The gene AccCyclin H mitigates oxidative stress by influencing trehalose metabolism in Apis cerana cerana.

BACKGROUND: Environmental stress can induce oxidative stress in Apis cerana cerana, leading to cellular oxidative damage, reduced vitality, and even death. Currently, owing to an incomplete understanding of the molecular mechanisms by which A. cerana cerana resists oxidative damage, there is no available method to mitigate the risk of this type of damage. Cyclin plays an important role in cell stress resistance. The aim of this study was to explore the in vivo protection of cyclin H against oxidative damage induced by abiotic stress in A. cerana cerana and clarify the mechanism of action. We isolated and identified the AccCyclin H gene in A. cerana cerana and analysed its responses to different exogenous stresses. RESULTS: The results showed that different oxidative stressors can induce or inhibit the expression of AccCyclin H. After RNA-interference-mediated AccCyclin H silencing, the activity of antioxidant-related genes and related enzymes was inhibited, and trehalose metabolism was reduced. AccCyclin H gene silencing reduced A. cerana cerana high-temperature tolerance. Exogenous trehalose supplementation enhanced the total antioxidant capacity of A. cerana cerana, reduced the accumulation of oxidants, and improved the viability of A. cerana cerana under high-temperature stress. CONCLUSION: Our findings suggest that trehalose can alleviate adverse stress and that AccCyclin H may participate in oxidative stress reactions by regulating trehalose metabolism. © 2023 Society of Chemical Industry.

RNAi-mediated silencing of AccCYP6k1 revealed its role in the metabolic detoxification of Apis cerana cerana.

Insect cytochrome P450 monooxygenases (P450s or CYPs) perform important functions in the metabolic detoxification of both endogenous and exogenous substrates. However, the mechanism of action of the P450 genes in bees is unclear. In this study, we investigated the effects of AccCYP6k1 on the metabolism and detoxification of Apis cerana cerana. Spatiotemporal expression profiling revealed that the expression of AccCYP6k1 was the highest in foragers (A15) and was mainly expressed in the leg, midgut and head. RT-qPCR results showed that AccCYP6k1 exhibited different expression patterns following exposure to xenobiotics. In addition, silencing AccCYP6k1 increased the pesticides sensitivity and affected the detoxification system and antioxidant process of A. cerana cerana. In brief, the induced expression of AccCYP6k1 is related to the resistance of A. cerana cerana, while knockdown AccCYP6k1 affect the pesticides resistance and metabolic detoxification system of A. cerana cerana. These findings not only support the theoretical basis of metabolic detoxification in bees but also provide a better understanding of P450-mediated resistance to pesticides in insects.

Identification of the cuticle protein AccCPR2 gene in Apis cerana cerana and its response to environmental stress.

Cuticular proteins (CPs) are known to play important roles in insect development and defence responses. The loss of CP genes can lead to changes in insect morphology and sensitivity to the external environment. In this study, we identified the AccCPR2 gene, which belongs to the CPR family (including the R&R consensus motif) of CPs, and explored its function in the response of Apis cerana cerana to adverse external stresses. Our results demonstrated that AccCPR2 was highly expressed in the late pupal stage and epidermis, and the expression of AccCPR2 may be induced or inhibited under different stressors. RNA interference experiments showed that knockdown of AccCPR2 reduced the activity of antioxidant enzymes, led to the accumulation of oxidative damage and suppressed the expression of several antioxidant genes. In addition, knockdown of AccCPR2 also reduced the pesticide resistance of A. cerana cerana. The overexpression of AccCPR2 in a prokaryotic system further confirmed its role in resistance to various stresses. In summary, AccCPR2 may play pivotal roles in the normal development and environmental stress response of A. cerana cerana. This study also enriched the theoretical knowledge of the resistance biology of bees.

FGF2 positively regulates osteoclastogenesis via activating the ERK-CREB pathway.

