Application of lignite fulvic acid in preparation of fish skin repairing medicine with micro-ecological purification function

Abstract

The invention discloses application of lignite fulvic acid in preparation of a fish skin repairing medicine with a micro-ecological purification function, and belongs to the technical field of aquatic biological medicine. The lignite fulvic acid prepared by a specific process is prepared into the medicated bath liquid with the final concentration of 50 mg / L, and a skin tissue PPAR signal channel is activated on a host endogenous level, so that lipid metabolism is regulated and controlled, and inflammatory response is inhibited; meanwhile, acinetobacter with a nitrogen metabolism function is directionally enriched on an exogenous layer of the water body, so that the content of ammonia nitrogen and nitrite is reduced. According to the invention, a dual synergistic repair mechanism of endogenous tissue regeneration and exogenous water quality purification is constructed, so that the fish full-thickness skin injury can be quickly healed under the concentration of 50 mg / L, and the water quality pH is synchronously stabilized and the osmotic pressure stress is reduced. The method is suitable for fish skin injury repair and culture microenvironment improvement.

Description

Technical Field

[0001] This invention relates to the field of aquatic biomedicine and aquaculture disease prevention and control technology, specifically to the application of lignite fulvic acid in the preparation of fish skin repair drugs with microecological purification functions. This invention particularly focuses on utilizing specific concentrations of lignite fulvic acid to achieve precise repair of full-thickness skin damage in fish and simultaneous improvement of the aquaculture environment through the synergistic effect of activating the host's endogenous PPAR signaling pathway and reshaping the exogenous aquatic microecology. Background Technology

[0002] With the global aquaculture industry shifting towards high-density, intensive models, and the widespread application of model organisms such as zebrafish in regenerative medicine and environmental toxicology research, the health management of fish surface barriers has become a core issue in aquatic biosafety. Significantly different from terrestrial vertebrates, fish skin lacks the protection of a keratinized dead skin layer; epidermal cells are in direct contact with the external liquid environment. They serve as an immune outpost against pathogen invasion and also perform crucial physiological functions such as osmotic pressure regulation, metabolic waste excretion, and auxiliary respiration.

[0003] Skin injuries caused by mechanical friction, biological parasite bites, or physicochemical irritants are extremely common during intensive aquaculture, long-distance transportation, or experimental operations. Once the integrity of the skin barrier is compromised, the rapid infiltration of external water and loss of electrolytes can lead to severe osmotic stress. Even more serious is the fact that opportunistic pathogens and water molds, which are widespread in aquaculture water, can use wounds as entry points to cause secondary infections, resulting in large-scale fish mortality and drug-induced economic losses.

[0004] Currently, interventions for fish skin injuries face a dual dilemma: on the one hand, long-term reliance on antibiotics has led to a severe drug resistance crisis and drug residue problems, seriously restricting the food safety of aquatic products; on the other hand, broad-spectrum oxidizing disinfectants that replace antibiotics (such as chlorine dioxide and potassium permanganate) lack selectivity. While high concentrations of oxidizing factors kill pathogens, they also produce significant cytotoxicity, directly burning the fragile new granulation tissue at the wound site and inhibiting the migration and proliferation of epithelial cells. Simultaneously, these disinfectants indiscriminately destroy beneficial bacteria responsible for the nitrogen cycle in the water, leading to the rapid accumulation of ammonia nitrogen and nitrite, resulting in "drug-induced water quality deterioration." This limitation of "treating the symptoms but not the underlying cause" often leads to the death of damaged fish due to increased environmental toxicity.

[0005] Fulvic acid, as a naturally sourced bioactive substance, has some applications in the aquaculture field, but existing technologies have significant blind spots: First, there is a lack of precise dosing guidelines, and the industry generally holds the misconception that "the higher the concentration, the better the effect," without considering the risks of a sharp drop in pH and osmotic pressure stress caused by high concentrations of organic acids; second, mechanism research is in a "black box" state, failing to reveal how fulvic acid regulates tissue repair through specific signaling pathways, nor clarifying how it positively synergizes with the succession of aquatic microecology during the treatment process.

