| Traumatic brain injury(TBI) is the most common diseases in neurosurgery, and is one of the leading reasons of death and disability for people under the age of 45 years. TBI is based on primary injury and gradually developed a mainly secondary brain injury of the sum of a series of complex molecular cascade events, and eventually lead to brain edema, neuron death,movement and impaired cognitive function of severe clinical disease. The mechanism of secondary damage including neuroinflammation, the accumulation of free radicals, calcium overload, mitochondrial dysfunction and cell apoptosis, however, evidence shown that aseptic nerve inflammation and excessive autophagy of neurons after TBI is also as an important factor of aggravating brain damage, which caused more attentions of researcher.TLR4 is a kind of transmembrane signal receptor by mediate natural immune, which associated with ligand binding involved in neuroinflammation regulation by launch a series of molecular cascade in the nervous system injury. Autophagy is a lysosomal degradation pathway which degrades damaged or superfluous amino acids, old organelles and intracellular pathogens. In recent years, autophagy is also known as a kind of innate immune defense mechanisms, while TLR4 may be served as a previously unrecognized environmental sensor for autophagy. Studies have shown that TLR4 via identifying ligand-LPS can trigger autophagic response in macrophages, while whether excessive activation of autophagy is associated with the activation of TLR4 after TBI; and whether TLR4 inhibitors can alleviate neurons autophagy after TBI and the downstream signaling molecules of TLR4, MyD88, TRIF is involved in the regulation of neurons autophagy. This paper is divided into three parts to elaborate the idea.Part one Changes of TLR4 expression and its roles in rats following TBIObjective: Application of electronic controlled cortical impact(eCCI)injury method reproduced TBI model by male SD rats, and to detect changes of TLR4 expression and its roles in hippocampal area by treatment TLR4 inhibitors TAK-242 following TBI.Methods: A total of 96 male SD rats were used and divided into four groups by randomized block method: Sham group, TBI group, DMSO group,and TAK-242 group. TBI models were reproduced according to the eCCI injury method. Each group was further divided into 6 h, 12 h, 24 h and 48 h subgroups after TBI, and to observe the following indexes: H ﹠ E stains observed pathological morphological changes of brain tissue. The cellular localization and expression of TLR4 was observed in hippocampal area by immunohistochemistry. The mRNA expression of TLR4 was observed in hippocampal area by Real-time fluorescence quantitative PCR. The expression of TLR4 protein was evaluated by Western blotting. Brain water content was measured by wet/dry method. Another 20 rats undergo morris water maze test to detect changes of the ability of learning and memory in space at 8, 9, 10 days. Using Image Proplus 6.0, Image J and Image Lab 4.1software analysis system for quantitative determination, and all data were expressed as mean ± standard error by SPSS 17.0 statistical software. P value of less than 0.05 was considered statistically significant.Results: 1 H ﹠ E staining: The histomorphology of brain tissue was normal and number of neurons cells was much in sham group. While in TBI and DMSO group there was obvious tissue edema at trauma focal and periphery, neurons swelling, nucleolus shrinkage and hyperchromatic significantly, a larger gap existed around cells, vasodilation and neuronal degeneration and necrosis in hippocampal area. When treated with TAK-242,these changes were weakened at the corresponding time points after TBI(P <0.05); 2 TLR4 results in immunochemistry: TLR4 was mainly located in the membrane of neural cells in hippocampal area. In sham group, we can occasionally see positive cells, and the positive cell was light stained.Compared with sham group, TLR4 positive cells began to increase significantly, color deepened and immune reactivity enhanced in TBI and DMSO group(P < 0.05). The immunoreactivity of TLR4 declined significantly in TAK-242 group(P < 0.05); 3 The Realtime PCR results:Compared with the sham group, the expression of TLR4 mRNA was significantly increased in the hippocampal in TBI group and DMSO group(P< 0.05), while the expression of TLR4 mRNA significantly decreased by treatment with TAK-242(P < 0.05); 4 Western blot results: Sham group has only a little expression of TLR4 protein and there were no changes in each point. Compared with sham group, the expression of TLR4 protein began to increase obviously at 6 h with peaking at 24 h after TBI, and still higher than the basic level till to 48 h in TBI and DMSO group(P < 0.05). While treated with TAK-242, expression of TLR4 protein was weakened compared with TBI and DMSO group at corresponding time points(P < 0.05); 5 Brain water content measurements: There was no difference in each point on brain tissue water content in sham group. Compared with sham group, the brain water content was significantly increased in TBI and DMSO group(P < 0.05), but the comparison of data in TBI group and DMSO group had no statistically significant(P > 0.05). TAK-242 treatment significantly attenuated brain water content compared with the TBI group; 6 Morris water maze results: Morris water maze test was performed at 8-10 days after