Fibroblast growth factor 2 (FGF2) plays crucial roles in the growth and development of several tissues. However, its function in bone homeostasis remains controversial. Here, we found that exogenous FGF2 supplementation inhibited the mineralization of bone marrow stromal cells (BMSCs), at least partially, via up-regulating the gene expression of osteoclastogenesis. The FGF receptor (FGFR) allosteric antagonist SSR128129E modestly, whereas the FGFR tyrosine kinase inhibitor AZD4547 significantly antagonized the effects of FGF2. Mechanistically, FGF2 stimulated ERK phosphorylation, and the ERK signaling inhibitor PD325901 strongly blocked FGF2 enhancement of osteoclastogenesis. Moreover, the phosphorylation of CREB was also activated in response to FGF2, thereby potentiating the interaction of p-CREB with the promoter region of Rankl gene. Notably, FGF2-deficient BMSCs exhibited higher mineralization capability and lower osteoclastogenic gene expression. Correspondingly, FGF2-knockout mice showed increased bone mass and attenuated expression of osteoclast-related markers, which were associated with moderate inhibition of the ERK signaling. In conclusion, FGF2 positively regulates osteoclastogenesis via stimulating the ERK-CREB pathway. These findings establish the importance of FGF2 in bone homeostasis, hinting the potential use of FGF2/ERK/CREB specific inhibitors to fight against bone-related disorders, such as osteoporosis.

Identification and characterization of an Apis cerana cerana nucleoside diphosphate kinase (AccNDPK) associated with oxidative stress.

Nucleoside diphosphate kinases (NDPKs) are widespread nucleotide-metabolizing enzymes that are involved in a variety of biological processes, including responses to oxidative stress. Although studies have been conducted on NDPKs in mammals and some plants, there is scant research on insect NDPKs, especially in honey bees. In the present study, we isolated AccNDPK from Apis cerana cerana. Sequence analysis showed that AccNDPK has high homology with many NDPKs and contains a highly conserved NDPK active site motif. Based on phylogenetic analysis, AccNDPK has a relatively recent evolutionary relationship with NDPKs in other hymenopteran insects. AccNDPK was found to be highly expressed in newly emerged honey bees and muscle tissues, and RT-qPCR analysis and bacteriostatic assays showed that the expression level of AccNDPK is affected by abnormal temperature, UV light, H2O2, heavy metals, and various pesticides. After AccNDPK knockdown, antioxidant-related genes, including AccCAT, AccCYP4G11, AccGSTS4, AccTpx1 and AccMsrA, were upregulated, whereas AccGSTD, AccGST1, AccHSP22.6 and AccTrx1 were downregulated. Furthermore, catalase (CAT), superoxide dismutase (SOD), and peroxidase (POD) activities were significantly increased, and the tolerance of bees to oxidative stress caused by cyhalothrin was reduced by silencing of AccNDPK. Given these findings, we speculate that AccNDPK plays an important role in the oxidative stress response of A. cerana cerana.

The interferon-inducible protein p205 acts as an activator in osteoblast differentiation of mouse BMSCs.

p205, an interferon-inducible protein, is induced in the course of osteogenesis in mouse bone marrow stromal cells (BMSCs). Knocking down p205 markedly impairs whereas overexpressing p205 enhances the osteoblast differentiation of BMSCs, as revealed by the altered expression of osteogenic genes, the change of ALP activity and the ARS-stained mineral nodules. The positive role of p205 in BMSC osteogenesis is probably due, at least in part, to the association of it with Id proteins. Further investigation indicated that p205 may disturb the formation of Runx2/Ids complex and free more Runx2 to induce the differentiation process. Taken together, our findings demonstrated for the first time that p205 functions as an activator in osteoblast differentiation.

Role of interferon-inducible protein 202 (p202) in the regulation of adipogenesis in mouse adipose-derived stem cells.

