of cerebrospinal fluid, routine biochemical examination of cerebrospinal fluid was performed immediately, and the remaining specimens were centrifuged at 4°C for 10 min at 4,000×r/min, and the supernatant was collected and stored immediately in a refrigerator at -80°C (discarded if there were red blood cells). The control group was treated in the same way as the surgical group.
Normal control group
Eighteen cases of outpatient health check-up population in the same period were selected as the control group, aged from 26 to 65 years old, with an average of 38.8 years old, 10 cases of men and 8 cases of women. The differences in their gender composition and mean age were not significant compared with the experimental group (P>0.05).
Experimental methods
The determination of specimens was carried out strictly according to the instructions of the kit, and the correlation curve was plotted according to the difference between the OD value of each well of the reagent plate and the OD value of the blank well as the vertical coordinate, and the concentration of the standard as the horizontal coordinate; the concentration of the specimen to be measured was found according to its OD value.
Statistical treatment
All data were expressed as mean ± standard deviation (±SD), and the data were analyzed by the statistical software SPSS17.0. t-test and one-way ANOVA were applied to compare the differences between groups and within groups at different time points, and P0.05 represented statistical differences.
Results
Levels of IL-6, IL-10, MMP-2, and MMP-9 in CSF of glioma patients were significantly different between groups for all measurements compared with the control group
Differences in the levels of IL-6, IL-10, MMP-2, and MMP-9 in CSF of high-grade and low-grade gliomas
Among the 36 glioma cases diagnosed according to the WHO classification criteria for brain tumors (1997), 17 cases of low to moderate malignant glioma (hereafter referred to as grade I-II group) (including 14 cases of astrocytoma grade I and II, 1 case of ventricular meningioma, and 2 cases of oligoblastoma) and 19 cases of high malignant glioma (hereafter referred to as grade III or higher) (including 14 cases of astrocytoma grade III and IV, and 2 cases of mesenchymal ventricular meningioma) were diagnosed. (including 14 cases of astrocytoma grade III and IV, 2 cases of mesenchymal ventricular meningioma, 1 case of mesenchymal oligoblastoma, and 2 cases of medulloblastoma.)
The levels of MMP-2, MMP-9 and IL-6 in cerebrospinal fluid were significantly higher in the high-grade glioma group than in the low-grade group. The levels of IL-10 in cerebrospinal fluid were not significantly different between the high-grade group and the low-grade group.
Changes of IL-6, IL-10, MMP-2, and MMP-9 over time in glioma patients after surgery
The levels of MMP-9 in the cerebrospinal fluid of glioma patients measured in this experiment decreased rapidly within 24 hours after surgery; the levels of MMP-2 and IL-10 did not change significantly at different time points after surgery; the levels of IL-6 in the cerebrospinal fluid decreased gradually after surgery
Discussion
Glioma is the most common malignant tumor of the human central nervous system, which often requires multiple surgeries due to its easy recurrence and may leave serious disabilities such as hemiplegia and aphasia, mental retardation and psychiatric disorders after surgery, bringing endless pain and disaster to families. High-grade gliomas, such as glioblastoma and mesenchymal glioma, exhibit diffuse infiltrative growth and have an average survival time of just over one year. Conventional treatments such as surgery and radiotherapy are dedicated to shrink the tumor, and tumor recurrence is almost inevitable. It is a current research hotspot to investigate glioma biology from key features such as aggressiveness and immunosuppression as an entry point, rather than just from gross excision of the tumor; although great progress has been made in glioma research, the median survival has only improved by 9.10 weeks [2]. Therefore, it becomes urgent and necessary to find new therapeutic targets based on the molecular mechanisms of glioma development.
