Application of trigonelline in prevention and treatment of animal urinary stone

Abstract

The invention relates to the technical field of biological medicines, in particular to application of trigonelline to prevention and treatment of animal urinary stones. The trigonelline provided by the invention can reduce the occurrence of animal kidney stone. The trigonelline provided by the invention has the functions of remarkably reducing the concentration of urea nitrogen, creatinine and calcium and phosphorus ions in serum of a glyoxylic acid-induced kidney stone mouse and reducing the concentration of serum oxalate. The trigonelline provided by the invention can promote the expression of MYO9A and Cabs1 proteins and inhibit the expression of SPP1, Flt1 and Cdc34 proteins by combining with kidney tissue ZFX proteins, thereby inhibiting abnormal cell proliferation, relieving cell oxidative stress, recovering epithelial barrier, promoting calcium ion transport and finally inhibiting the occurrence of kidney stones.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to application of trigonelline in preventing and treating animal urinary stones. Background Art

[0002] Urinary stones are a common urinary tract disease in animal clinics, with an increasing incidence year after year, particularly in companion animals (such as dogs and cats) and commercial animals (such as cattle and sheep). According to statistics, the prevalence of urinary stones in dogs can reach 12%-15%, of which calcium oxalate stones account for over 60%. Due to their unique urine concentrating mechanism, cats have a significantly higher proportion of uric acid stones than other species. The formation of urinary stones is closely related to metabolic abnormalities, dietary structure, genetic factors, and oxidative stress. The pathological process involves multiple steps, including crystal nucleation, growth, aggregation, and damage to renal tubular epithelial cells.

[0003] Current clinical treatments mainly include surgical lithotripsy, medical litholysis, and dietary adjustments, but they still have the following limitations: (1) Surgical intervention is highly invasive, with a postoperative complication rate (such as infection and renal impairment) as high as 25%, and a recurrence rate of over 30% within 1 year; (2) Commonly used drugs such as potassium citrate can alkalinize urine and inhibit uric acid stones, but long-term use can easily lead to hypokalemia and gastrointestinal irritation; (3) Specific inhibitors for calcium oxalate stones (such as thiazide diuretics) have the risk of electrolyte imbalance and are ineffective against existing stones. Therefore, the development of new, highly effective, low-toxic, and multi-target drugs for the prevention and treatment of stones has become an urgent need in the field of veterinary medicine.

[0004] In recent years, natural products have become a hot topic in the research of stone treatment drugs due to their wide availability, diverse structures, and high safety. For example, flavonoids (such as quercetin) can alleviate renal tubular damage through antioxidant effects, but their poor water solubility and low bioavailability (<5%) limit their clinical application. Polyphenols (such as curcumin) can inhibit calcium oxalate crystal growth, but high doses can easily cause hepatotoxicity. Trigonelline (TRL) is a pyridine alkaloid widely found in coffee beans (0.6%-1.2% dry weight), fenugreek seeds (0.3%-0.5%), and legumes. Studies have shown that TRL has significant antioxidant and anti-inflammatory effects, regulates glucose and lipid metabolism, and can enhance cellular antioxidant defenses by activating the Nrf2 / ARE pathway. However, existing research has focused on the application of TRL in diabetes, neuroprotection, and anti-cancer treatments, while its association with kidney disease, particularly urinary stones, has not yet been revealed. It is worth noting that TRL contains a pyridine ring and a methyl group in its molecular structure, which may interfere with stone formation through the following mechanisms: (1) coordinating with calcium ions or oxalate ions to directly inhibit crystal nucleation; (2) regulating urine pH and ion concentration to reduce calcium oxalate supersaturation; and (3) alleviating oxidative stress damage to renal tubular epithelial cells, blocking the pathological basis of crystal adhesion. Despite this, there are currently no patents or literature reporting the specific application and mechanism of TRL in the prevention and treatment of urinary stones. Summary of the Invention

