C1q/CTRP1 exerts neuroprotective effects in TBI rats by regulating inflammation and autophagy

Objective C1q/CTRP1 is a newly discovered adiponectin protein, which is highly expressed in adipose and heart tissues. Recent studies have revealed that C1q/CTRP1 can regulate metabolism and inhibit inflammation. CTRP1 is also expressed in brain tissues and vascular cells of human and rat, and research on cerebral hemorrhage and cerebral ischemia-reperfusion injury demonstrates that the CTRP family can attenuate secondary brain injury and exert neuroprotective effects. Thus, this study was designed to explore the role of CTRP1 in traumatic brain injury (TBI) and the underlying mechanism. Main methods Rats were assigned into rCTRP1 group, vehicle group, and sham group. Modified Feeney’s method was used to establish a closed traumatic brain injury model. Morris water maze was used for directional navigation, reverse searching and space exploration tests in rats. In addition, Golgi-Cox staining was utilized to visualize neurons, dendrites and dendritic spines. ELISA was conducted to detect the levels of inflammatory factors (IL-6 and TNF-α). Finally, Western blot was adopted to detect the relative expression of p-mTOR and autophagy-related proteins (Beclin-1 and LC3-II). Results CTRP1 improved the behavioral and histopathological outcomes, inhibited the inflammatory response, activated mTOR and decreased autophagy-associated protein synthesis in TBI rats. Conclusion CTRP1 exerts neuroprotective effects in TBI rats by regulating inflammation and autophagy and has potential therapeutic properties after TBI.


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Traumatic brain injury (TBI) is the main cause of traumatic death and disability globally. The common 39 causes of TBI include violence, traffic accidents, tumble, falls, and sports accidents [1]. TBI is generally 40 divided into primary and secondary brain injury. To be specific, primary TBI is caused by direct external 41 force acting on the central nervous system. Primary TBI often leads to neurometabolic disorders, 42 hippocampal synapse damage, neuron, astrocytic degeneration (CA1/CA3 layer, dentate gyrus) and 43 glutamate excitotoxicity. In addition, with the subsequent destruction of the blood-brain barrier (BBB) 44 and the persistence of neurogenic inflammation [2], there are secondary changes in nerve tissues, 45 including a series of pathophysiological changes, energy metabolism disorders and inflammatory 46 response [3][4][5], which generally aggravate neuronal necrosis, dendrite and synapse damage [2]. Both primary and secondary TBI can directly cause cognitive and behavioral dysfunction in patients, which 48 seriously affects the patient's quality of life [6,7]. Therefore, how to attenuate the damage of primary 49 and secondary TBI on the nerve tissue and to enhance the neuroprotective effects has become the present 50 research focus of neuroscience and trauma science.

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Based on the above research, this study aimed to confirm the effectiveness of CTRP1 on neuroprotection, 63 and to investigate its effect on memory, cognitive and behavioral functions in TBI rats. In addition, we 64 further revealed the mechanism of the neuroprotective effects of CTRP1 in TBI rats.  were maintained under SPF condition, with the temperature of 25±1 o C, the relative humidity of 40%-60% 72 and the light/dark cycle of 12 h/12 h. Rats were freely accessible to food and water. The rats were 73 adaptively fed for one week before the experiment. All surgery was performed under sodium 74 pentobarbital anesthesia, and all efforts were made to minimize suffering.

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The modified Feeney's method was used to establish the closed TBI model 16 . In brief, rats were fasted 77 and deprived of water for 12 h before operation. The head was fixed on the stereotaxic apparatus, 78 followed by shearing and disinfection of the operation area. Afterwards, the scalp was cut along the 79 midline sagittal of the head to expose the right parietal skull. To penetrate the skull and open a circular 80 window 5 mm in diameter while preventing endocranial injury, a dental drill was used on the skull at a bleeding and a few seconds of apnea were shown in rats after the hit. Based on the neurological 88 severity score (NSS) at 0.5 h and 1 h after injury, the rats with common TBI were screened, excluding 89 eight rats that died or did not present moderate TBI.  were trained for five consecutive days before the experiment. Then, at 24 h, 72 h and 1 w after TBI, 108 rats were allowed to seek the platform in the 1-min test. After the water maze, rats were sacrificed.

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Hippocampal tissue samples were immediately removed and stored at -80°C.      The expression of CTRP1 in rat hippocampus was detected at 24 h after TBI. The results showed that 159 the expression of CTRP1 increased in rCTRP1 group and vehicle group compared with sham group (p 160 <0.001). In addition, the expression of CTRP1 was significantly higher in rCTRP1 group than that in 161 vehicle group (p<0.001) (Fig 1A B).

Spatial orientation ability 166
The escape latency is defined as the time from entering water to boarding the platform of rats, which is 167 one of the commonly indicators of spatial orientation ability. In this study, the escape latency time was 168 detected in rats of three different groups at 24 h, 72 h, and 1 w after craniocerebral trauma. As a result, 169 the escape latency was significantly longer in rCTRP1 group and vehicle group than that in sham group 170 at the first two time points (p<0.005), and there was no significant difference in the escape latency 171 between rCTRP1 group and sham group at 1 w after craniocerebral trauma. However, the time was 172 significantly shorter in rCTRP1 group than that in vehicle group at 1 w after TBI (p<0.05) (Fig 2A).

Reverse searching ability 179
The second quadrant of the platform was changed to the fourth quadrant, followed by re-examination of 180 the escape latency of rats, which was used as an indicator of working memory ability. The escape latency 181 of reserve searching was significantly prolonged in rCTRP1 group and vehicle group than that in sham 182 group at 24 h and 72 h after craniocerebral trauma (p<0.05), and there was no significant difference in 183 the time between rCTRP1 group and sham group at 1 w after craniocerebral trauma. However, the time 184 was significantly shorter in rCTRP1 group than that in vehicle group at 1 w after TBI (p<0.05) (Fig 2B).

Spatial exploration ability 186
The number of entries into the target quadrant (TA) after removing the underwater platform is one of the 187 commonly used indicators to detect the spatial exploration ability of rats. In this study, the number of 188 entries into the TA was significantly less in rCTRP1 group and vehicle group than that in sham group at 189 24 h and 72 h after TBI (p<0.05), and there was no significant difference in the number between rCTRP1 190 group and sham group at 1 w after craniocerebral trauma. While the number significantly increased in 191 rCTRP1 group compared to vehicle group at 1 w after TBI (p<0.05) (Fig 2C). compared to those in vehicle group at 1 w after TBI (p<0.05) (Fig 3ABC, Fig 4A). The dendritic spines 199 decreased in rCTRP1 group and vehicle group than those in sham group at 24 h and 72 h after TBI 200 (p<0.005), and there was no significant difference in those between rCTRP1 group and sham group at 1 201 w after craniocerebral trauma. However, the dendritic spines significantly increased in rCTRP1 group 202 compared to those in vehicle group at 72 h and 1 w after TBI (p<0.05) (Fig 3 DEF, Fig 4B).

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In this study, we find that the CTRP1 recombinant protein can improve the behavioral and 287 histopathological outcomes, inhibit inflammatory response, activate mTOR and decrease autophagy-288 associated protein synthesis in TBI rats. Therefore, CTRP1 exerts neuroprotective effects in TBI rats by 289 regulating inflammation and autophagy and has potential therapeutic properties after TBI.