Study On The Protective Effect And Mechanism Of Salidroside Analog On Nerve Cell

Objective Neurodegenerative disease is a complex disease pattern and the damaged nerve cell is the main cause. Recently, salidroside(Sal)has attracted more and more attention due to its widely pharmacological activities. However, the drug efficacy of Sal has not always done that well. The hydrophilic property of salidroside affects its efficacity since hydrophilic molecules are hard to penetrate across the cytoplasmic membrane and blood brain barrier, which affects its’ function. As of now,there are only few reports on the protective effects of salidroside analogue. In our previous studies, we have synthesized a series of analogues of salidroside. In this study, we established nerve cell damage model to imitate the damaged nerve cell status in vivo, and salidroside analogues were applied to the model to detect their protective effects.The results revealed that MADP and compound 6 possess higher protective activity with comparison to salidroside, which laid the basic foundation for further development of new drugs for the treatment of nervous system degenerative diseases.Methods In this study, two kinds of cell damage models were established. In the first model, the cultured hippocampal neurons were pretreated with MADP or salidroside at the concentrations of 120 and240 μM for 24 h, and then the neurons were exposed to 125 μM glutamate for 15 min. After excitotoxicity, cells were further culturedwith fresh medium for 24 h. Cell morphology was observed by light microscopy; MTT assay and LDH release were used to evaluate cell viabilities; Hoechst 33342 staining, TUNEL assay and Annexin V-FITC/PI double staining were applied to detect apoptotic cells;dynamic change of intracellular free [Ca2+]i was measured by Fluo-4/AM;QPCR and Western blot were used to detect the expression of apoptosis related genes and proteins. In the second model, the cultured PC12 cells were pretreated with compound 6, 7, 11, 12(75, 150, 300 μg/mL) and Sal(300 μg/mL) for 24 h, and then hypoglycemia and serum limitation was applied and incubated for another 24 h. Cell morphology was observed by light microscopy; survived cells were tested by MTT, apoptosis cells were detected by Hoechst33342 staining, apoptosis proteins Bcl-2/Bax were measured by Western blot, the caspase-3 activities was detected by caspase-3 activity kit.Results In the glutamate damage model, the results of MTT and LDH release showed that under the same condition, cells pretreated with MADP had higher viability than Sal. Hoechst 33342, TUNEL, Annexin V-FITC/PI experiments showed that cell apoptosis rate in MADP group was lower than that in Sal group. For Fluo-4/AM staining, [Ca2+]i had stronger fluorescence intensity in Sal group; MADP had less Caspase-3mRNA than that in Sal group; result of Western blot showed that expression of Bcl-2/Bax and p-ERK didn’t change in both groups, while p-Akt and p-JNK expression was higher in MADP group than that in Sal group. In the hypoglycemia and serum limitation model, most compounds showed protection on cell viability, especially compound 6;pretreating cells with compound 6 could improve the morphology of damaged cells; the result of MTT, Hoechst 33342, Western blot and caspase-3 displayed that compound 6 could suppress apoptosis induced by hypoglycemia and serum limitation in a dose-dependent manner. At the concentration of 300 μg/mL, compound 6 showed the most efficient protective effects.Conclusions The protective effects of MADP and compound 6 are stronger than that of Sal.