[0005] The present invention aims to provide a metabolite, trigonelline, that can alleviate animal urinary stones, reduce animal urine oxalate excretion, promote fecal oxalate excretion, and reduce animal kidney oxidative stress. The trigonelline provided by the present invention is derived from the metabolism of nicotinic acid in the colon tissue of small-tailed Han sheep to produce trigonelline. The trigonelline is mainly produced by Prevotella copri through the metabolism of L-aspartate to nicotinic acid, which is then utilized by nicotinic acid N-methyltransferase 1 (NANMT1) in the colon epithelial tissue of small-tailed Han sheep to ultimately produce trigonelline. Trigonelline is negatively correlated with the concentration of oxalate in urine, can upregulate the expression of SLC26A6 protein in the colon epithelial cells of small-tailed Han sheep, reduce the expression of SLC26A3 protein, promote the transport of oxalate from serum to colonic contents, and reduce the accumulation of oxalate in the renal tubules. Trigonelline can restore the weight loss of mice caused by intraperitoneal injection of glyoxylic acid, reduce kidney weight, lower the concentrations of urea nitrogen, creatinine, calcium and phosphorus ions in serum, inhibit the deposition of calcium oxalate crystals in the kidneys, and promote the accumulation of oxalate in feces, confirming that trigonelline can significantly inhibit the occurrence of urinary stones in animals.

[0006] In order to achieve the above-mentioned purpose of the invention, the present invention provides the following technical solution: use of trigonelline in the preparation of a drug for preventing or treating animal urinary stones, wherein the chemical formula of the metabolite is C7H7NO2; PubChem SID: 4250; CAS: 535-83-1.

[0007] Preferably, the animal is a mammal, a ruminant or a rodent.

[0008] Preferably, the ruminant is a cow or a sheep; the mammal is a cat or a dog; and the rodent is a mouse or a rabbit.

[0009] Preferably, the urinary stones are metabolic stones, oxidative damage stones or drug-induced stones.

[0010] Preferably, the type of urinary stones is calcium oxalate stones.

[0011] The present invention also provides a pharmaceutical composition for preventing and treating animal urinary stones, comprising trigonelline and an adjuvant.

[0012] Preferably, the adjuvant is citrate, an antioxidant or a diuretic.

[0013] Preferably, the mass ratio of trigonelline to adjuvant is 1:0.1-2.

[0014] Preferably, the citrate is potassium citrate; the antioxidant is α-lipoic acid; and the diuretic is hydrochlorothiazide.

[0015] The present invention also provides the use of trigonelline in preparing functional food for preventing animal urinary stones.

[0016] Preferably, it can also be used as a feed additive, wherein the concentration of trigonelline in the feed additive, medicine and food is 20-100 μM, and the feeding or administration interval is 24 hours.

[0017] The trigonelline provided by the present invention can be added to conventional feed components for ruminants such as cattle and sheep, and companion animals such as dogs and cats. For example, it can be added to medicines, foods, feed additives, and probiotics, and can also be added with appropriate amounts of excipients such as excipients, diluents, fillers, and absorption enhancers. It can also be prepared into a variety of dosage forms, such as powders, tablets, granules, microcapsules, and liquid preparations.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] The trigonelline provided by the invention can reduce the occurrence of kidney stones in animals.

[0020] The trigonelline provided by the present invention has the function of significantly reducing the concentrations of urea nitrogen, creatinine and calcium and phosphorus ions in the serum of mice with kidney stones induced by glyoxylic acid, and reducing the serum oxalate concentration.

[0021] The trigonelline provided by the present invention can promote the expression of MYO9A and Cabs1 proteins and inhibit the expression of SPP1, Flt1, and Cdc34 proteins by binding to the ZFX protein in kidney tissue, thereby inhibiting abnormal cell proliferation, alleviating cell oxidative stress, restoring the epithelial barrier, promoting calcium ion transport, and ultimately inhibiting the occurrence of kidney stones.

[0022] The trigonelline provided by the present invention can also directly reduce the adhesion of calcium oxalate crystals to mouse renal tubular epithelial cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are merely embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying any creative work.

