). A similar result was also observed in western blot analysis that the protein expression of BCL2 was significantly (P < 0.05) lower in the VDRA siRNA 3+LPS and VDRB siRNA 1+LPS than that in the NC siRNA+LPS group ( Figure 3D, F).

Autophagy-related parameters after VDR RNAi in vivo
The injection of LPS prominently (P < 0.05) reduced the gene expression of MAP1LC3B and ATG16L1 in the intestine of the turbot. Compared with the NC siRNA+LPS group, the knockdown of VDRA&B led to a significant (P < 0.05) reduction of gene and protein expression of MAP1LC3B and ATG16L1( Figures 3C, D, F).

Discussion
According to the previous diagnostic criteria of enteritis in Atlantic salmon (Salmo salar L.) (37) and turbot (38), typical intestinal enteritis histomorphology was observed in the VD 3 deficiency group in terms of shortened mucosal fold, disordered goblet cells, and widened lamina propria. Besides, a reduced expression of tight junction proteins, decreased mucin secretion, overexpression of pro-inflammatory cytokines, and suppressed immune response were also involved in the pathological process of enteritis in fish (3,4,6,39). In the present study, suppressed secretion of intestinal mucus and lower expression of tight junction protein led to the intestinal mucosal barrier dysfunction in turbot, which was also observed in vitro (reduction in the gene expression of tight junction proteins). Moreover, the mRNA levels of pro-inflammatory and antiinflammatory cytokines in VD 3 deficiency treatment were significantly enhanced or suppressed in vivo and in vitro, respectively. In addition, the in vivo results showed that VD 3 deficiency contributed to the reduced intestinal immune-related enzyme activities of ACP, ALP, and LYZ activities. These results showed that the deficiency of VD 3 in diet could induce intestinal enteritis of turbot, which was consistent with the previous studies that diets with sufficient VD 3 are the basis for maintaining intestinal immune function, and VD 3 deficiency provoked an intestinal inflammatory response in European sea bass (Dicentrarchus labrax L.) and Chinese mitten crabs (5,8).
In the present study, VD 3 deficiency in diet activated the NF-kB signalling pathway of turbot by blocking NF-kB nuclear translocation and reducing IkBa phosphorylation. Generally, activation of NF-kB signalling activates the inflammasome by promoting the expression of the inflammasome adaptor ASC, which may also lead to increased expression and secretion of interleukins (40). In the present study, dietary administration of VD 3 suppressed intestinal inflammation by down-regulating the NF-kB/inflammasome signalling. In male Sprague-Dawley rats, the treatment of VD 3 suppressed the exercise-induced muscle inflammation through the modulation of MAPK and NF-kB involved with VDR (41). The study in abalone (Haliotis discus hannai) also showed that VD 3 could inhibit inflammation by significantly decreasing the phosphorylation of IKK and IkB and further blocking nuclear translocation of NF-kB (32).
A steady accumulation of studies showed that apoptosis and autophagy are often regulated by similar pathways and usually cooperated in a balanced interplay or facilitated cellular destruction in a complementary fashion (42). Effects of VD 3 on apoptosis are diverse in different studies because of the different experimental procedures, concentrations of VD 3, and the species of animals. On the one hand, VD 3 could increase apoptosis in the treatment of cancer, obesity, and inflammatory bowel disease (43-45). On the other hand, studies in the murine showed that the exogenous VD 3 attenuated the cell apoptosis in LPS-induced lung injury and hippocampal apoptosis induced by kainic acid and pentylenetetrazol (46,47). DNA fragmentation assay is an apparent indicator of apoptosis, and one of the most distinctive characteristics is the 180 to 200 bp fragments (35). Under the inflamed circumstance in the intestine, diets with VD 3 supplementation prominently decreased the relative density of the DNA ladder, especially the 180 to 200 bp DNA band, indicating that dietary VD 3 could reduce the intestinal apoptosis of turbot in this study. Similarly, the TUNEL assay showed that VD 3 reduced the fluorescence intensity of positive apoptotic nuclei, indicating that the administration of VD 3 in turbot was crucial to ameliorate DNA damage in apoptosis. In the process of apoptosis activation, BCL2 blocked the release of cytochrome C by preventing the pores in the mitochondrial outer membrane formed by the combo of BAX and BCL2 Antagonist/Killer (BAK) proteins, which inhibited the activation of the caspases for dismantling the cell (48-52). In this study, VD 3 deficiency resulted in the higher expression of CASP3 and BAX, and the lower expression of BCL2. Similar results were also observed in the study on MCF-7 breast cancer cell line that the regulation of VD 3 on apoptosis was BAX and BCL2 depended (53). The present results indicated that intestinal apoptosis could be enhanced in response to the aggressive intestinal inflammation induced by VD 3 deficiency, and this process relied on activating BAX/BAK combo and inhibiting BCL2. A u t o p h a g y w i t h i n t h e e p i t h e l i u m c o n t r o l l e d inflammation-induced apoptosis and barrier integrity to limit chronic intestinal inflammation (54). ATG16L1 contributed to the addition of lipid moieties to the ubiquitin-like molecule MAP1LC3B, which promoted autophagosome formation and function (55). The present results showed that VD 3 deficiency suppressed the expression of ATG16L1 and MAP1LC3B, and confocal images of MAP1LC3B fluorescence in vitro showed that the fluorescence reaction was significantly reduced without VD 3 stimulation. And the reduction of intestinal autophagy status could be alleviated by the addition of VD 3. Taken these results together, VD 3 can suppress the activation of apoptosis but enhance autophagy in turbot. Moreover, the mutually exclusive cellular states of apoptosis and autophagy were also observed in abalone, in which dietary VD 3 can significantly increase the expression of autophagy-related proteins but decrease the expression of apoptosis-related proteins (32).
Previous studies in mammals demonstrated that the VDR was involved in the inhibition of NF-kB nuclear translocation, which suppressed the expression of pro-inflammatory cytokines (56,57). VDR also plays an important role in apoptosis. For example, the VDR/ERK signalling pathway inhibited the apoptotic cascade in hippocampal CA1 neurons of global cerebral ischemia rats (58). VDR can also inhibit high glucose-induced endothelial cell apoptosis by inhibiting oxidative stress (59). In addition, VDR may play a crucial role in vitamin D regulation of autophagy in hepatitis C virus viral infection (60). The current results showed that the gene expression of VDR was up-regulated by dietary VD 3, and the knockdown of VDR in turbot aggravated the LPS-induced intestinal inflammatory response and apoptosis, while the reduced autophagy led by the VDR silencing was further declined. Thus, VD 3 /VDR is involved in alleviating the intestinal inflammatory response of turbot and maintaining the dynamic balance of apoptosis and autophagy.

