Abstract:The half-smooth tongue sole (Cynoglossus semilaevis) is a major mariculture species in northern China, is highly favored for its rapid growth. However, under high-density intensive farming, bacterial diseases break out frequently, which have become a major bottleneck restricting the sustainable development of this industry. Among the most frequently encountered and economically devastating pathogens are Vibrio anguillarum, Vibrio vulnificus, Edwardsiella tarda, and Mycobacterium marinum. Infection can provoke a vigorous liver inflammatory response, and excessive inflammation often leads to pathological apoptosis and consequent liver damage. SHP-1 (Src homology 2 domain-containing protein tyrosine phosphatase-1) is a non-receptor protein tyrosine phosphatase that functions as a negative regulator of immune signaling. It suppresses inflammation through dephosphorylation and modulates apoptosis by regulating Bcl-2 family members in mammals. In teleost fish, research on SHP-1 is relatively limited, existing reports indicate that teleost SHP-1 participates in the negative feedback control of interferon responses, the maintenance of myeloid cell homeostasis, and certain immunosuppressive processes. Our previous work demonstrated that tongue sole SHP-1 exerts anti-inflammatory effects by inhibiting the NF-κB pathway during V. anguillarum infection and is involved in T-cell immunity against M. marinum infection. However, whether SHP-1 contributes to the regulation of apoptosis induced by bacterial infection remains unknown. To investigate the role of SHP-1 in bacterium-induced apoptosis, the present study employed a comprehensive experimental strategy integrating in vivo bacterial challenge models with in vitro cellular assays to systematically dissect the role of SHP-1 in bacterium-induced apoptosis. For the in vivo experiments, healthy half-smooth tongue sole was subjected to single-pathogen infection with V. anguillarum, Vibrio vulnificus, E. tarda, or M. marinum, as well as a mixed-infection group (equal volumes of V. anguillarum, E. tarda, and M. marinum suspensions) to mimic the complex infection environment encountered in aquaculture. The experimental groups were as follows: V. anguillarum group (VA, 1.6×10? CFU/g), Vibrio vulnificus group (VV, 2.5×10? CFU/g), E. tarda group (ET, 6×10? CFU/g), M. marinum group (MM, 1.5×10? CFU/g), mixed-infection group (Mix, equal-volume mixture of VA, ET, and MM, total bacterial load of 1×10? CFU/g), and corresponding PBS control groups. Liver tissues were collected at 24 h post-infection (72 h for the MM group) and stored at -80°C. The expression changes of shp-1 and key genes of apoptosis (bax, bcl-xl, caspase-3, caspase-7, cyt-c and apaf1) in the liver after infection by different pathogenic bacteria were detected by qRT-PCR technology. To further explore the role of SHP-1 in apoptosis, this study used the liver cell line of tongue sole and primary macrophages as the research objects of in vitro cell experiments. Overexpression and knockdown of shp-1 were achieved respectively through shp-1 plasmid transfection and siRNA interference. To induce apoptosis, cells were stimulated with either lipopolysaccharide (LPS, 10 μg/mL) or lipoteichoic acid (LTA, 10 μg/mL) for 24 hours. The functional consequences of shp-1 manipulation on the mitochondrial apoptosis pathway were evaluated by combining qRT-PCR profiling of the above-mentioned genes with Hoechst 33342 fluorescent staining to visualize nuclear morphological changes indicative of apoptosis. The results showed that there were differences in the effects of different pathogenic bacteria infection on the expression of shp-1 in the liver of tongue sole. The expression of shp-1 was significantly upregulated in the VA, VV and Mix groups, while it showed a downward trend in the ET and MM groups; among all the infection groups, the expressions of pro-apoptotic genes bax and caspase-7 were generally upregulated, the expression of anti-apoptotic gene bcl-xl was generally downregulated, while caspase-3 was consistently downregulated in all groups, suggesting that there might be functional differentiation of caspase-7 in tongue sole. Moreover, cyt-c expression increased in the VA, VV, ET, and Mix groups but decreased in the MM group; apaf1 was elevated in the VA, VV, and MM groups yet reduced in the ET and Mix groups. These findings indicate that the expression changes of these genes exhibit pathogen-specific patterns, notably, the robust and consistent upregulation of caspase-7 observed in all groups raises the intriguing possibility that caspase-7 may partially compensate for caspase-3 effector functions in tongue sole. In both tongue sole liver cells and macrophages, LPS stimulation induced upregulation of bax, cyt-c, apaf1, caspase-3, and caspase-7, together with downregulation of bcl-xl. Shp-1 overexpression reversed these changes, upregulating bcl-xl and suppressing the expression of pro-apoptotic genes, whereas shp-1 knockdown further exacerbated the pro-apoptotic expression profile. Hoechst 33342 staining confirmed that shp-1 overexpression alleviated LPS-induced nuclear condensation, while shp-1 knockdown aggravated these morphological hallmarks of apoptosis. The results under LTA stimulation were consistent with those of LPS, indicating that SHP-1 has a broad-spectrum inhibitory effect on mitochondrial apoptosis induced by bacterial components in liver cells and macrophages. In conclusion, SHP-1 mitigates pathological apoptosis during bacterial infection in the tongue sole. This study provides new experimental evidence for understanding immune regulatory mechanisms in teleosts and highlights the application potential of SHP-1 as a promising target for molecular breeding of disease-resistant tongue sole.
