Hui Chang

Cisplatin, which kills cancer cells mainly through DNA crosslinking, has been widely used as a first-line chemotherapeutic agent although it also causes severe side effects. To improve anticancer outcomes, various types of cisplatin-based nanomedicines have been developed, either through direct incorporation or coordination of cisplatin within nanoparticles (NPs). During the formulation and characterization of cisplatin-loaded NPs, quantitative determination of cisplatin is crucial for both clinically used and newly developed NPs. While NPs facilitate cisplatin delivery, the use of different nanomaterials inevitably complicates its determination and increases the cost of quantification. Currently, there is still a significant demand for an accurate, simple, and cost-effective method to determine cisplatin in NPs, which would facilitate the screening and quality control of cisplatin-based nanomedicines. This review aims to discuss the main strategies for quantifying cisplatin, following a summary of the main types of cisplatin-loaded NPs. Application examples of cisplatin determination in NPs are provided, and the key features of each quantification strategy are compared. In addition, NP-based electrochemical sensors are included as an emerging approach for characterizing cisplatin loaded in NPs. Rational selection of an appropriate cisplatin determination method for NPs according to the quantification principle and specific drug-delivery settings is highly recommended.

Whether regulation of protein synthesis, autophagy, and regeneration are involved in slow/fast muscles in two disuse models (hindlimb unloading Sprague-Dawley rats ( Rattus norvegicus domestica (Berkenhout, 1769))) (HLU) and hibernating ground squirrels ( Spermophilus dauricus Brandt, 1843 (HIB)) is still unclear. Our results showed (1) fiber cross-sectional area was reduced in soleus (SOL) and extensor digitorum longus (EDL) of HLU whereas no change in HIB. The satellite cells/fiber was reduced and myonuclei/fiber was increased in SOL of HLU, while the percentage of satellite cells was significantly reduced in EDL of HIB. (2) Protein levels of phosphorylated- (P-)Akt, mTORC1, P-mTORC1, and P-S6K1 were reduced in SOL of HLU, whereas phosphorylated S6K1 was increased only in EDL of HIB. (3) Myostatin decreased in EDL of HLU but decreased in SOL of HIB. (4) Beclin1 increased in EDL of HLU and in SOL of HIB. (5) The activity of cathepsin L increased in SOL of HIB. (6) Collagen III increased in both SOL and EDL of HLU, but myogenin and collagen III increased in SOL, and collagen III was reduced in EDL of HIB. Taken together, Akt-mTORC1, Beclin1, and myogenin signaling showed muscle-specific responses in slow-twitch versus fast-twitch muscles, which may contribute to disuse atrophy in non-hibernators and anti-atrophy in hibernators.

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.

Hibernating mammals are natural models of resistance to ischemia, hypoxia‐reperfusion injury, and hypothermia. Daurian ground squirrels (spermophilus dauricus) can adapt to endure multiple torpor‐arousal cycles without sustaining cardiac damage. However, the molecular regulatory mechanisms that underlie this adaptive response are not yet fully understood. This study investigates morphological, functional, genetic, and metabolic changes that occur in the heart of ground squirrels in three groups: summer active (SA), late torpor (LT), and interbout arousal (IBA). Morphological and functional changes in the heart were measured using hematoxylin‐eosin (HE) staining, Masson staining, echocardiography, and enzyme‐linked immunosorbent assay (ELISA). Results showed significant changes in cardiac function in the LT group as compared with SA or IBA groups, but no irreversible damage occurred. To understand the molecular mechanisms underlying these phenotypic changes, transcriptomic and metabolomic analyses were conducted to assess differential changes in gene expression and metabolite levels in the three groups of ground squirrels, with a focus on GO and KEGG pathway analysis. Transcriptomic analysis showed that differentially expressed genes were involved in the remodeling of cytoskeletal proteins, reduction in protein synthesis, and downregulation of the ubiquitin‐proteasome pathway during hibernation (including LT and IBA groups), as compared with the SA group. Metabolomic analysis revealed increased free amino acids, activation of the glutathione antioxidant system, altered cardiac fatty acid metabolic preferences, and enhanced pentose phosphate pathway activity during hibernation as compared with the SA group. Combining the transcriptomic and metabolomic data, active mitochondrial oxidative phosphorylation and creatine‐phosphocreatine energy shuttle systems were observed, as well as inhibition of ferroptosis signaling pathways during hibernation as compared with the SA group. In conclusion, these results provide new insights into cardio‐protection in hibernators from the perspective of gene and metabolite changes and deepen our understanding of adaptive cardio‐protection mechanisms in mammalian hibernators.

