The Role And Mechanism Of Gap Junctional Intercellular Communication In Damage Of Rat Liver Induced By Cadmium

Gap junctional intercellular communication (GJIC) is an important kind of communication exists widely among mammalian cells, it is actively involved in virtually all aspects of the cellular life cycle, ranging from cell growth to cell death. Cadmium (Cd) is a serious environmental toxicant with harmful effects on health in both animals and humans, targeting multiple organ systems especially kidney and liver. Some researches show that cadmium could induce apoptosis, down-regulate GJIC hepatocytes, even affect expression and distibution of the gap junction protein. So far, the role of GJIC in damage of rat liver induced by cadmium is still unclear. In the current experiment, the model of BRL 3A cells were exposed to Cd (2.5,10 μmol/L) and 50 μmol/L Salidroside (Sal) for 6,12h in vitro, the model of female Sprague-Dawley (SD) rats weighing 80-100 g were divided randomly into three groups as follows. (1) Control group:10 rats consuming DDW as their drinking water. (2) Cadmium group: 10 rats consuming a solution of Cd (50 mg/L) as their drinking water. (3) Cadmium+Sal group: 10 rats treated daily with Sal (35 mg/kg body weight, intragastric gavage, i.g.) and consuming a solution of Cd (50 mg/L) as their drinking water. All rats were sacrificed 12 weeks after initial treatment. This study will elucidate the change of gap junction intercellular communication in the process of rat liver damage induced by cadmium, and its role in cellular signal transduction and autophagy, and the protection of salidroside in the process of the above, further reveal the mechanism of liver damage induced by cadmium via various cell and molecular biological methods.1. Salidroside protects against Cd’s cytotoxicity in rat hepatocytes and BRL 3AcellsIn this study, we examined cell viabilities, cell index (CI), organosimatic index (OSI), internal cellular structures, pathologic histology to establish the model of the toxicity of cadmium both in vitro and in vivo. Results showed that 2.5,5,10,20 μmol/L Cd altered cell morphology with cell round and shrunken, nuclei shrinkage, chromatin karyopyknosis, mitochondrial swelling and mitochondrial cristae fused partly and became blurry or were even missing. Cd also induced decrease in cell viability and CI in a time and dose-dependent manner. Sal (25.50 and 100 μM) did not alter cell viability compared with control. These results made us choose 50 μM Sal for subsequent studies. the addition of Sal was associated with a slower decrease of the CI compared with Cd group. these results demonstrated that Sal can ameliorate the effect of Cd in BRL 3Acells. Cd induced liver steatosis, liver cells arranged in disorder, central venous congestion in vivo. Liver steatosis and the cellular organelle ultrastructure injuries were partly rescued by treatment with Sal.2. GJIC has a dual effect on Cd-induced cytotoxic injuryIn this study, we examined GJIC, the expression of Cx43, Cx32 and Cx43 mRNA, Cx32 mRNA, immuno-labeled Cx32, and assessd the effect of Cd damaged cells on healthy cells in transwell co-culture system, to investigate the effects of GJIC on Cd induced liver injury in rats. We found that treatment with Cd resulted in inhibition of GJIC in both BRL 3A cells and rat liver tissue, while Sal had a protective effect on GJIC. The relative expression level of Cx32 was significantly (p< 0.05 or p< 0.01) decreased following Cd treatment compared with the control group, while co-treatment with Sal significantly attenuated the Cd-induced decrease of Cx32 mRNA. In the control group, Cx32 was seen at the gap junctions between adjacent hepatocytes; however, the gold particles appeared decreased in number and scattered after Cd treatment. Notably,Sal appeared to suppress the Cd-induced Cx32 mRNA decrease. In transwell co-culture system, Cd-exposed cells exhibited decreased viability, nuclei chromatin condensation and even nuclear fragmentation. Co-treatment with GA exacerbated the reduction in cell viability, as well as nuclei injury, compared with the cells treated only with Cd. However, in the transwell co-culture system, GA had a protective effect on BRL 3A cells co-cultured with Cd-exposed cells. Sal can ameliorate these damage induced by Cd in vitro and in vivo. These results demonstrated that GJIC inhibition has dual effects:(ⅰ) it restricts release of Ca2+ from the cell, which exacerbates the [Ca2+]i elevation and cytotoxicity induced by Cd; (ⅱ) protects healthy cells from their dangerous neighbors by blocking intercellular communication.3. GJIC regulates Cd-induced hepatotoxicity via calcium signallingIn this