Background: Both pathological conditions and hibernation can affect the barrier function of small intestine mucosa. However, the effect of hibernation on the barrier function of colonic mucosa remains unclear. Methods: We investigated morphological changes in colonic mucosa, the concentrations of specific proteins and molecules, and the enzymatic activity of diamine oxidase (DAO), in serum and colonic tissue; the expression of tight junction proteins and mucin, and the changes in inflammatory, farnesoid X receptor (FXR)–small heterodimer partner (SHP), and apoptosis-related molecules that could play a role in gut permeability changes in Daurian ground squirrels in summer active (SA), late torpor (LT), and interbout arousal (IBA) periods. Results: The results show that hibernation reduced the thickness of the colonic mucosa and the depth of the crypt, decreased the number of goblet cells (GCs), and damaged the structure of some microvilli. The concentrations of proteins and molecules, and the enzymatic activity of DAO, were all increased in the serum and colon, and the localization of tight junction proteins and mucin in the colonic mucosa were altered (compensatory response). Although the ground squirrels ate during the interbout arousal period, the changes remained similar to the response to torpor. Inflammation, apoptosis–anti-apoptosis, and FXR–SHP signaling may be involved in the possible changes in intestinal gut permeability during the torpor–arousal cycle in Daurian ground squirrels. In addition, periodic interbout arousal may play an inflammation-correcting role during the long hibernation season of Daurian ground squirrels.

Hibernators are a natural model of vascular ischemia–reperfusion injury; however, the protective mechanisms involved in dealing with such an injury over the torpor–arousal cycle are unclear. The present study aimed to clarify the changes in the thoracic aorta and serum in summer-active (SA), late-torpor (LT) and interbout-arousal (IBA) Daurian ground squirrels (Spermophilus dauricus). The results show that total antioxidant capacity (TAC) was unchanged, but malondialdehyde (MDA), hydrogen peroxide (H₂O₂), interleukin-1β (IL-1β) and tumor necrosis factor α (TNFα) were significantly increased for the LT group, whereas the levels of superoxide dismutase (SOD) and interleukin-10 (IL-10) were significantly reduced in the LT group as compared with the SA group. Moreover, the levels of MDA and IL-1β were significantly reduced, whereas SOD and IL-10 were significantly increased in the IBA group as compared with the SA group. In addition, the lumen area of the thoracic aorta and the expression of the smooth muscle cells (SMCs) contractile marker protein 22α (SM22α) were significantly reduced, whereas the protein expression of the synthetic marker proteins osteopontin (OPN), vimentin (VIM) and proliferating cell nuclear antigen (PCNA) were significantly increased in the LT group as compared with the SA group. Furthermore, the smooth muscle layer of the thoracic aorta was significantly thickened, and PCNA protein expression was significantly reduced in the IBA group as compared with the SA group. The contractile marker proteins SM22α and synthetic marker protein VIM underwent significant localization changes in both LT and IBA groups, with localization of the contractile marker protein α-smooth muscle actin (αSMA) changing only in the IBA group as compared with the SA group. In tunica intima, the serum levels of heparin sulfate (HS) and syndecan-1 (Sy-1) in the LT group were significantly reduced, but the serum level of HS in the IBA group increased significantly as compared with the SA group. Protein expression and localization of endothelial nitric oxide synthase (eNOS) was unchanged in the three groups. In summary, the decrease in reactive oxygen species (ROS) and pro-inflammatory factors and increase in SOD and anti-inflammatory factors during the IBA period induced controlled phenotypic switching of thoracic aortic SMCs and restoration of endothelial permeability to resist ischemic and hypoxic injury during torpor of Daurian ground squirrels.

Hibernating mammals experience severe hemodynamic changes over the torpor–arousal cycle, with oxygen consumption reaching peaks during the early stage of torpor to re-enter arousal. Melatonin (MT) can improve mitochondrial function and reduce oxidative stress and inflammation. However, the regulatory mechanisms of MT action on the vascular protective function of hibernators are still unclear. Morphology, hemodynamic, mitochondrial oxidative stress, and inflammatory factors of the carotid artery were assessed in ground squirrels who were sampled during summer active (SA), late torpor (LT), and interbout arousal (IBA) conditions. Changes were assessed by methods including hematoxylin and eosin staining, color Doppler ultrasound, ELISA, Western blots, and qPCR. Changes in arterial blood and serum melatonin were also measured by blood gas analyzer and ELISA, whereas mitochondrial oxidative stress and inflammation factors of primary vascular smooth muscle cells (VSMCs) were assessed by qPCR. (1) Intima-media carotid thickness, peak systolic velocity (PSV), end diastolic blood flow velocity (EDV), maximal blood flow rate (Vmax) and pulsatility index (PI) were significantly decreased in the LT group as compared with the SA group, whereas there were no difference between the SA and IBA groups. (2) PO₂, oxygen saturation, hematocrit and PCO₂ in the arterial blood were significantly increased, and pH was significantly decreased in the LT group as compared with the SA and IBA groups. (3) The serum melatonin concentration was significantly increased in the LT group as compared with the SA and IBA groups. (4) MT treatment significantly reduced the elevated levels of LONP1, NF-κB, NLRP3 and IL-6 mRNA expression of VSMCs under hypoxic conditions. (5) Protein expression of HSP60 and LONP1 in the carotid artery were significantly reduced in the LT and IBA groups as compared with the SA group. (6) The proinflammatory factors IL-1β, IL-6, and TNF-α were reduced in the carotid artery of the LT group as compared with the SA and IBA groups. The carotid artery experiences no oxidative stress or inflammatory response during the torpor–arousal cycle. In addition, melatonin accumulates during torpor and alleviates oxidative stress and inflammatory responses caused by hypoxia in vitro in VSMCs from ground squirrels.