The interferon-inducible protein 202 (p202) has emerged as a key regulator of cell proliferation and differentiation. To explore the role of p202 in adipocyte differentiation, p202 mRNA and protein levels in differentiating mouse adipose-derived stem cells (mASCs) were examined, and were found to continuously increase during mASC adipogenesis. The nuclear and cytoplasmic distribution of p202 in the differentiation process was also determined. In addition, suppression and overexpression of p202 impaired and enhanced the differentiation process, respectively. Further, results of co-immunoprecipitation and co-immunofluorescence showed the interaction and intracellular co-localization of p202 with C/EBPß, C/EBPα, and PPARγ at intermediate and/or late differentiation stages. Knockdown of p202 interfered with the elevated expression of C/EBPß, C/EBPα, and PPARγ. In conclusion, the temporal and spatial profiles of p202 and the observed manner in which p202 affected the expression of these transcription factors provided evidence that p202 plays a role during mASC adipogenesis.

Cholesterol loading affects osteoblastic differentiation in mouse mesenchymal stem cells.

Effects of cholesterol on osteoblastic differentiation was evaluated in mouse bone marrow derived mesenchymal stem cells (MSCs). Cholesterol-treated MSCs showed a stimulated differentiation process with induced mRNA and protein levels of osteogenic lineage markers, increased alkaline phosphatase (AKP) activity and more mineralized nodules. However, the stimulation extent was reduced when incubating the cells with cholesterol plus the ACAT (acyl-CoA:cholesterol acyltransferase) inhibitor Sandoz58035 or SiRNA-ACAT1 [which blocks the esterification of free cholesterol (FC) to cholesteryl ester (CE)], indicating the osteogenic potency of cholesterol was mostly due to CE levels. The key role of BMP2 and Runx2 in the effects of cholesterol on MSC osteogenesis was elucidated. These results point to cholesterol as a modulator of osteoblastic differentiation, which separate cholesterol itself from other components of modified lipoproteins.

Cholesterol suppresses adipocytic differentiation of mouse adipose-derived stromal cells via PPARγ2 signaling.

The effects of cholesterol on cell proliferation and adipocytic differentiation have been evaluated for the first time in mouse adipose-derived stromal cells (mASCs). Cholesterol loading by using cholesterol:methyl- ß-cyclodextrin (Chol:MßCD) complexes promoted cellular levels of free cholesterol (FC) and cholesteryl ester (CE), induced high cell proliferation of mASCs dose-dependently. Compared with control cells, cholesterol-treated mASCs showed an impaired differentiation process in both dose- and time-dependent manners, based on reduced oil red O-stained lipid droplets, SREBP-1, PPARγ2 and aP2 expression levels. The involvement of SREBP-1-mediated PPARγ2 in the effects of cholesterol on mASC adipogenesis was elucidated. These results point to cholesterol as a modulator of adipogenesis, which separate cholesterol itself from other components of modified lipoproteins.

[Effects of different planting patterns on farmland soil quality in Yellow River alluvial plain of Shandong Province].

Taking Chiping County in the Yellow River alluvial plain of Shandong Province as study area, a systematical survey was conducted on the 20 parameters of farmland soil physical and chemical properties under wheat/corn rotation, open air vegetable planting, sunlight greenhouse vegetable planting, and plastic shed vegetable planting, aimed to evaluate the effects of different planting patterns on the farmland soil quality in the plain. Significant differences (P < 0.05 or P < 0.01) were observed in the soil pH, soil moisture content, and the contents of soil organic matter, N, P, K, available S and Zn, and total salt under different planting patterns. The soil available P under all tested planting patterns and the soil available S under sunlight greenhouse vegetable planting presented a comparatively higher variability. Different planting patterns had significant effects on the soil quality, with the trend of protected vegetable planting > open air vegetable planting > wheat/ corn rotation. The effects were higher on soil chemical properties than on soil physical properties, and higher on soil organic matter and macronutrients than on soil secondary nutrients. Soil micronutrients were less affected. The main causes for these were the straw-returning of wheat and corn, the application of sulfur-containing and zinc fertilizers, and the long-term high rate fertilization of protected vegetable planting.