HNF4α improves hepatocyte regeneration by upregulating PXR.

Hepatocyte nuclear factor 4 alpha (HNF4α) and the pregnane X receptor (PXR) are involved in hepatocyte regeneration. It is not clear whether HNF4α is involved in hepatocyte regeneration through the regulation of PXR. This study aims to explore the regulatory relationship between HNF4a and PXR, and whether it affects hepatocyte regeneration. A mouse PXR gene reporter and an HNF4α overexpression plasmid were constructed and transfected into mouse hepatoma cells (Hepa1-6). Overexpression of HNF4α, detection of the PXR gene reporter fluorescence value, PXR gene, and protein expression analysis were conducted to explore the regulatory relationship between HNF4α and PXR. Apoptosis and cell cycle data were measured to verify whether HNF4α is involved in hepatocyte regeneration through PXR. The luciferase gene reporter assay results indicated when HNF4α was overexpressed, the fluorescence value of the PXR gene reporter was higher than that in the control at 24 h. With increasing HNF4α expression, the PXR gene and protein expression increased, indicating that HNF4α binds to the PXR promoter and upregulates PXR expression. Apoptosis and cell cycle analysis results demonstrated that when the expression of HNF4α increased, the expression of PXR increased, the apoptosis rate decreased, and the proliferation rate increased. Meanwhile, when the upward trend of PXR gene expression was inhibited by ketoconazole, the proliferation rate decreased. By inhibiting HNF4α and creating a partial hepatectomy (PHx), we demonstrated that HNF4α can upregulate PXR to promote liver regeneration in vivo. Therefore, HNF4α is shown to improve hepatocyte regeneration by upregulating PXR, which provides a reference for future research on the combined application of drugs for the treatment of liver injury.

Isolation and Identification of a Tibetan Pig Porcine Epidemic Diarrhoea Virus Strain and Its Biological Effects on IPEC-J2 Cells.

Porcine epidemic diarrhoea virus (PEDV) is a coronavirus that can cause severe watery diarrhoea in piglets, with high morbidity and mortality rates, seriously hindering the healthy development of the global swine industry. In this study, we isolated a strain of PEDV from Tibetan pigs and named it CH/GS/2022. Subsequently, we screened the apoptosis signals of PEDV-infected IPEC-J2 cells and studied the correlation between apoptosis signals and cell apoptosis. The results showed that different infections of PEDV induced different degrees of apoptosis in cells, and PEDV-induced cell apoptosis was dose-dependent. We then detected the expression of the p53, p38, JNK, Bax, and Bcl-2 genes in the apoptosis signal pathway. The results showed that 24 h after PEDV infection, the expression of the p53, p38, JNK, and Bax genes in IPEC-J2 cells increased significantly, while the expression of the Bcl-2 gene decreased significantly (p < 0.05). Subsequently, we used Western blot to detect the protein levels of these five genes, and the results showed that PEDV infection upregulated the expression of p53, p38, JNK, and Bax proteins (p < 0.05) while downregulating the expression of Bcl-2 protein (p < 0.05). Thus, it was initially inferred that PEDV infection could regulate cell apoptosis by activating the p53, p38, and JNK signalling pathways. Finally, we further investigated the apoptosis of the cells through the use of inhibitors. The results indicated that the p53 inhibitor Pifithrin-α has a significant inhibitory effect on the expression of the p53 protein after PEDV infection and can reverse the expression levels of Bax and Bcl-2 proteins. This suggested that p53 is involved in PEDV-induced cell apoptosis. Similarly, the p38 MAPK inhibitor SB203580 has an inhibitory effect on the expression of the p38 protein and can reverse the expression levels of Bax and Bcl-2 proteins. This suggested that p38 is also involved in PEDV-induced cell apoptosis. On the other hand, the JNK inhibitor SP600125 has no inhibitory effect on the expression of the JNK protein after PEDV infection, but the expression levels of Bax and Bcl-2 proteins have changed. Furthermore, it is noteworthy that SP600125 can inhibit the activity of apoptotic proteins but not their levels, resulting in reduced cell apoptosis. These preliminary results indicated that JNK may be involved in PEDV-induced IPEC-J2 cell apoptosis.

Molecular detection and phylogenetic analysis of porcine circovirus type 3 in Tibetan pigs on the Qinghai-Tibet Plateau of China.