Loss of bone mass can occur in mammals after prolonged disuse but the situation for hibernators that are in a state of torpor for many months of the year is not yet fully understood. The present study assesses the bone remodeling mechanisms present in Daurian ground squirrels (Spermophilus dauricus) during hibernation as compared with a model of hindlimb disuse. Differences in microstructure, mechanical properties, bone remodeling-related proteins (Runx2, OCN, ALP, RANKL, CTK and MMP-9) and key proteins of Wnt/β-catenin signaling pathway (GSK-3β and phospho-β-catenin) were evaluated in ground squirrels under 3 conditions: summer active (SA) vs. hibernation (HIB) vs. hindlimb unloaded (HLU). The results indicated that the body weight in HLU ground squirrels was lower than the SA group, and the middle tibia diameter in the HLU group was lower than that in SA and HIB groups. The thickness of cortical and trabecular bone in femurs from HLU ground squirrels was lower than in SA and HIB groups. Most parameters of the tibia in the HLU group were lower than those in SA and HIB groups, which indicated cortical bone loss in ground squirrels. Moreover, our data showed that the changes in microscopic parameters in the femur were more obvious than those in the tibia in HLU and HIB ground squirrels. The levels of Runx2 and ALP were lower in HLU ground squirrels than SA and HIB groups. The protein levels of OCN were unchanged in the three groups, but the protein levels of ALP were lower in the HLU group than in SA and HIB groups. RANKL, CTK and MMP-9 protein levels were significantly decreased in tibia of HLU ground squirrels as compared with SA and HIB groups. In addition, the protein expression levels of RANKL, CTK and MMP-9 showed no statistical difference between SA and HIB ground squirrels. Thus, the mechanisms involved in the balance between bone formation and resorption in hibernating and hindlimb unloading ground squirrels may be different. The present study showed that in femur, the Wnt signaling pathway was inhibited, the protein level of GSK-3β was increased, and the protein expression of phospho-β-catenin was decreased in the HIB group as compared with the SA group, which indicates that the Wnt signaling pathway has a great influence on the femur of the HIB group. In conclusion, the natural anti-osteoporosis properties of Daurian ground squirrels are seasonal. The squirrels do not experience bone loss when they are inactive for a long time during hibernation, but the mechanisms of anti-osteoporosis did not work in HLU summer active squirrels.

This study compared the effects on bone metabolism and morphology of pathological obesity induced by excessive fat intake in a non-hibernator (mice) versus

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.

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.

Background This study compared effects on bone metabolism and morphology of pathological obesity induced by excessive fat intake in a non-hibernator (mice) versus healthy obesity due to pre-hibernation fattening in a hibernator (ground squirrels). Methods Kunming mice were fed a high-fat diet for 3 months to provide a model of pathological obesity (OB group). Daurian ground squirrels fattened naturally in their pre-hibernation season (PRE group) were used as a healthy obesity model. Body weight and adipose tissue wet weight were measured. Micro-computed tomography and three-point bending tests were used to determine the microstructure and mechanical properties of bone. Western blots were used to analyze protein expression levels related to bone formation (RunX2, OCN, ALP), bone resorption (RANKL, Cathepsin K, MMP9) and Wnt signaling (P-β-catenin, GSK-3β). Results Micro-CT showed that there was no obvious bone loss in the OB group, compared with controls, whereas bone formation in the PRE group was enhanced, compared to summer-active squirrels. Results of three-point bending tests showed that stiffness of the femur in OB mice was significantly enhanced, whereas the mechanical properties of bone in the PRE group did not change. Analysis of bone protein expression showed significantly increased expression levels of ALP, OCN, RANKL, MMP9, Cathepsin K, GSK-3β and P-β-catenin in OB mice, but RunX2 expression did not change. By contrast, PRE ground squirrels, showed significantly increased expression of most proteins except for OCN that decreased significantly, and P-β-catenin that did not change. Conclusion For non-hibernating mice, moderate obesity had a certain protective effect on bones, demonstrating two-way regulation, enhancing both bone loss and bone formation, so that bone metabolism was at a higher level of metabolic balance. For pre-hibernating ground squirrels, the healthy obesity acquired before hibernation had a positive effect on the microstructure of bones and also enhanced the expression levels of proteins related to bone formation, bone resorption and Wnt signaling.

To determine that differential bone remodeling mechanism (especially Wnt signaling) in hindlimb unloaded rats and hibernating Daurian ground squirrels, the bone microstructure, mechanical properties, and expression levels of bone remodeling related proteins and key proteins of Wnt/β-catenin signaling were analyzed in this study. The thickness of cortical and trabecular bone was decreased in femur of hindlimb unloaded rats, while it was maintained in femur of hibernating ground squirrels. Interestingly, the ultimate bending energy and ultimate normalized displacement were reduced and the bending rigidity was increased in tibia of hibernating ground squirrels. Besides, the protein level of Runx2 was decreased in femur and tibia of unloaded rats, while it was maintained in tibia and even increased in femur of hibernating ground squirrels. The protein levels of RANKL and MMP-9 were increased in femur and tibia in unloaded rats, while they were maintained in both femur and tibia of hibernating ground squirrels. The protein level of GSK-3β was increased in femur and tibia of unloaded rats, while it was maintained in both femur and tibia of hibernating ground squirrels. The phospho-β-catenin expression was increased in both femur and tibia of unloaded rats, while it was only decreased in femur, but maintained in tibia of hibernating ground squirrels. In conclusion, the femur and tibia in hindlimb unloaded rats showed obvious bone loss, while they mitigated disuse-induced bone loss in hibernating ground squirrels, involving differential protein expression of key molecules in bone remodeling. In comparison with hindlimb unloaded rats, promoting osteoblast differentiation through activating canonical GSK-3β/β-catenin signaling involving Runx2 might be an adaptation to natural disuse in femur of hibernating Daurian ground squirrels. However, there was no statistical change in the protein levels of bone formation related proteins, GSK-3β and phospho-β-catenin in tibia of hibernating Daurian ground squirrels.