[0006] Therefore, developing a precise drug delivery program that can both initiate endogenous regeneration in the host through specific molecular pathways and directionally reshape the aquatic microecology to purify water quality is key to breaking through the current bottlenecks in fish skin repair technology. Summary of the Invention

[0007] The purpose of this invention is to address the technical shortcomings of existing fish skin damage repair drugs, such as high cytotoxicity, easy disruption of aquatic microecology, lack of precise dosing guidance, and unclear repair mechanisms. This invention provides an application of lignite fulvic acid in the preparation of fish skin repair drugs with microecological purification functions. The aim is to achieve a dual synergistic effect of initiating endogenous regeneration in the host and purifying the exogenous aquatic environment through specific drug intervention, thereby improving the healing quality of full-thickness skin damage.

[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows: The application of lignite fulvic acid in the preparation of a fish skin repair drug with microecological purification function is characterized in that the drug uses lignite fulvic acid as the sole active ingredient, and during the application process, the drug is added to the aquaculture water, and the final concentration of lignite fulvic acid in the aquaculture water is controlled to be 50 mg / L.

[0009] Furthermore, the application exerts its repair function through the following dual synergistic mechanism: Endogenous signaling pathway regulation mechanism: The drug specifically activates the peroxisome proliferator-activated receptor (PPAR) signaling pathway in damaged fish skin tissue at a specific working concentration of 50 mg / L. By inducing the upregulation of genes related to this pathway, it regulates local lipid metabolism and energy supply in the wound area and effectively suppresses the inflammatory response, thereby initiating and accelerating skin re-epithelialization and dermal remodeling.

[0010] Exogenous microecological purification mechanism: At a concentration of 50 mg / L, the drug induces directional succession in the microbial community of aquaculture water, selectively enriching *Acinetobacter* spp., which has heterotrophic nitrification function, through pressure. The increased relative abundance of this spp. significantly reduces ammonia nitrogen and nitrite load in the water and maintains the pH value within the neutral physiological range of 7.0-7.8, thereby creating a low-toxicity, stable external microenvironment for wound healing in fish.

[0011] Furthermore, the lignite humic acid is prepared from lignite as raw material through a peroxide-hydrogenation degradation process in the presence of a catalyst.

[0012] Furthermore, the fish skin injury encompasses full-thickness skin defects caused by mechanical friction, biological invasion, or physical and chemical stress; the drug is administered via a continuous 21-day water immersion treatment.

[0013] Compared with the prior art, the beneficial effects of the present invention are reflected in the following aspects: (1) Breakthrough in the nonlinear technical barrier of dose-effect: Based on gradient experiments, this invention confirms that lignite fulvic acid has an "effective window" in fish skin repair. 50 mg / L is the key critical point for this application. At this concentration, the activation efficiency of the PPAR pathway reaches its peak. However, below this concentration (such as 25 mg / L), it is difficult to trigger the signal cascade reaction. Above this concentration (such as 75 mg / L), the environmental stress caused by high osmotic pressure leads to apoptosis of new cells, and the healing rate decreases.

[0014] (2) Achieved deep synergy between repair and water protection: This invention abandons the traditional disinfectant approach of "killing bacteria first, then treating diseases, and then polluting the water". By reshaping the microecology of the water body (enriching Acinetobacter spp.), it actively improves water quality while the drug exerts its therapeutic effect, avoiding the secondary impact of drug-induced ammonia nitrogen poisoning on injured fish and greatly improving the survival rate of fish.