Conclusion
The present study showed that VD 3 deficiency in diet could induce intestinal enteritis of turbot. The inflammatory response caused by VD 3 deficiency was regulated by the NF-kB/ inflammasome pathway; interestingly, the regulation of VD 3 on intestinal inflammatory response may be related to intestinal epithelial cell apoptosis and autophagy, establishing an antagonism of apoptosis and autophagy. Further, the regulation of VD 3 on the inflammatory response, apoptosis, and autophagy was VDR-depended. Besides, excessive VD 3 (4000 IU/kg) did not adversely impact turbot as in mammals, and further research relating to VD 3 metabolism is needed to explain these results.

Data availability statement
The original contributions presented in the study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding authors.

Ethics statement
The animal study was reviewed and approved by Institutional Animal Care and Use Committee of the Ocean University of China.Derek Kirkup is a film composer with a unique understanding of story, producing evocative and heartfelt music with a diverse array of influences.
Living and breathing music from a young age, he was writing and producing layered, multi-track recordings by the age of 13, using only a twin tape deck. Throughout his teens he continued to develop as songwriter and producer, creating lush soundscapes of organic and electronic instruments, drawing influence from atmospheric artists such as Nine Inch Nails and Sigur Ros.
Before specialising in music for narrative film, he spent five years as a Samaritan, listening to, understanding and empathising with the real-life protagonists in some of the deepest and most powerful stories around. He gathered extraordinary insights through the lives of others, learning to confront, explore and empathise with the essential, emotional struggle of the human underneath.
Later he would build upon this understanding within the context of story by studying scriptwriting and working as a scriptreader for Hollywood studio Eclectic Pictures, (producers of Lovelace, Septembers of Shiraz), all the while learning and developing his sound as composer and producer.
All of this experience has helped shape a highly sensitive composer, able to dig into a story and write music from the very core outwards.
Derek’s recent credits include Carl Mackenzie’s acclaimed drama about child abduction, “The Interrogation of Olivia Donovan”, and the short horror “Hushy Bye”, directed by the