Expression and significance of IL-6 and IL-10 in glioma patients
The brain has long been considered an “immune immune immune organ” due to the relative impermeability of the blood-brain barrier to immune cells, the lack of a lymphatic system in the central nervous system, and the relative inactivity of microglia and intrinsic macrophages in the central nervous system. The presence of active immunity in the CNS would provide a new direction for the treatment of glioma, i.e., the development of more effective glioma treatment options using specific immunotherapy. For researchers, the ability of glioma immunotherapy to kill target glioma cells without damaging normal tissue appears more attractive and promising for research. However, recent phase I and phase II clinical trials conducted by Wheeler et al. have shown unsatisfactory treatment outcomes despite the safety of dendritic cell vaccines in patients with recurrent glioblastoma [3]. It is now known that the failure of immunotherapy and of the immune system to target tumor cells is mainly due to the dysregulation of some immune cytokines associated with glioma; these cytokines lead to immunosuppression and immune evasion in patients with glioma and promote tumor cell proliferation and angiogenesis, thus increasing tumor aggressiveness, promoting tumor progression, as well as leading to resistance to various treatments tolerance.
IL-6 and IL-10 have received much attention as new targets for the treatment of gliomas. In recent years, it has been shown that high-grade glioma cells secrete more of these cytokines than low-grade glioma cells and that there is a correlation between the expression level and the degree of tumor malignancy [4]. We compared the levels of IL-6 and IL-10 expression in the CSF of patients with different grades of glioma, respectively, and also found a correlation between their levels and the degree of malignancy. Recently, Samaras et al. provided immunohistochemical staining results showing that IL-6 expression in GBM is mainly confined to tumor cells and macrophages and is located within large ischemic necrotic areas.
IL-6 is a pleiotropic cytokine that functions in many physiological and pathological processes, including inflammatory responses, bone metabolism, C-reactive protein synthesis, and hematopoiesis. IL-6 is secreted by many cell types and acts on a variety of target cells, regulating cell development, differentiation, and induction of specific genes associated with several diseases. IL-6 is also a potential stimulator of VEGF expression, which is a major pro-endothelial cytokine in the development of glioma.
In this experiment, we measured IL-6 levels in cerebrospinal fluid of experimental and control groups by ELISA, determined its changes in different periods before and after surgery, and performed statistical analysis. The results showed that IL-6 levels were significantly higher in CSF of experimental group compared with control group, which confirmed the existence of abnormal activation of inflammatory factors in glioma patients and suggested a state of chronic inflammatory damage in the organism of glioma patients. We also compared the levels of IL-6 in CSF of patients with different grades of glioma, which showed a positive correlation with the degree of malignancy of glioma. The gradual decrease of IL-6 levels in cerebrospinal fluid after surgery may be due to a decrease in secretory release and an increase in catabolism, etc. Currently, the role of inflammatory cytokines in neurological injury and repair remains controversial.
Increased IL-10 expression has now been found in a variety of tumors, including gliomas. It has been demonstrated [5] that IL-10 is expressed at higher levels in malignant tumors than in benign tumors, and IL-10 was found to enhance the proliferation and invasiveness of glioma cell lines in in vitro experiments, and this effect was dose-related. They concluded that in glioma patients, the main sources of IL-10 are microglia and macrophages are rather than tumor cells [6]. Several studies have found that IL-10 reduces antigen presentation by monocytes by downregulating the levels of major histocompatibility antigen complex II (MHC II), thereby impeding antigen-activated proliferation of T cells and secretion of other inflammatory mediators. In animal models, IL-10, however, enhances specific antitumor immune responses, which has led to controversy about the role of IL-10 on the immune system. There are data demonstrating that IL-10 plays a very important role in the generation of anti-GBM effector T cells as an immunomodulator rather than an immunosuppressant [7].