[0015] (3) It has extremely high clinical applicability and safety: The specific process used in this invention produces natural lignite humic acid, which has excellent pH buffering capacity and will not cause water acidification. The dosage is accurate and the operation is simple, with no risk of antibiotic resistance or environmental residues, and it meets the standards of modern green aquaculture. Attached Figure Description

[0016] Figure 1 This is an evaluation chart of the effect of different concentrations of lignite humic acid on the repair of full-thickness skin damage in zebrafish in the embodiments of the present invention; wherein, 1A is a morphological photograph and 1B is a bar chart of wound healing rate.

[0017] Figure 2The diagram shows the transcriptomic analysis of skin tissue in the 50 mg / L concentration group in this embodiment of the invention; wherein, 2A is a volcano diagram of differentially expressed genes between the treatment group and the model group; 2B is a bubble diagram of GO functional enrichment of differentially expressed genes; 2C is a bubble diagram of KEGG pathway enrichment, which clearly indicates the significant activation of the PPAR signaling pathway.

[0018] Figure 3 The figures are dynamic monitoring curves of the physicochemical indicators of the aquaculture water in each experimental group in this embodiment of the invention; wherein, 3A is the curve of pH value change; 3B is the curve of electrical conductivity (EC) change, showing the osmotic stress risk of the high concentration group; 3C is the curve of ammonia nitrogen (NH3-N) concentration change; and 3D is the curve of nitrite (NO2-N) concentration change.

[0019] Figure 4 This is a diagram of microbial omics analysis of water bodies based on 16S rRNA gene sequencing in an embodiment of the present invention; wherein, 4A is the total colony count; 4B is the alpha diversity; 4C is the genus-level species distribution; 4D is the LEfSe differential species analysis, indicating the specific enrichment of Acinetobacter in the 50 mg / L group. Detailed Implementation

[0020] The present invention will be further described in detail below with reference to specific embodiments. Those skilled in the art should understand that the embodiments described below are for illustrative purposes only and should not be considered as limiting the scope of protection of the present invention. This embodiment uses zebrafish as a model organism to verify the universal mechanism of the drug of the present invention for skin repair in fish. Example 1 Preparation and characterization of lignite humic acid feedstock

[0021] The lignite humic acid used in this invention is prepared from lignite using a hydrogen peroxide oxidation-hydrogenation degradation method. The specific process follows the method disclosed in Chinese Invention Patent ZL200810233669.X: lignite is pulverized and mixed with hydrogen peroxide solution, and an oxidative degradation reaction is carried out in the presence of a catalyst. Subsequently, the mixture is centrifuged, purified, and dried to obtain lignite humic acid powder. Testing showed that the raw material had a pH value (0.5% aqueous solution) of 3.28, a moisture content of 14.28%, and an ash content of 17.79%. The heavy metal content (Pb 9.87 mg / kg, Cd 0.43 mg / kg, Hg < 0.01 mg / kg) was all below the national fishery water quality standards, meeting safety requirements. This specific process preserves the highly active carboxyl and phenolic hydroxyl functional groups in the molecule, laying a material basis for its subsequent activation of biological signaling pathways. Example 2 Fish full-thickness skin damage repair experiment and concentration gradient screening

[0022] This embodiment aims to establish the precise concentration window for fulvic acid treatment of full-thickness skin lesions in fish, and to disprove the conventional technical bias that "strong antibacterial activity is equivalent to rapid healing" by combining morphological and microbiological data. A standardized model was constructed using healthy adult zebrafish (3.0-3.5 cm in length). A full-thickness defect with a diameter of 1.5 mm and reaching the dermis was created below the dorsal fin using a low-power laser. The experiment included a blank control group, a model group, a 25 mg / L group, a 50 mg / L group, and a 75 mg / L group.

