| Objective: To investigate the protective effect of zinc-rich food on cleft palate of offspring mice induced by high temperature/lipopolysaccharide(LPS)exposure in the second trimester of pregnancy,and to clarify whether the antagonistic effect of zinc-rich and its cellular and molecular mechanism are related to classical Wnt signaling pathway.Methods: C57BL/6J pregnant mice were randomly divided into 6 groups,namely normal control group(N),zinc-rich control group(ZR),LPS group,zinc-rich+LPS group(ZR+LPS),high temperature group(HT),and zinc-rich+high temperature group(ZR+HT).Pregnant mice in group N were treated with normal zinc feed(30mg/kg),and were injected with normal saline or placed in room temperature water on day8.5(ED8.5).Pregnant mice in the ZR group were fed free zinc-rich diet(100mg/kg).Pregnant mice in the LPS group were injected with LPS(0.5?g/g)through the neck at ED8.5.Pregnant mice in ZR+LPS group were fed free zinc-rich feed(100mg/kg)from ED0.5-ED14.5,and were injected with LPS through the neck(0.5?g/g)by ED8.5.Pregnant mice in group HT were placed in a 42.5? water bath for 9 minutes at ED8.5,dried with a dryer,and repeated after 6 hours.Pregnant mice in ZR+HT group were fed free zinc-rich feed(ED0.5-ED14.5),and ED8.5was treated with high temperature(the same method as group HT).Palatal tissue was obtained after euthanasia of ED14.5 pregnant mice.Palatal development was observed by hematoxylin-eosin(HE)staining,and stillbirth rate was calculated.The palatal development of newborn mice in each group was observed and the incidence of cleft palate was calculated.Cell proliferation and apoptosis were observed by PCNA and TUNEL staining at ED14.5.The expression levels of Wnt pathway and related factors were detected by immunofluorescence staining,Western blot and Real-time fluorescence quantitative PCR at ED14.5.Results:(1)The incidence of cleft palate in group N was 5% and the stillbirth rate was0%;The incidence of cleft palate in group ZR was 2.4%,and the stillbirth rate was 0%,there was no statistical difference compared with group N(P >0.05).The incidence of cleft palate in the LPS group was 27.8%,and the stillbirth rate was 13.8%,there was a statistical difference compared to group N(P<0.01).The incidence of cleft palate in ZR+LPS group was 10%,and the stillbirth rate was 5%,there was a statistical difference compared to LPS group(P<0.05).The incidence of cleft palate in HT group was 32.4%,and the stillbirth rate was 18.9%,there was a statistical difference compared to group N(P<0.01).The incidence of cleft palate in ZR+HT group was 7.1%,and the stillbirth rate was 2.4%,there was a statistical difference compared to group HT(P<0.01).(2)The results of HE staining showed that in group N,the lingual tissue was decreased,and bilateral lateral palatine was completely lifted up to the horizontal direction,showing horizontal growth to the contralateral side.There was no significant difference in palatal development between group ZR and group N.Compared with group N,the lingual tissue of the LPS group decreased,but the lateral palatal process did not rise completely to the horizontal direction,and the palatal process was shorter than that of group N.Compared with the LPS group,the tongue tissue of the ZR+LPS group decreased significantly,and both sides of the lateral palatal process reached a certain height and grew horizontally to the contralateral side.Compared with group N,the elevation of lateral palatine in group HT was significantly delayed,while the elevation of lateral palatine on the other side was already increased,but the palatine was shorter and the distance of bilateral palatine was longer than that in group N.Compared with group HT,both lateral palatal processes in group ZR+HT had been lifted above the tongue body.Although the palatal development on one side was relatively short,the overall palatal development had been significantly improved.(3)TUNEL staining results: ED14.5,there were fewer apoptotic cells in group N and group ZR,the difference between the two groups was not statistically significant(P>0.05).Compared with group N,partial apoptotic cells could be seen in the lateral palatal mesenchymal cells of the LPS group,the difference was statistically significant(P<0.01).Compared with the LPS group,the apoptotic cells in the ZR+LPS group were significantly reduced and the difference was statistically significant(P<0.01).Compared with group N,a large number of apoptotic cells were observed in the lateral palatal tissue cells of group HT,the difference was statistically significant(P<0.01).Compared with group HT,the apoptotic cells in group ZR+HT were reduced and the difference was statisticallysignificant(P<0.01).(4)PCNA staining results: Compared with group N,the tissue proliferation index of LPS group and group HT were significantly reduced and the difference was statistically significant(P<0.01).The proliferation index of ZR+HT group was significantly higher than that of HT group,and that of ZR+LPS group was significantly higher than that of LPS group,the difference was statistically significant(P<0.01).(5)Immunofluorescence results: The expression levels of GSK-3?,?-catenin,Sp5 and Wnt3 A in HT and LPS groups were significantly lower than those in group N,the difference was statistically significant(P<0.01).ZR+HT group compared with HT group,ZR+LPS group compared with LPS group,GSK-3?,?-catenin,Sp5 and Wnt3 A were significantly increased,and the difference was statistically significant(P<0.05).(6)Western blot results: The expression levels of GSK-3?,?-catenin,LEF-1,Sp5,Sp8 and MT were decreased in group HT compared with group N,and the difference was statistically significant(P<0.01);Expression levels of ?-catenin,Sp5,Sp8 and MT were significantly decreased in the LPS group compared with group N,and the difference was statistically significant(P<0.01).The expression levels of GSK-3? and LEF-1 showed no significant change and no statistical significance(P>0.05).Compared with group HT,the expression levels of GSK-3?,?-catenin,LEF-1,Sp5,Sp8 and MT in group ZR+HT were significantly increased,and the difference was statistically significant(P<0.05).Compared with LPS group,MT in ZR+LPS group was significantly increased,and the difference was statistically significant(P<0.01).The expression levels of GSK-3?,?-catenin,LEF-1,Sp5 and Sp8 were slightly increased,the difference was not statistically significant(P>0.05).(7)Real-time PCR results: Compared with group N,GSK-3? mRNA,?-catenin mRNA,LEF-1 mRNA,Sp5 mRNA and MT mRNA expression levels in group HT and LPS were significantly decreased,and the difference was statistically significant(P<0.01).ZR+HT group compared with HT group,ZR+LPS group compared with LPS group,GSK-3?mRNA,?-catenin mRNA,LEF-1 mRNA,Sp5 mRNA and MT mRNA were significantly increased,and the difference was statistically significant(P<0.05).Conclusion:(1)Zinc-rich(100mg/kg)can effectively reduce fetal stillbirth induced by LPS/ high temperature exposure in the second trimester.(2)Zinc-rich(100mg/kg)can effectively prevent cleft palate induced by LPS/ high temperature exposure in the second trimester of pregnancy.(3)The protective effect of zinc-rich(100mg/kg)on the cleft palate model induced by LPS/ hyperthermia in mice may be related to the promotion of palatal mesenchymal cell proliferation.(4)The protective mechanism of zinc rich(100mg/kg)on the cleft palate induced by high temperature /LPS in mice may be related to the activation of the classical Wnt/?-catenin signaling pathway and the up-regulation of Sp5,Sp8,MT and other factors. |
PDF Full Text Request