Mechanisms Of Maternal High Fructose Exposure During Pregnancy And Lactation Activating The Hypothalamic Hippo/AT1R/TLR4 Signaling Pathway Resulting In Offspring Hypertension And Impaired Hypothalamic Development Programming

Objective: In recent years,the excessive consumption of sugar-sweetened beverages(SSBs)has led to severe public health issues,particularly among young people.Fructose is the primary sweetening additive in SSBs,and its excessive intake is closely associated with hypertension.Hypertension is a metabolic syndrome characterized by a complex metabolic disease that results from the interaction of endocrine dysfunction,environmental factors,and genetic factors,leading to elevated arterial blood pressure.The hypothalamus plays a critical role in coordinating and integrating central blood pressure control neural networks.During hypothalamic development,neural activity is easily altered by external stimuli,leading to long-term and persistent physiological abnormalities.Poor dietary habits during pregnancy and lactation may lead to abnormal intrauterine environments and have long-term adverse effects on the development of offspring’s hypothalamus.Studies have found that the Hippo signaling pathway is an essential regulator of homeostasis in the heart,cardiovascular system,and vasculature,and its activation may be accompanied by damage to microvascular development.Angiotensin-II(Ang-II)is an important target for cardiovascular diseases and hypertension,mainly acting through its receptor AT1 R in the hypothalamus.This receptor’s activity has been proven to be closely related to hypertension,regulating the structure and function of vascular cells,and its overexpression activates the Toll-like receptor 4(TLR4)signaling pathway.The activation of the TLR4 signaling pathway is also closely related to hypertension.There are few reports on the impact of maternal fructose exposure on the development of offspring’s hypothalamus leading to hypertension.This study aims to investigate the mechanisms related to maternal high-fructose exposure during pregnancy and lactation,activating the Hippo/AT1R/TLR4 signaling pathway,leading to hypertension in offspring and affecting hypothalamic developmental programming.Methods: Establish a maternal fructose exposure model during pregnancy and lactation,divided into a control group,13% fructose group(F13),and 40% fructose group(F40).From Gestation Day 0(GD0)to Postpartum Day 21(PND21),the experimental groups were given fructose solution,while from PND21 to Postpartum Day60(PND60),all groups were given deionized water.The tail-cuff method was used to measure the blood pressure of offspring on PND21 and PND60.Oxford Nanopore Technologies(ONT)full-length RNA sequencing was employed to study the changes in hypothalamic developmental programming in PND60 offspring.Western blot analysis and immunofluorescence were used to determine the expression changes of the Hippo/AT1R/TLR4 pathway in the offspring’s hypothalamus and the main functional areas.PCR was used to verify the transcriptomic sequencing results and the changes in the pathway-related genes in the offspring’s hypothalamus.Results: 1.Maternal fructose exposure led to a significant increase in systolic blood pressure(SBP),diastolic blood pressure(DBP),and mean arterial pressure(MAP)in PND60 offspring(P<0.05),with no changes in PND21 offspring blood pressure;2.Hypothalamic developmental programming was impaired in PND60 offspring,with varying degrees of changes at the transcriptomic level;3.Western blot results showed that compared to the control group,the expression levels of WWC2,p-LATS1,and pYAP proteins in the F13 and F40 groups were significantly reduced(P<0.05)in both PND21 and PND60 offspring’s hypothalamus,but the expression levels of the total proteins LATS1 and YAP did not change;Additionally,there were no changes in IκBαexpression levels in the hypothalamus of PND21 and PND60 offspring,while compared to the control group,the expression levels of TLR4,p-IκBα,and NF-κB p65 in the F13 and F40 groups were significantly increased(P<0.05);4.Immunofluorescence results indicated that in the hypothalamus of PND21 and PND60 offspring,compared to the control group,the expression levels of AT1 R and NF-κB p65 in the PVN region of the F13 and F40 groups showed a clear increasing trend;5.PCR results were consistent with transcriptomic sequencing results,and the expression levels of Hippo signaling pathway-related genes were significantly reduced.Conclusion: This study found that high-fructose exposure during maternal pregnancy and lactation leads to hypertension and impaired hypothalamic developmental programming in PND60 offspring.The primary mechanism may be through the activation of the Hippo/AT1R/TLR4 signaling pathway,ultimately leading to increased expression of AT1 R and NF-κB p65 in the offspring’s hypothalamic PVN.