TBI. The average latency in searching safety island was significant increase in TBI and DMSO groups compared with the sham group(P < 0.05). Search moving tracks and average latency was improved significantly at 8-10 days after TBI in TAK-242 group than TBI and DMSO group(P < 0.05).Summary: ECCI method has been applied successfully to establish the rat model of focal TBI. The level of TLR4 expression was significantly increased in hippocampal area after TBI in rats, and which aggravates the secondary brain injury. Treatment with TAK-242 can reduce the activation of TLR4, attenuate brain edema, improve learning and memory ability and afford neuroprotective effects.Part two The effect of TLR4 antagonist on neuronal autophagy in hippocampal area after TBI in ratsObjective: To investigate activation of neuronal autophagy in rat hippocampal area after TBI, and whether or not TLR4 regulates expression levels of neuronal autophagy in the hippocampus.Methods: The eCCI injury method was used to reproduce TBI model. A total of 95 male SD rats were randomly divided into four groups: sham group,TBI group, 3-MA group and TAK-242 group. Each group was further divided into 6 h, 12 h, 24 h and 48 h subgroups after TBI, and to observe the following indexes: Transmission electron microscopy was used to detect ultrastructure changes of the hippocampus nerve celland autophagosome. The cellular localization and expression of LC3 and Beclin1 were observed by immunohistochemistry. The autophagy related protein, LC3 and Beclin1 were tested in hippocampal area by Western blot. The colocalization of LC3 and NeuN(Neuronal marker) in hippocampal area was observed by immunofluorescence microscope. Quantitative analysis and statistical method are the same as mentioned in part one.Results: 1 Transmission electron microscopy results: There were lots of swelling and degeneration of mitochondria around the nucleus, and can detected autophagosome which was double-membranes structures surrounding the injured mitochondria at 24 h after TBI; 2 LC3 and Beclin1 results in immunochemistry: LC3 was mainly located in the cytoplasm of in hippocampal neural cells. In sham group, we can occasionally see positive cells, and the positive cell was light stained. The positive cell of LC3 was increased significantly at 6 h after TBI, color deepened and immune reactivity enhanced, with peaked at 24 h, then slightly decreased at 48 h, but still higher than sham group(P < 0.05). When treated with 3-MA or TAK-242, expression of LC3 was weakened compared with TBI groups at corresponding time points(P < 0.05). Beclin1 was also mainly located in the cytoplasm of in hippocampal neural cells and occasionally see positive cells in sham group.Compared with sham group, the positive cells of Beclin1 were increasedsignificantly at 6 h after TBI, color deepened and immune reactivity enhanced,and maintain higher expression levels to 48 h(P < 0.05). Compared with TBI group, expression of Beclin1 was weakened in 3-MA and TAK-242 groups at corresponding time points(P < 0.05); 3 LC3 results in western blot: There were no changes of LC3 Ⅱ /LC3 Ⅰ in each point in sham group. Compared with sham group, the LC3Ⅱ/LC3Ⅰratio in TBI group began to increased with the passage of time gradually and maintain higher expression levels until 48 h(P < 0.05). When treated with 3-MA or TAK-242, these changes were weakened at corresponding time points than TBI group(P < 0.05); 4 Beclin1 results in western blot: The sham group had only a little expression of Beclin1 and there were no changes at corresponding time. The protein expression of Beclin1 was increased significantly at 6 h after TBI, with peaked at 24 h, then slightly decreased at 48 h, but still higher than sham group(P < 0.05).Compared with TBI groups, the protein expression of Beclin1 were significantly decreased at each time points in 3-MA and TAK-242 groups; 5LC3 and NeuN(Neuronal marker) fluorescent double labeling results in hippocampal area: In hippocampal CA1 area of TBI group, we can see there were large number of overlapping of red(LC3) and green(NeuN) fluorescent,presented as orange color at 24 h, suggesting that autophagy is located in neurons. While when treated with 3-MA or TAK-242, red fluorescent(LC3)was significantly weaker and the overlapping of red and green fluorescent decreased signifcantly, illustrating that the level of neuronal autophagy in hippocampal area decreased.Summary: Neuronal autophagy could be detected in hippocampal area at6-48 h after TBI. Treatment with TAK-242 or 3-MA can exert the same effect,which significantly attenuate the level of neuronal autophagy expression.Therefore, TLR4 might mediate autophagy expression of hippocampal neurons through some way after TBI.Part three Study on the molecular mechanisms on neuronal autophagy in hippocampal area by TLR4 mediated MyD88 / TRIF pathway after TBIObjective: MIP, MyD88 inhibitory peptide, and Resveratrol, TRIF inhibitor, were used as treatment drugs. To identify whether or not TLR4 mediated MyD88 or TRIF signal transduction pathways are involved in neuronal autophagy in hippocampal area after TBI.Methods: TBI model was used as mentioned in part one. A total of 85 male SD rats were randomly divided into five groups: sham group, TBI group,TAK-242, MIP group and RV group. Each group was further divided into 12 h,24 h and 48 h subgroups after TBI, and to observe the following indexes: