Aquaculture as a fast-growing industry provides almost half of fish on our dining table. However, the bacterial disease in aquaculture causes enormous lost every year. To cure bacterial disease, antibiotics have been applied into feedings of cultured animals. The usage of antibiotics has brought many problems, including water pollution, drug residue and growing of antibiotic resistant bacteria. Traditional Chinses medicine (TCM) has been used for thousands of years with various pharmacology functions. Additionally, the TCM has characters of low toxicity, low price and environmentally friendly, which makes it an ideal replacement of antibiotic in aquaculture. A high throughput screening method, called starting growth time (SGT), was applied as screening platform to find the TCM extracts with anti-bacterial effect. Four kinds of pathogenic bacterial strains that commonly found in South China sea, e.g. Aeromonas hydrophila, Edwardsiella tarda, Vibrio alginolyticus and Vibrio harveyi, were chosen as experimental bacteria. According to our screening result, the water extract of S̲cutellaria b̲aicalensis a̲erial part, named as SBA, inhibited the growth of experimental bacteria in time- and dose- dependent manners. The chemical components of SBA were further tested by HPLC, and scutellarin and baicalin were tended to be the main components. The chemical profile of SBA was similar with its medicinal part, e.g. the root of S. baicalensis (Scutellaria Radix). The stem and leaf (aerial part) S. baicalensis are considered as waste during the production of root. Thus, the usage of SBA as an additive in aquaculture feeding could be a new way to recycle the waste. Siganus fuscescens (rabbit fish) was chosen as experimental animal to test the effect of SBA in fish. SBA was included in the feeding of rabbit fish. The total fish output and its nutritive value were determined. Feeding the fish with different doses of SBA for a month, the body length and weight were significantly increased after intake of standard feed containing 1% SBA. In parallel, the expressions of alkaline phosphatase and growth-related factors in bone, liver and muscle of 1% SBA-fed fish were markedly increased, suggesting the beneficial effects of SBA. The composition of amino acid and fatty acid in fish muscle, after intaking of 1% SBA containing feed, was altered. In SBA-fed fish muscle, the amounts of threonine and methionine were increased; while the amount of leucine was decreased, as compared with control group. The amounts of fatty acids, including docosahexaenoic acid, phosphatidylcholine and phosphatidylethanolamine, were increased in the 1% SBA-fed fish, while the amounts of triglycerides were decreased. The results indicated the growth promoting activity of SBA in an in vivo culture of rabbit fish, as well as to increase the nutritive values of the meat. SBA was included in the feeding of rabbit fish and tested its anti-bacterial and anti-inflammatory effect in vivo under bacterial contamination. Here, SBA was mixed into the feedings of rabbit fish that was cultured with a mixture of pathogenic bacteria. After 2 days of feeding, the mRNAs from fish tissues were extracted. The expression of inflammatory cytokines in these tissues were measured by qPCR. The expression levels of COX-2, IL-1β and TNF-α were markedly reduced in the present of SBA. The gut microbiota of rabbit fish, after intake of the SBA, was determined as well. The gut content of fish was collected for the extraction and sequenced of 16S rDNA. The results indicated that the intake of SBA showed strong anti-bacterial effect, which inhibited the growth of pathogenic bacteria, i.e. Deltaproteobacteria and Fusobacteria. In contrast to antibiotic (enrofloxacin), the growth of probiotics, e.g. Erysipelotrichia, was promoted after intake of SBA. Two cell lines isolated from rabbit fish were established to reveal the anti-inflammatory mechanism of SBA. One epidermal cell line was derived from dorsal fin of rabbit fish and named as r̲abbit f̲ish f̲in (RFF) cell line. Another cell line was isolated and from the head kidney of rabbit fish, corresponding to a new macrophagic cell line, named as r̲abbit f̲ish m̲acrophage (RFM). These cell lines were tested for growth in different temperatures and serum concentrations, and the best growing condition was at 20% serum at 28 °C. In cultured RFF and RFM cells, amplification of 18S rDNA from genomic DNA confirmed their proper identities. After 30
th passage of cultures, the RFF cells were exposed to challenge of inflammation, triggered by LPS, and hypoxia, mimicked by CoCl
2. Cultured RFF cells showed robust sensitive responses to inflammation and hypoxia in directing the expressions of cytokines and hypoxia inducible factor-1α (HIF-1α). Application of SBA inhibited the expression of LPS-induced inflammatory cytokines, i.e. IL-1β, IL-6, as well as the signaling of NF-κB. SBA suppressed the LPS-induced transcription of NF-κB subunit, triggered by LPS. The application of CoCl
2 in cultured RFF cells triggered the hypoxia-induced cell death and up regulation of HIF-1α. As expected, the applied SBA in cultures prevented the hypoxia-induced signaling. The isolated RFM cell was identified by immunostaining of CD 68, confirmed the identity as macrophagic cell. Cultured RFM cells were exposed to challenge of inflammation, as triggered by LPS, showing highly sensitive responses to inflammation, including release of nitric oxide, expression of cytokine, and activation of phagocytosis. In order to test the effect of SBA, the SBA and its effective flavonoids, i.e. baicalin and scutellarin, were applied in LPS-treated RFM cells. Application of SBA, baicalin or scutellarin, inhibited the expressions of LPS-induced inflammatory cytokines, i.e. IL-1β, TNF-α, as well as the signaling of transcription factor NF-κB. Our results show the establishment of RFF and RFM cell lines may be served as an ideal in vitro model in drug screening relating to inflammation and hypoxia. The application of SBA on other aquaculture fish species, e.g. Epinephelus fuscoguttatus (groupers) or Oreochromis niloticus (tilapia) was carried out as well. Here, the addition of SBA in their feeding could promote the growth of groupers and tilapia. In addition, the addition of SBA enhanced the immune function by promoting the activity of lysozyme and expression of anti-inflammatory cytokines, including IL-4, IL-10 and TGF-β. These findings proved that application of SBA as a feeding additive can be extended to other fish species. Our investigations showed the anti-bacterial and anti-inflammatory effect of SBA in vitro and in vivo, as well as its potential effect to promote the uptake of nutrients. The superiority of using this herbal extract in comparing to antibiotic was illustrated here, and which further suggested the possible development of recycling the waste product of S. baicalensis in fish aquaculture. Thus, the re-cycle of waste products during the farming of S. baicalensis herb in serving as fish feeding should be encouraged.
| Date of Award | 2021 |
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| Original language | English |
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| Awarding Institution | - The Hong Kong University of Science and Technology
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| Supervisor | Karl Wah Keung TSIM (Supervisor) |
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