[0024] Figure 1 The phenotypic determination results of sheep with urinary stones in Example 1 of the present invention; A is the grouping and number selection of Small Tail Han sheep, and the comparison of kidney tissue morphology between sheep with urinary stones and healthy sheep; B is the comparison of bladder tissue morphology and crystal particles in the bladder of healthy and sheep with urinary stones; C is the H&E staining section observation results of the kidneys of healthy and sheep with urinary stones; D is the stone composition analysis measured using Fourier transform infrared spectroscopy; E is the kidney weight of healthy and sheep with urinary stones; F is the determination of oxalate concentration in urine, serum and feces of healthy and sheep with urinary stones; GI is the concentration of calcium, phosphorus ions, urea nitrogen and creatinine in urine and serum of healthy and sheep with urinary stones; J is the determination of kidney tissue protein expression levels in healthy and sheep with urinary stones.

[0025] Figure 2In Example 2 of the present invention, metagenomics and non-targeted metabolomics analysis showed that trigonelline is a metabolite that can potentially prevent and treat urinary stones; A is the principal component analysis (PCoA) of hindgut microorganisms of healthy and urinary stone-affected sheep; B is the difference in hindgut microorganisms of healthy and urinary stone-affected sheep analyzed by Lefse; C is the difference in P. copri abundance in the hindgut of the two groups of sheep; D is the correlation analysis of P. copri and oxalate concentrations in sheep serum and feces; E is a circle map of the P. copri genome ; F is a schematic diagram of the multi-step synthesis of nicotinic acid from L-aspartic acid by P. copri, and the further synthesis of trigonelline from nicotinamide N-methyltransferase in the intestinal tissue; G and H are the relative concentrations of nicotinic acid and trigonelline in the serum of the two groups of sheep, respectively; I is the determination of the NNMT gene expression level in the colon tissue of the two groups of sheep; J is the determination result of the niacin concentration in the culture medium of P. copri cultured in vitro; K and L are the correlation analysis of the concentrations of niacin and trigonelline in feces and the oxalate content in urine and feces.

[0026] Figure 3 This is a display of the results of Example 3 of the present invention, which shows that trigonelline inhibits the occurrence of urinary stones by promoting the transport of oxalate to the intestine; A is a schematic diagram of oxalate metabolism in animals; B is a schematic diagram of an in vitro Transwell cell assay; C is the measurement results of oxalate transport after inhibiting SLC26A6 gene expression; D is the measurement of LDH concentration in liver tissue of two groups of sheep; E is the measurement of oxalate transporter expression levels in colon tissue of two groups of sheep; and F is the measurement of oxalate concentration in feces of two groups of sheep.

[0027] Figure 4 4 is an illustration of the therapeutic effect of trigonelline on mice with glyoxylic acid-induced kidney stones in Example 4 of the present invention; A is a flowchart of the mouse experiment; B is the weight change of mice in each group; C is the kidney weight measurement of mice in each group; DG is the measurement of creatinine, urea nitrogen and calcium and phosphate ion concentrations in mouse serum; H is the H&E histomorphological observation of kidney tissue of mice in each group (scale bar is 100 μm); I and J are the measurement of oxalate concentrations in serum and feces; K is the summary NNMT gene expression level in the colon of mice in each group; L is the measurement of protein expression level in kidney tissue of mice in each group.

[0028] Figure 5 This is an illustration of the results of Example 5 of the present invention, in which trigonelline regulates renal protein expression and inhibits calcium oxalate crystal deposition; A is a volcano plot of differential proteins between the TRL group and the CON group determined by renal tissue proteomics; B shows the predicted binding conformation of TRL and ZFX protein; C is a Veen plot of proteins downregulated in both the PC group and the TRL group; D is a Veen plot of proteins upregulated in both the PC group and the TRL group; E and F are Figure 5Heatmap analysis results of 35 and 17 proteins in the enrichment center in C and D; G, H, and I are the protein expression levels of mouse renal tubular epithelial cells (TCMK-1) after treatment with TRL, calcium oxalate, and Si-ZFX in vitro; J is the dual luciferase reporter result of transcription factors binding to the Cdc34, Cabs1, Flt1, MYO9A, and SPP1 promoters; K is the adhesion of calcium oxalate to TCMK-1 cells in vitro. DETAILED DESCRIPTION

[0029] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0030] The reagents required for solution preparation in the following examples were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. Small-tailed Han sheep, both healthy and suffering from urinary stones, were collected primarily from a farm in Tang County, Hebei Province. Kits for quantifying oxalate concentrations in serum and cell suspensions were purchased from Nanjing Jiancheng Bioengineering Research Institute.