The cellular localization and expression of MyD88 and TRIF were observed by immunohistochemistry. The protein expression of TLR4, MyD88, TRIF and Beclin1 were tested in hippocampal area by Western blot. The colocalization of TLR4, Beclin1 and NeuN(Neuronal marker) in hippocampal area was observed by immunofluorescence microscope. Quantitative analysis and statistical method are the same as mentioned in part one.Results: 1 MyD88 and TRIF results in immunochemistry:MyD88 was mainly located in the cytoplasm of in hippocampal neural cells. In sham group,we see a small amount positive cell, and the positive cell was light stained.Compared with sham group, the positive cell of MyD88 was increased significantly at 12 h after TBI, color deepened and maintain higher expression levels to 48 h(P < 0.05). Compared with TBI group, expression of MyD88 was weakened in TAK-242 and MIP groups at corresponding time points(P <0.05). That in RV group was similar to TBI group, and there were no significantly difference between the two groups(P > 0.05). TRIF was mainly located in the cytoplasm of in hippocampal neural cells. In sham group, we can occasionally see positive cells, and there were no changes at corresponding time. Compared with sham group, we see a large number of TRIF-positive cells at each time points in TBI group, color deepened and the immunoreactivity enhanced(P < 0.05). That in TAK-242 and MIP groups were similar to TBI group, and there were no significantly difference between the two groups(P > 0.05). That in RV group was similar to TBI group.Compared with TBI groups, expression of TRIF was weakened in RV groupsat corresponding time points(P < 0.05); 2 TLR4 results in western blot: The sham group had only a little expression of TLR4 and there were no changes at corresponding time. Compared with sham group, the protein expression of TLR4 were increased significantly in TBI group, and maintain higher expression levels until 48 h(P < 0.05). Compared with TBI group, the protein expression of TLR4 was significantly decreased at each time points in TAK-242 group(P < 0.05). That in MIP group and RV group was similar to TBI group, the protein expression of TLR4 was higher levels at each time points; 3 MyD88 results in western blot: The sham group had only a little expression of MyD88 and there were no changes at corresponding time.Compared with sham group, the protein expression of MyD88 was increased significantly in TBI group(P < 0.05). Compared with TBI group, the protein expression of MyD88 was significantly decreased at each time points in TAK-242 and MIP group(P < 0.05). That in RV group was similar to TBI group; 4 TRIF results in western blot: The sham group had only a little expression of TRIF and there were no changes at corresponding time.Compared with sham group, the protein expression of MyD88 was increased significantly in TBI, TAK-242 and MIP groups(P < 0.05), but there were no significantly difference between any two groups(P > 0.05). That in RV group was similar to sham group; 5 Beclin1 results in western blot: The protein expression of Beclin1 was relatively low in sham group and there were no changes at corresponding time. Compared with sham group, the protein expression of Beclin1 were increased significantly at 12 h after TBI, and maintain higher expression levels until 48 h(P < 0.05). Compared with TBI group, the protein expression of Beclin1 was significantly decreased at each time points in TAK-242 and MIP group(P < 0.05), there were no significantly difference between the two groups(P > 0.05). That in RV group was similar to TBI group; 6 TLR4 and NeuN(Neuronal marker) fluorescent double labeling results in hippocampal area: we can clear see the red fluorescence of TLR4 labeled by DyLight 594 accumulated in the cytomembrane and the green fluorescence of NeuN labeled by DyLight 488 located in neuronalnucleus. In hippocampal CA1 area of TBI group, we can see there is a large number of overlapping of red and green fluorescent, presented as orange color at 24 h. While when treated with TAK-242, the overlapping of red and green fluorescent decreased significantly; 7 Beclin1 and NeuN(Neuronal marker)fluorescent double labeling results in hippocampal area: the red fluorescence of Beclin1 labeled by DyLight 594 accumulated in the cytoplasm and the green fluorescence of NeuN labeled by DyLight 488 located in neuronal nucleus. In hippocampal CA1 area of TBI group, we can see red fluorescent of Beclin1 increased significantly, and overlapping of red and green fluorescent,presented as orange color at 24 h. While in TAK-242 group and MIP group,red fluorescent of Beclin1 was decreased significantly. That in RV group was similar to TBI group, overlapping presented as orange color, illustrating that TAK-242 and MIP can inhibit neuronal autophagy.Summary: TLR4 mediated MyD88/TRIF signal pathways were activated after TBI in rats, which common involved in the secondary brain damage. TAK-242 reduced neuronal autophagy via TLR4/MyD88/Beclin1 signaling pathways in hippocampal area after TBI.Conclusions: The activation of TLR4 aggravated secondary brain injury after TBI in rat, and TAK-242 can alleviate neuronal autophagy in hippocampal area via TLR4/MyD88/Beclin1 signal pathways, attenuate brain edema, improve the learning and memory ability and could afford neuroprotective effects. |
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