study, we examined the intracellular free Ca2+ concentration ([Ca2+]i), the expression of calcium signalling related proteins and genes, CI. Further more, we used the GJIC inhibitor GA and calcium signalling related inhibitors, to investigate the effects of GJIC on Cd induced hepatotoxicity via calcium signalling. We found that treatment with Cd resulted in a prolonged and sustained rise in [Ca2+]i levels, which was inhibited by co-administration of Sal. Intracellular calcium chelator BAPTA-AM prevented the Cd-induced decrease in CI. The endoplasmic reticulum (ER) Ca2+-ATPase inhibitor TG exacerbated Cd induced the decrease of cell viability, whereas the extracellular calcium chelator EGTA prevented Cd-induced thr decrease of cell viability. Cd markedly down-regulated the CaM expression and Sal prevented it in vitro and in vivo. Co-administration of Cd and GA was associated with an even higher concentration of [Ca2+]i compared to Cd treatment alone.Co-treatment with Cd and GA, or co-treatment with Cd and TG, significantly (p<0.01,p< 0.05) exacerbated [Ca2+]i elevation compared with the group of treatment with Cd alone, we demonstrated that Cd induces cytotoxicity in BRL 3 A cells by inhibiting GJIC and triggering elevation of [Ca2+]i by stimulating Ca2+ release from intracellular stores, and LA may be a functional chemopreventative agent which could ameliorate the cytotoxic effects of Cd via GJIC and Ca2+ signaling.4. GJIC regulates Cd-induced hepatotoxicity via MAPK pathwaysIn this study, we examined the expression of MAPK pathways related proteins and genes, CI and GJIC, and we used the GJIC inhibitor GA and ERK inhibitor U0126, p38 inhibitor SB202190, JNK inhibitor SP600125, further more we assessd the role of MAPK pathways in Cd exposed cells induced healthy cells damage in transwell co-culture system, to investigate the effects of GJIC on Cd induced hepatotoxicity via MAPK pathways. We found that the phosphorylation levels of ERK, JNK and p38 were increased after Cd treatment, significantly so for ERK (p< 0.01). Sal co-treatment significantly (p< 0.01) inhibited the up-regulation of phosphorylated ERK, JNK and p38. Co-treatment with U0126 significantly (p< 0.05) blocked decreases in Cd-induced Cx32 mRNA and SB202190 also restored Cx32 levels to some extent, whereas SP600125 had no effect on the expression of Cx32 mRNA, and alterations in Cx32 mRNA expression were found to be in accordance with GJIC function. U0126 and SB202190 had a protective effect on Cd-exposed cells, with a decrease in nuclei chromatin condensation and nuclear fragmentation, while SP600125 had little effect in transwell co-culture systrm. Meanwhile, GA had no effect on the Cd-induced up-regulation of phosphorylated ERK. JNK and p38.5. GJIC regulates Cd-induced autophagy in BRL 3A cellsIn this study, we examined the CI, GJIC and the expression of GJIC related proteins Cx43, autophagy related protein and genes LC3, Atg-5, Atg-7 and Beclin-1.We also assessed the level of autophagy by monodansylcadaverine (MDC) staining. Further more, we used the prototypical gap junction blocker carbenoxolone disodium (CBX), the autophagic inducer rapamycin (RAP), and the autophagic inhibitor chloroquine (CQ), to investigate the effects of GJIC on Cd induced autophagy in BRL 3A cells. After stained with MDC, control cells displayed diffuse staining, whereas Cd treatment increased MDC fluorescent intensity in a concentration-dependent manner. The expression of LC3-II significantly increased (p< 0.01) in a concentration-dependent manner after Cd treatment, correspondingly the mRNA expression of autophagy-related proteins Atg-5, Atg-7, Beclin-1 was increased. The autophagic inducer RAP prevented the CI decrease induced by Cd, while the autophagic inhibitor CQ caused a further reduction in CI. These results demonstrate that autophagy has a protective effect on Cd-induced cytotoxicity in BRL 3A cells. CBX further promoted Cd-induced autophagy, with MDC fluorescence intensity increasing in the co-treatment group. Correspondingly, the mRNA expressions of autophagy-related genes Atg-5, Atg-7, Beclin-1 were significantly increased (p< 0.01), and the same did the LC3-II expression. Co-treatment with CQ significantly blocked(p< 0.01) the Cd-induced decrease in Cx43 expression, whereas RAP further reduced Cx43 expression. In addition, RAP significantly decreased(p< 0.05) Cx43 expression. Correspondingly, alterations in GJIC function were found to be in accordance with Cx43 expression. These results demonstrated that autophagy serves an important protective function in Cd-induced cytotoxicity in BRL 3A cells at an early stage, meanwhile, autophagy exacerbates Cd-induced GJIC inhibition.