YD improved the intestinal physical barrier
The intestinal barrier represents a critical defense mechanism in fish physiology (Lin, et al., 2020). Preserving the structural and functional integrity of the gut is paramount for overall body health (Messina, et al., 2019; Veeraval, et al., 2020). Central to this process are tight junctions and the renewal of intestinal epithelial cells, which collectively maintain tissue homeostasis (Veeraval, et al., 2020). The impact of drugs and nutrition on intestinal structure and function has been widely acknowledged (Yang, et al., 2019; Zhang, et al., 2018). In our study, we observed that exposure to HC led to increased inflammation and mucosal loss in the gut. However, the application of YD notably mitigated these detrimental effects of HC. Earlier investigations have also pointed out HC-induced impairments in intestinal structure in largemouth bass and snakehead fish (Channa argus)(Ding, et al., 2019). The role of the epidermal growth factor receptor (EGFR) was evident, as it promotes the renewal and repair of intestinal epithelial cells, thereby preserving the integrity of the intestinal structure (Van Emburgh, et al., 2014). YD treatment was found to elevate EGFR mRNA levels, thereby ameliorating the damage caused by HC. Consequently, YD appears to support the renewal and repair of intestinal epithelial cells as a defense against the adverse effects of HC. Moreover, certain herbal compounds like curcumin and indole compounds have been shown to enhance tight junction proteins and reduce gut permeability (Ghosh, et al., 2014; Sugimoto, et al., 2016). The tight junction, composed of various proteins such as ZOs, occludin, and claudins, forms a robust physical barrier between adjacent epithelial cells in the intestine (Schumann, et al., 2017; Turner, et al., 2014). This barrier serves to regulate permeability, allowing selective nutrient absorption while preventing the entry of harmful substances into the body circulation (Gil-Cardoso, et al., 2016). Measuring the activity of plasma DAO (diamine oxidase), a highly active intracellular enzyme in the intestinal mucosa, is commonly used to assess intestinal permeability (Zhou, et al., 2021). Our findings demonstrated that HC reduced the relative expression of tight junction genes and increased plasma DAO activity, indicative of heightened intestinal permeability. Similar results were observed in Chinese perch (Siniperca chuatsi) and largemouth bass (Zhang, et al., 2020; Zhou, et al., 2021). Remarkably, YD treatment led to a notable decrease in plasma DAO activity and an increase in mRNA levels of tight junction genes, effectively reducing intestinal permeability.
Apoptosis, a programmed cell death process primarily regulated by the caspases family, plays a pivotal role in the renewal of intestinal epithelial cells(D'Arcy, 2019; Fritsch, et al., 2019). However, when apoptosis becomes excessive, it can lead to damage in the intestinal physical barrier (Fritsch, et al., 2019). The delicate balance of apoptosis is governed by the bcl2 family, with bcl2 promoting the process, while bax inhibits it (D'Arcy, 2019). In the death receptor pathway, caspase 8 serves as a key inducer, activating caspase 3 and ultimately promoting apoptosis (Zhao, et al., 2021a). In our study, we found that HC notably upregulated the expression of pro-apoptosis genes (bcl2, caspase8 and caspase3), indicating an acceleration of apoptosis. Conversely, YD application significantly suppressed the expression of these genes, suggesting that YD mitigates the apoptotic process induced by HC. These findings align with previous research demonstrating HC's ability to promote apoptosis in intestinal epithelial cells of largemouth bass and other carnivorous fish(Zhao, et al., 2021a). The efficacy of YD in alleviating apoptosis and repairing intestinal injury induced by various harmful factors has been attributed to its effective components, such as emodin and chlorogenic acid (Li, et al., 2022; McDonald, et al., 2022). In our current study, YD administration was associated with enhanced tight junction integrity and renewal of intestinal cells, achieved through the reduction of apoptosis. This effect helped to alleviate the structural damage to the gut caused by HC.
In summary, our study elucidates the beneficial effects of YD in mitigating HC-induced damage and apoptosis, highlights the importance of YD in the intestinal barrier.