BACKGROUND: Porcine circovirus type 3 (PCV3) has been confirmed to infect pigs, posing a health risk and making pigs more susceptible to other pathogens. After the first report of PCV3 infection in the United States, its prevalence was determined in pigs suffering from clinical digestive or respiratory diseases in several other regions, including the Sichuan and Gansu provinces of China. In this study, we describe the frequency of PCV3 detection in Tibetan pigs inhabiting three different provinces surrounding the Qinghai-Tibet Plateau of China. METHODS: A total of 316 samples from diarrheic animals and 182 samples from healthy animals were collected in a randomized manner. Conventional PCR was applied for PCV3 DNA detection. The conserved regions of the PCV3 gene were analyzed with MEGA 7.1 software to design specific primers to sequence entire Cap genes in PCV3 strains, and the sequences were then used to confirm the subtypes of PCV3 in the positive samples. Prediction of the amino acid sequences by nucleotide sequence translation was also performed to compare the point mutations in the entire Cap protein. Twenty PCV3 whole-genomic sequences were used for genome phylogenetic analyses of PCV3 and sequence alignments with 22 other reference strains. RESULTS: We found that the prevalence of the virus was significantly higher in samples from pigs with diarrhoea than that in samples from healthy pigs. Phylogenetic analysis of Cap proteins demonstrated that the 20 PCV3 strains formed three clades, including PCV3a (8/20, 40.00%), PCV3b (5/20, 25%) and PCV3c (7/20, 35.00%). The complete genome sequence revealed that these strains formed one branch in the phylogenetic tree. Sequence analysis showed that the Cap proteins of the 20 different viral strains shared between 95.84 and 99.18% nucleotide identity. Cap protein sequence analyses showed that the positivity rate of PCV3a was highest in the samples from pigs with diarrhoea. In comparison, PCV3c was the most elevated subtype in the healthy samples. There was no mutation at a specific site in the amino acid sequences of the entire Cap protein from different PCV3 subtype strains from heathy samples. There was a mutation at site 113 in PCV3a, site 129 in PCV3b, and site 116 in PCV3c. CONCLUSION: Our present data provide evidence that PCV3 is prevalent in Tibetan pigs at high altitudes in China, and the higher prevalence rates of the PCV3a and PCV3b subtypes in samples from pigs with diarrhoea further indicate that the genotypes should not be neglected during surveys of the pathogenicity of PCV3. Phylogenetic and genetic diversity analyses suggested that the continuous evolution, adaptation and mechanisms of pathogenicity of PCV3 in Tibetan pigs living in this special environment should be further studied.

Hypoxia-Inducible Factor 1α Affects Yak Oocyte Maturation and Early Embryonic Development by Regulating Autophagy.

In animal assisted reproductive technology, the production of high-quality oocytes is crucial. The yak, having lived in the Qinghai-Tibet Plateau for an extended period, has reproductive cells that are regulated by hypoxia-inducible factor 1α (HIF-1α). This study aimed to investigate the impact of HIF-1α on yak oocyte maturation and early embryonic development in vitro through the regulation of autophagy. The in vitro maturation process of yak oocytes involved the addition of the HIF-1α inducer DFOM and the inhibitor LW6 to examine their effects on yak oocyte maturation, early embryonic development, cell autophagy, cytochrome P450s (CYP450s) enzyme expression, and cumulus diffusion factors. The findings revealed that DFOM significantly upregulated the expression of HIF-1α, resulting in increased the cumulus diffusion area, elevated first polar body expulsion rate of oocytes, enhanced mitochondrial and actin levels, decreased ROS production, and reduced early apoptosis levels of oocytes. Moreover, DFOM promoted the expression of autophagy-related proteins, CYP450s enzymes, and cumulus diffusion factors, thereby enhancing oocyte maturation and early embryonic development. Conversely, LW6 exhibited opposite effects. The inhibition of autophagy levels with 3-MA during DFOM treatment yielded similar outcomes. Furthermore, reducing autophagy led to increased apoptosis levels at all stages of early embryonic development, as well as a significant decrease in total cell number and ICM/TE ratio of blastocysts. Studies have shown that during the in vitro maturation of yak oocytes, HIF-1α can affect the cumulus expansion area of oocytes by regulating autophagy, the first polar body excretion rate, mitochondrial level, actin level, ROS and early apoptosis level, the CYP450s enzyme, and the expression of cumulus expansion factors, thereby improving the in vitro maturation and early embryonic development of yak oocytes. These findings offer valuable insights into the reproductive regulation mechanism of yaks in hypoxic environments and suggest potential strategies for the advancement of yak assisted reproductive technology.