[0023] Combination Figure 1 The effect evaluation data shown will be analyzed. For example... Figure 1 The morphological photographs shown in Figure A visually record the dynamic repair process of the dorsal wounds in each group of zebrafish from day 0 to day 21. By day 21, the wounds in the 50 mg / L group were almost completely closed, with intact epithelial tissue coverage and new scale formation, and no obvious scarring. In contrast, the model group still had obvious defects, and the healing degree of the 25 mg / L and 75 mg / L groups was visibly weaker than that of the 50 mg / L group. Figure 1 B further provides quantitative statistical evidence, with the graph showing the linear changes in wound healing rate over time for each group (Note: the healing rate was calculated using the wound area on day 2 as a baseline and normalized, n=12). Statistical results show that on day 21, the endpoint of the experiment, the healing rate of the 50 mg / L treatment group reached its highest value, significantly better than the model group and other concentration groups, exhibiting a typical "inverted U-shaped" optimal efficacy range.

[0024] To explore the microbiological mechanisms behind the therapeutic effect, this embodiment simultaneously monitored the bacterial load in the aquaculture water. For example... Figure 4 The total colony count (CFU) chart shown in Figure A indicates that the 25 mg / L group maintained the lowest colony count on the nutrient agar medium throughout the entire cycle, demonstrating the strongest environmental antibacterial ability; while the 50 mg / L group had a relatively high colony count, comparable to the 75 mg / L group. Figure 1 B's healing rate data and Figure 4 A comparative analysis of the antibacterial data of group A revealed a key counterintuitive phenomenon: the 25 mg / L group, which had the strongest antibacterial ability, showed a significantly slower tissue repair rate than the 50 mg / L group. This finding confirms that the 50 mg / L concentration did not work through simple antibacterial action, but rather triggered a crucial physiological regeneration mechanism. Example 3 Validation of endogenous PPAR pathway activation based on transcriptomics

[0025] To reveal the endogenous driving force behind the efficient repair achieved by the 50 mg / L fulvic acid group, high-throughput transcriptome sequencing (RNA-Seq) was performed on the blank group, the model group, and the skin tissue at the healing site on day 21 in this embodiment. The analysis results are as follows: Figure 2 As shown.

[0026] first, Figure 2 A shows the statistics of differentially expressed genes (DEGs). The model group compared with the blank control group (Model vs Control) showed extensive changes in gene expression, confirming the successful construction of the skin injury model and triggering a systemic response at the transcriptome level; while the 50 mg / L treatment group compared with the model group (Day 21 vs Model) also showed a significant number of upregulated and downregulated genes, confirming that fulvic acid treatment successfully induced gene expression reprogramming in the damaged tissue.

[0027] Further functional analysis, such as Figure 2 As shown in bubble plot B (GO functional enrichment), fulvic acid treatment effectively repaired multiple functional disorders, including inflammatory response, extracellular matrix (ECM) tissue remodeling, and lipid metabolism, thus normalizing the physiological functions of damaged tissues. The most crucial mechanism was discovered in... Figure 2 C (KEGG pathway enrichment bubble diagram). The figure clearly indicates that the peroxisome proliferator-activated receptor signaling pathway (PPAR signaling pathway) was significantly enriched in the treatment group (P<0.05). Combined with the known functions of the PPAR pathway in lipid metabolism, anti-inflammation, and cell differentiation, this chain of molecular evidence clearly reveals the endogenous mechanism of this invention: 50 mg / L fulvic acid, as a signaling molecule, coordinates lipid oxidation metabolism in the damaged area by activating the PPAR pathway, providing key energy for epithelial cell migration, and effectively quelling excessive acute inflammation, achieving a leap from "simple repair" to "precise regeneration". Example 4 Verification of exogenous water body microecological regulation and water quality purification

[0028] This embodiment combines microbiome analysis to investigate the regulatory effect of fulvic acid on the aquaculture microenvironment. Figure 4 ) and water quality physicochemical index monitoring ( Figure 3 ).

[0029] Microbial community structure analysis, such as Figure 4 As shown. Figure 4 The Alpha diversity index of B reflects the differences in community richness and evenness among the groups; Figure 4 C shows the species distribution at the genus level. The most crucial finding comes from... Figure 4The LEfSe differential species analysis of D, as shown in the figure, intuitively reveals that in the 50 mg / L treatment group for 21 days (50-21d), Acinetobacter had the highest LDA score, making it the most significant biomarker in this group.