YD promoted the stability of intestinal microbiota
The intestinal microbiota plays a crucial role in shaping the bio-function of fish(Kokou, et al., 2019). The development of microbiota communities is influenced by both stochastic and deterministic processes, and factors such as nutrition and drugs exert significant effects on their composition (Kokou, et al., 2019; Lewis, et al., 2015; Maslowski, et al., 2011). A stable microbial community is generally characterized by high abundance and diversity, with richness assessed using the Chao1 index, diversity represented by the Shannon index (higher values indicate higher diversity), and the Simpson index indicating lower diversity (Butt, et al., 2019). In our study, we observed that HC reduced the richness and diversity of intestinal microbial communities in comparison to the NC group, consistent with findings reported by Huang et al (Huang, et al., 2021). However, when 0.5-2% YD was applied in combination with HC, it led to an elevation in richness and diversity of intestinal microbial communities (Huang, et al., 2021), demonstrating that YD has the potential to enhance the stability of these communities. Interestingly, YD has been shown to remodel microbial communities and increase butyrate content in the rat intestine, which subsequently improved liver injury induced by carbon tetrachloride (Liu, et al., 2019). Conversely, when 4% YD was used, it decreased the richness and diversity of intestinal microbial communities, potentially due to the presence of antibacterial substances (such as emodin and geniposide) in YD (XunLi, et al., 2019; Zhou, et al., 2019). Excessive YD application may inhibit the growth of both probiotics and harmful bacteria, leading to the reduction of richness and diversity of microbial communities.
In line with other studies in largemouth bass, the dominant phyla of intestinal microbial communities were found to be Firmicutes, Fusobacteriota and Proteobacteria (Huang, et al., 2021; Zhou, et al., 2021). Similarly, the dominant families in the intestine included Mycoplasma, Cetobacterium, Clostridium, Clostridium-senu-stricto, romboutsia, peptostreptococcaceae, plesiomonas. While Mycoplasma was previously considered a harmful bacterium, recent evidence from microbial sequencing technologies has suggested its dominance in fish intestines (Nayak, 2010). However, the specific bio-function of Mycoplasma in fish still requires further exploration. In our study, the mycoplasma is the maximum bacteria and it shared more than 80% abundance in wild salmon intestine microbiota and dominated the microbiota of long-jawed mudsucker (Gillichthys mirabilis) (Bano, et al., 2007; Holben, et al., 2002). However, the bio-function of mycoplasma in fish still needs to be explored. Cetobacterium, found in several fish species, has been associated with promoting carbohydrate utilization and insulin sensitivity through the activation of the parasympathetic nervous system(Wang, et al., 2021). In our study, the abundance of Cetobacterium in high carbohydrate groups (including YD treatment groups) was significantly higher than in the NC group, possibly indicating adaptive remodeling of intestinal microbiota in response to a high carbohydrate diet.
Interestingly, Alsobacter, Sediminibacterium and Norank-f-sulfobacillaceae showed a strong positive correlation with increasing YD concentration, with the highest abundance observed in the H + YD4 group. Alsobacter, with its function in nitrogen removal, has been detected in the benthic zone of breeding ponds and tilapia intestines, but its specific role in the gut remains to be explored (Sun, et al., 2018). Both Sediminibacterium and Norank-f-sulfobacillaceae have been recognized as probiotics capable of maintaining intestinal homeostasis and reducing the abundance of harmful bacteria (Ma, et al., 2020; Wu, et al., 2019). Thus, YD appears to increase the abundance of beneficial intestinal probiotics, thereby alleviating gut injuries through the improvement of the intestinal environment.
In conclusion, our study sheds light on the complex dynamics of the intestinal microbiota in fish, demonstrating how YD application can influence the richness, diversity, and abundance of specific microbial communities. This understanding holds promise for harnessing YD's potential as a beneficial agent in promoting gut health and mitigating gut-related issues in fish populations.
