The Molecular Mechanisms Of Left-handed Camphor Prevents Autophagy By Targeting MiR-140 In PC12 Cells

ObjectiveAutophagy is a double-edged sword:on the one hand, excessive autophagy can induce cell death, and interact with the signal of apoptosis; on the other hand, autophagy is an important process of self repair. Appropriate activation of autophagy can help the damaged cells to clear the damaged organelles and abnormal proteins, thereby protecting the cell damage. Autophagy as a new kind of intervention of ischemic brain damage targets is more and more consensus, in recent years, there are also some preclinical research intervention autophagy as targets to treat diseases of the central nervous system, it can also be a stroke of basic research and potential clinical application of the new hot spot. Further research and exploration of the molecular biological mechanism of autophagy will help to find new drug intervention molecular targets.The previous experimental research group found L-camphor can inhibit apoptosis through GABAA receptors, and can prevent the overactivation of autophagy process during cerebral ischemia-reperfusion injury by targeting P53. A large number of studies have found that miRNA is involved in the immune escape of cancer cells and unlimited proliferation of cancer cells. Therefore, combine the two to explore L-camphor whether by targeting to specific miRNAs to regulate autophagy, which help to find a new drug for molecular intervention targets and for the development of medicine to provide new ideas.Methods1. Internal carotid artery to establish ischemia-reperfusion injury models was exercised to Intraluminal filament occluding the right middle cerebral artery (MCA)in SD rats, and then neurobehavioral behaviors were evaluated the models establishment. We observe the effect of L-camphor after 24h can improve cerebral ischemia and reperfusion model of neurological score. Identify with the qRT-PCR methods, we can find the influence of L-camphor on miRNA and which may be associated with autophagy pathways.2. By constructing miR-140 promoter luciferase reporter gene and transfecting PC12 cells, to observe the effect of L-camphor on miR-140 promoter transcriptional activity when the cells was stimulated with serum starvation.3. By the construction of a specific expression of rno-miR-140-3p mimics and inhibitors, and with the bioinformatics prediction about miR-140 to detect the target, and also through the construction of BCL-XL-3’UTR region containing binding sites of miR-140 dual luciferase reporter gene to further determine the function of miR-140.4. By the construction of a specific expression of rno-miR-140-3p mimics and inhibitors, Using immunofluorescence and Western blot method to detect the expressions of autophagy related proteins(LC3 and P62) and apoptosis related proteins(BCL-XL and caspase-3) in the serum starvation cells for observing the protective effection of L-camphor on serum starvation cells model.5. We intracerebroventricular inject rno-miR-140-3p agomir and rno-miR-140-3p antagomir into the MCAO rat model, observe the effect of miR-140 protein on apoptosis of MCAO rat model.Results1. L-camphor can improve cerebral ischemia and reperfusion injury models of behavioral symptoms, qRT-PCR results showed that L-camphor can inhibit cerebral ischemia and reperfusion of mir-140 expression.2. L-camphor can effectively down-regulated the transcriptional activity of miR-140 promoter in PC12 cells which Serum starvation induced.3. The potential target of rno-miR-140-3p is BCL-XL, which is predicted by bioinformatics. After Western Blot and Luciferase reporter gene, it was confirmed that rno-miR-140-3p could inhibit the expression of BCL-XL.4. Rno-miR-140-3p mimics can inhibit induced the expression of Bcl-XL and p62, promote the expression of Caspase-3 and LC3 in PC12 cells which the serum starvation induced. L-camphor and rno-miR-140-3p inhibitor can promote the expression of Bcl-XL and p62, inhibit the expression of Caspase-3 and LC3. When L-camphor and rno-miR-140-3p inhibitor combination, this effect is more apparent.5. Rno-miR-140-3p agomir can inhibit BCL-XL protein and increase caspase-3 protein in the MCAO model. rno-miR-140-3p antagomir can increase BCL-XL protein and inhibit Caspase-3 protein in the MCAO model. ConclusionL-camphor has neuroprotective effect by inhibiting autophagy. One of the mechanisms is upregulating of Bcl-XL protein by target to inhibit mir-140 expression, thereby inhibiting cell apoptosis and autophagy. Finally L-camphor plays a neuroprotective role.