In this experiment, IL-10 levels were measured by ELISA and statistically analyzed, and the results showed a significant increase in IL-10 levels in the cerebrospinal fluid of the experimental group compared to the control group, which also confirmed the presence of inflammatory factor activation in glioma patients. We also compared the level of IL-10 in CSF of patients with different grades of glioma, and the data showed no significant difference, it cannot be used as a reference indicator for tumor grade assessment. no significant change of IL-10 in cerebrospinal fluid of glioma patients before and after surgery for 120 hours may be due to its low clearance efficiency or continuous release, no change of its increase or significant decrease was found, or it may be that our study’s time was short and to be continued in the next step.
Expression and significance of MMP-9 and MMP-2 in glioma patients
MMPS is a family of proteases whose main role is to degrade structures such as extracellular matrix and basement membrane, and the participation of Ca2+ and Zn2+ plasma is required for this enzymatic function. The expression of MMPS increases significantly under various conditions such as inflammatory response and tumor invasion and metastasis, and many factors such as its expression level, regulation by TIMPS and other inhibitory factors and its own negative feedback regulation may affect the role of MMPS.
Although both MMP-9 and MMP-2 are classified in the matrix metalloproteinase family, their expression levels in the same glioma patients are very different. Our analysis of the reasons for this difference may be closely related to their different origins, but their exact origin is not clear and may also be related to factors such as molecular weight, release and clearance kinetics. One study [8] showed that their expression increases significantly after 2 h of cerebral ischemia-reperfusion injury, and we suggest that this protease can lead to the opening of the BBB by disrupting the tight junctions between endothelial cells and breaking down the basement membrane of brain microvessels. The disruption of the blood-brain barrier and the increased permeability of brain microvessels cause brain edema, and it can be hypothesized that MMP-2 and MMP-9 are closely related to clinical prognosis.
MMP-9 is the largest molecular weight enzyme in the family of MMPs and can catabolize various proteins such as gelatin, type IV and V collagen and elastin [9]. In this experimental study, we found that the level of MMP-9 in the cerebrospinal fluid of glioma patients was significantly higher than that of the control group, and the expression of MMP-9 in the high-grade group was significantly higher than that of the low-grade group, and the level of MMP-9 in the cerebrospinal fluid increased with the increase of tumor malignancy, and the expression of MMP-9 may become an important indicator of the malignancy, invasive ability, and prognosis of glioma.
It is noteworthy in the study of MMP-9 expression that its expression in cerebrospinal fluid decreases abruptly after surgery, a characteristic not shown in other biomarkers tested, which may be related to the release of tumor-bearing status and suggests that it may originate from the tumor cells themselves or from the tumor stimulating the production of surrounding normal cells. status is closely related and can be used as a reference indicator for prognosis of glioma patients. Whether it has correlation with tumor size needs further study, and if it has correlation, it will provide greater clinical reference value.
MMP-2 is an important member of the MMP family that hydrolyzes proteins such as elastin, laminin and fibronectin, which are the main components of the tight junctions between endothelial cells and the basement membrane of capillary walls. The breakdown of these proteins causes increased permeability of the basement membrane and disruption of the BBB structure, resulting in pathological damage such as brain tissue edema and increased intracranial pressure.
In this experimental study, we found that MMP-2 was lowly expressed in normal controls and significantly increased in gliomas, and the difference in expression between high-grade and low-grade groups was statistically significant, but the difference was not prominent, suggesting that MMP-9 may play a more important role in the aggressive growth of gliomas.
In conclusion, MMP-9, MMP-2, IL-6 and IL-10 may play a dominant or decisive role in the immunity and invasion of gliomas. The levels of IL-10 in cerebrospinal fluid of glioma patients were not significantly different between the high-grade and low-grade groups, and could not be used as a reference indicator of glioma grade, whereas the expression of MMP-2, MMP-9 and IL-6 were significantly different between the high-grade and low-grade groups, and could be used as a reference indicator of glioma tumor grade. MMP-9 levels in the cerebrospinal fluid of glioma patients decreased significantly within 24 hours after surgery, which may be related to the release of tumor-bearing status and can be used as a biochemical indicator to predict the clinical prognosis of patients.