[0030] To verify the ecological effects and safety of enrichment of this specific bacterial genus, it is necessary to combine... Figure 3 A comprehensive assessment of the water quality chemical indicators is conducted. For example... Figure 3 C (ammonia nitrogen) and Figure 3 As shown by D (nitrite), although the 25 mg / L group showed the best ammonia nitrogen control (consistent with its low colony count), the ammonia nitrogen and nitrite levels in the 50 mg / L group were also better than the model group and remained within safe ranges. This chemical phenotype is highly consistent with the known heterotrophic nitrification-aerobic denitrification physiological characteristics of environmental Acinetobacter. This confirms that the enrichment of Acinetobacter in the 50 mg / L system plays a positive role in nitrogen cycling and purification, rather than pathogenicity, thereby creating a low-toxicity environment for wound healing through exogenous microecological regulation. Example 5 Comparative Analysis of the Risks of High-Concentration Environmental Stress

[0031] This embodiment further combines Figure 3 The physicochemical indicators in the study elucidated the mechanism of the decreased efficacy in the high-concentration group (75 mg / L) and defined the application boundaries of the present invention.

[0032] like Figure 3 As shown in Figure A, although the addition of fulvic acid caused a brief drop in pH, it quickly recovered and stabilized within the neutral physiological range of 7.0-7.8, demonstrating good buffering properties and providing a suitable acid-base environment for wound healing. However, Figure 3 The conductivity curve of group B reveals the risk of high concentrations: the conductivity of the water in the 75 mg / L group remained consistently high, significantly higher than that in the 50 mg / L group. High conductivity corresponds to a high osmotic pressure environment, which imposes continuous osmotic regulatory pressure on the damaged skin barrier of freshwater fish. The energy consumption caused by this environmental stress explains why... Figure 1 In group B, the healing rate was actually lower in the 75 mg / L group than in the 50 mg / L group. Therefore, considering... Figure 3 Physicochemical data and Figure 1 The efficacy data confirmed that 50 mg / L is the optimal critical concentration that precisely balances the activity of PPAR molecules, the microecological purification function, and the steady state of water osmotic pressure.

[0033] In summary, this invention, through a rigorous chain of experimental evidence, demonstrates that 50 mg / L lignite fulvic acid has a technical contribution to fish skin repair independent of its antibacterial effect. This specific technical window not only solves the problems of ambiguous drug concentration and unclear mechanism in existing technologies, but also achieves a highly efficient and green repair effect through the dual synergy of "endogenous pathway activation + exogenous microecological remodeling," possessing significant inventiveness and industrial applicability.

Claims

1. The application of lignite humic acid in the preparation of a fish skin repair drug with microecological purification function, characterized in that, The drug uses lignite fulvic acid as its active ingredient, and during application, the final concentration of lignite fulvic acid in the aquaculture water is controlled to be 50 mg / L.

2. The application according to claim 1, characterized in that, The lignite humic acid is prepared from lignite through a peroxide-hydrogenation degradation process.

3. The application according to claim 1, characterized in that, The drug is used to promote the regeneration and repair of full-thickness skin damage by activating the peroxisome proliferator-activated receptor (PPAR) signaling pathway in damaged skin tissue of fish.

4. The application according to claim 1, characterized in that, The microecological purification function refers to the drug's ability to induce the microbial community in aquaculture water to evolve towards enrichment of Acinetobacter.

5. The application according to claim 4, characterized in that, The increase in the relative abundance of Acinetobacter was accompanied by a decrease in the concentrations of ammonia nitrogen and nitrite in the culture water.

6. The application according to claim 1, characterized in that, The drug is used to maintain the pH value of the aquaculture water in the range of 7.0 to 7.8 24 hours after administration.

7. The application according to claim 1, characterized in that, The skin damage in fish includes full-thickness skin defects caused by mechanical, biological, or physicochemical factors.

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