Cldn4 overexpression promotes penile cavernous smooth muscle cell fibrotic response via the JNK signaling pathway.

BACKGROUND: Erectile dysfunction (ED), defined as the inability to achieve or maintain a penile erection sufficient to satisfy sexual behavior, is prevalent worldwide. AIM: Using previous research, bioinformatics, and experimental confirmation, we aimed to discover genes that contribute to ED through regulating hypoxia in corpus cavernosum smooth muscle cells (CCSMCs). METHODS: We used the Gene Expression Omnibus to acquire the sequencing data of the corpus cavernosum transcriptome for diabetic ED and nerve injury type ED rats. We intersected the common differentially expressed genes. Further verification was performed using single cell sequencing. Real-time quantitative polymerase chain reaction and immunofluorescence were used to investigate whether the differentially expressed genes are found in the corpus cavernosum. We used induced hypoxia to assess cell viability changes, and we developed a lentivirus overexpressing Cldn4 for in vitro and in vivo experiments to measure changes in JNK signaling, fibrosis, hypoxia, and erectile function. OUTCOMES: Our results indicate that targeting the JNK pathway and decreasing local hypoxia may be better options for therapeutic intervention to improve erectile function. RESULTS: We identified Cldn4 and found its expression increased in the corpora cavernosa of the 2 datasets. In addition, we found that hypoxia can increase the expression of Cldn4, activate the JNK signaling pathway, and exacerbate fibrosis in CCSMCs. Cldn4 overexpression in CCSMCs activated the JNK signaling pathway and increased fibrotic protein expression. Last, rat corpus cavernosum overexpressing Cldn4 activated the JNK signaling pathway, increased local fibrosis, and impaired erectile function. CLINICAL IMPLICATIONS: Through bioinformatics and in vitro and in vivo experiments, we found that Cldn4 has a negative effect on ED, and targeting Cldn4 may provide new ideas for ED treatment. STRENGTHS AND LIMITATIONS: Although we have identified Cldn4 as a potential target for ED treatment, we have only conducted preliminary validation on CCMSCs, and we still need to further validate in other cell lines. CONCLUSION: CCSMC hypoxia leads to increased Cldn4, in both nerve injury and diabetic ED rat models, and promotes fibrosis by activating the JNK signaling pathway.

Neuroprotective effect of magnesium-L-sulfonate on focal cerebral ischemia in rats and its mechamism / 中华行为医学与脑科学杂志

Objective To investigate the effects of magnesium sulfate and magnesium L-sulfonate on the neurobehavioral response and the expression of induced nitric oxide synthase (iNOS) in neurons after acute cerebral ischemia in rats. Methods One hundred and twelve male Sprague-Dawley ( SD) rats were double-blinded randomly divided into sham-operated group, middle cerebral artery occlusion ( MCAO ) group,MgSO4 treatment group,L-MgT treatment group. Each group was further divided into 6 h,12 h and 24 h subgroups according to the different detection time points. Rat MCAO models were produced following the Longa's method. And the Longa score,limb-placing test,rotarod test,and sticky tape test were performed to evaluate the neurological damage,autonomous movement and coordinate perception in the 24 h subgroup. At the end of the experiment,2,3,5-triphenyltetrazolium chloride ( TTC) was used to evaluate the area of cerebral infarction at 24 h reperfusion. Immunohistochemical staining was employed to determine the altera-tions in iNOS expression in neurons 6 h,12 h and 24 h after reperfusion. Results In behavioral evaluation:Longa score:Normal performance was observed in sham-operated group. Compared with the MCAO group,the scores of MgSO4 treatment group(1. 71±0. 18) and L-MgT treatment group(1. 14±0. 14) were decreased (t=0. 548,3. 873,all P<0. 05),and the score of L-MgT treatment group was lower than that of the MgSO4 group(t=2. 828,P<0. 05). Limb symmetry score:There was no statistically significant difference between MgSO4 group and MCAO group,but there was a statistically significant difference between L-MgT group and MCAO group (t=7. 071,P<0. 05). The roding experiment:The time of MgSO4 group and the L-MgT group were significantly different from that of the MCAO group (t=9. 588,20. 776,P<0. 05),and the time of the L-MgT group was significantly higher than that of the MgSO4 group (t=4. 983,P<0. 05). The right limb strip removal experiment: The time of MgSO4 group and L-MgT group were statistically different from that of MCAO group (t=6. 135,5. 825,P<0. 05),and the time of L-MgT group was increased compared with that of MgSO4 group(t=4. 507,P<0. 05). TTC test:No infarction was formed in the sham group. Compared with MCAO group ((36. 82±1. 35)%),the cerebral infarction volume of MgSO4 group ((17. 39±1. 72)%) and L-MgT group ((10. 81 ± 1. 35)%) significantly decreased, with statistically significant differences ( t=8. 874,11. 105,P<0. 05). Compared with MgSO4 group,cerebral infarction volume in L-MgT group was sig-nificantly reduced,with statistical significance (t=2. 593,P<0. 05). HE staining:There was no statistically significant difference in cell morphology between MgSO4 group and MCAO group at each time point,but the cell morphology of L-MgT group was intact compared with that of MCAO group. INOS staining at 24 h:There was no statistically significant difference in the positive cell density between the MgSO4 group and the MCAO group,but the L-MgT group (cortex:(196. 7±8. 1);striatum:(153. 3±3. 8)) positive cell density was lower than that of the MCAO group (cortex:(375. 0±6. 7),striatum:(358. 3±4. 5)),and the difference was sta-tistically significant (t=11. 113,36. 231,P<0. 05). Conclusion L-MgT may have a significantly protective effect on MCAO rats,and its mechanism may be related to the level of iNOS in neurons.