YD relief the ERS, oxidant stress and inflammation
The intestine serves as a crucial digestion and absorption organ, adapting to complex and fluctuating environments (Maslowski, et al., 2011). Unsuitable nutritional factors often lead to endoplasmic reticulum stress (ERS) and oxidative stress, ultimately resulting in inflammation (Zhao, et al., 2021a; Zhou, et al., 2021). The endoplasmic reticulum (ER) plays a vital role in protein synthesis, folding, assembly, and transportation (Ma, et al., 2017; Redhai, et al., 2021). Intestinal epithelial cells face significant challenges due to high-intensity secretion, leading to the accumulation of misfolded and unfolded proteins in the ER, triggering ERS (Kaser, et al., 2010). Glucose-regulated protein 78 (GRP78/Bip) plays a crucial role in folding, assembly, and proper modification of peptide chains and serves as a marker of ERS (Ma, et al., 2017). Stable complexes of GRP78 with transcription factors 6 (ATF6), protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK), and inositol-requiring enzyme 1 (IRE1) are dissociated during ERS occurrence (Redhai, et al., 2021). ATF6 is subsequently transferred to the Golgi membrane where it is spliced to activate the unfolded protein response (UPR) target genes (Iurlaro, et al., 2016). Additionally, PERK dimer activates Eif2 to reduce protein synthesis and promote the transcription of UPR target genes. Moreover, IRE1 dimer splices and activates XBP1 mRNA, promoting the transcription of UPR target genes (Cao, et al., 2014). In our present study, the mRNA levels of GRP78 and ATF6 in the HC group were significantly higher than in the NC group, indicating that HC induced ERS. Previous studies on yellow catfish (Pelteobagrus fulvidraco) and largemouth bass are consistent with the findings in our current study. Remarkably, YD treatment significantly decreased the expression of ERS-related genes (GRP78, IRE1, ATF6, Eif2α, XBP1, and chopα), demonstrating that YD could effectively alleviate the ERS induced by HC. The intestine's critical functions in digestion and absorption make it highly susceptible to stress induced by unsuitable nutrition factors, leading to ERS and inflammation. The ER's role in protein synthesis and folding makes it particularly vulnerable to misfolded protein accumulation. However, YD treatment shows promising results in mitigating ERS by regulating the expression of ERS-related genes.
The intestinal anti-oxidant system serves as a critical defense mechanism in protecting the intestine from the damaging effects of excessive reactive oxygen species (ROS) and hydroxyl radicals (Han, et al., 2021). The Keap1-Nrf2/ARE (anti-oxidation reaction element) pathway plays a pivotal role in the removal of ROS and hydroxyl radicals, with Keap1-Nrf2 being the key regulatory factor in this process (Zhao, et al., 2021b). MDA is a product of lipid peroxidation and is commonly used as a marker of oxidative damage(Zhao, et al., 2021a). In our present study, we observed that HC significantly increased the content of MDA and the mRNA level of keap1 while notably decreasing the enzyme activities of CAT and GPX. These findings demonstrate that HC reduces the intestinal anti-oxidative ability by inhibiting the Keap1-Nrf2/ARE pathway, aligning with previous studies (Zhao, et al., 2021a; Zhou, et al., 2021). Interestingly, YD treatment significantly decreased the content of MDA and noticeably increased the enzyme activities of GPX and CAT, along with an upregulation of the relative expression of CAT. These results indicate that YD plays a prominent role in alleviating oxidant damage induced by HC. Previous research has reported that the main active ingredients in YD, such as geniposide, emodin, chrysophanol, and aloe emodin, have significant curative effects in scavenging oxidation free radicals (Huang, et al., 2017; Liang, et al., 2022). Therefore, YD effectively alleviates intestinal oxidation injuries induced by HC by increasing the enzyme activities of CAT and GPX, as well as by reducing the mRNA level of keap1.
In the complex internal environment, intestinal cells often activate an immune response, leading to increased production of inflammatory factors and immune-related enzyme activities (Kaser, et al., 2010). While suitable inflammation is beneficial for removing harmful substances, persistent and excessive inflammation can lead to tissue impairment (Ma, et al., 2017). In our present study, HC notably increased the activities of immune enzymes and the mRNA level of pro-inflammatory genes, while decreasing the mRNA level of anti-inflammatory genes, indicating that HC induced intestinal inflammation. This observation is in line with previous findings of HC-induced intestinal inflammation in largemouth bass and Nile tilapia (Oreochromis niloticus) (Li, et al., 2020; Zhao, et al., 2021a). Remarkably, our results show that YD treatment notably decreased the number of inflammation cells and the mRNA level of pro-inflammatory genes, while significantly increasing the mRNA level of anti-inflammatory genes, demonstrating that YD has the ability to alleviate intestinal inflammation caused by HC. Previous studies have demonstrated that YD effectively reduces intestinal inflammation with high expression of pro-inflammatory genes (TNFα and NF-κB) caused by dampness-heat syndrome (Li, et al., 2017). Additionally, the main effective contents of YD, such as Dahuang and Zhizi, and their extractions have been shown to prominently decrease the mRNA levels of pro-inflammatory genes and reduce inflammation factors (Chen, et al., 2020; Xin, et al., 2022). Therefore, YD alleviates intestinal inflammation due to its anti-inflammatory effects mediated by its effective contents. In conclusion, the activation of immune response in intestinal cells can lead to inflammation, and HC-induced inflammation in the intestine can be effectively mitigated by YD treatment. YD's anti-inflammatory effects, attributed to its active contents like Dahuang and Zhizi, make it a promising therapeutic agent for promoting intestinal health and reducing inflammation-related issues in fish populations.