[0031] Example 1 Urinary stones induce urinary system pathological changes in sheep and change their normal physiological state

[0032] The present invention screens sheep with urinary stones from a group of small-tailed Han sheep rams with the same breeding environment and similar genetic background in an intensive fattening period. The selection criteria are to observe the sheep's urination, find sheep with frequent urination, dribbling urine, and hematuria, and palpate the sheep's bladder for granular abnormalities. Finally, rams with urinary stones (US, n=12) and healthy rams (HS, n=12) are selected for use in the present invention. After slaughter, bladder urine and bladder urinary stone particles from the US group are collected to analyze urine oxalate concentration and urinary stone composition; kidney tissue is collected from the HS and US groups for histomorphology and protein expression level determination; colon contents are collected from the HS and US groups for metagenomic and non-targeted metabolomics sequencing; serum samples are obtained from the HS and US groups by venous blood sampling for serum biochemical index analysis and non-targeted metabolomics sequencing; and colon tissue is collected from the HS and US groups for protein expression level determination.

[0033] Figure 1 A shows the phenotypic differences in kidney tissue between sheep in the US and HS groups; Figure 1 B shows the presence or absence of granular stones in the bladders of sheep in the US and HS groups and the differences in bladder tissue morphology; Figure 1 C shows the differences in kidney tissue morphology between sheep in the US and HS groups; Figure 1 D shows the composition analysis results of sheep stone particles in the US group determined by Fourier transform infrared spectroscopy; Figure 1 E shows the difference in kidney tissue weight between sheep in the US and HS groups; Figure 1F shows the differences in urine, serum, and fecal oxalate concentrations between sheep in the US and HS groups; Figure 1 GI showed the differences in the concentrations of calcium, phosphorus, urea nitrogen, and creatinine in urine and serum between sheep in the US and HS groups; Figure 1 J shows the differences in protein expression in kidney tissues of sheep in the US and HS groups.

[0034] Results showed that the renal pelvis of sheep in the US group showed erosion and hemorrhage, and a large number of sand-like stone particles, mainly composed of monohydrated calcium oxalate, were present in their bladders. Kidney weight was significantly increased in the US group (P < 0.01), and a large number of crystals were deposited in the renal tissue. Interestingly, oxalate concentrations in urine and serum of sheep in the US group were significantly increased (P < 0.01), but oxalate concentrations in feces were significantly decreased (P < 0.05). Serum biochemical parameters showed that the concentrations of calcium, phosphate, urea nitrogen, and creatinine in urine and serum of sheep in the US group were significantly increased. Renal protein expression analysis showed that the expression of CD44, SPP1, and NOX2 proteins was upregulated, while SOD2 expression was downregulated in the US group.

[0035] Example 2: Metagenomics and non-targeted metabolomics analysis showed that trigonelline is a potential metabolite for the prevention and treatment of urinary stones

[0036] Based on the phenotype shown in Example 1, the sheep colon microorganisms and their metabolites were further determined by metagenomics to determine whether they were factors regulating the progression of urinary stones.

[0037] Fecal microbial DNA was extracted from the colonic contents of 24 sheep in the HS and US groups using the microbead milling method developed by Yu and Morison. DNA concentration and purity were determined using a NanoDrop 2000 spectrophotometer (A260 / A280 ratio 1.8-2.0), and DNA integrity was verified by 1% agarose gel electrophoresis. Paired-end sequencing libraries were constructed from qualified samples using the NEXTFLEX RapidDNA-Seq library construction kit. 150 bp paired-end sequencing was performed on the Illumina NovaSeq 6000 platform. Sequencing quality was assessed using a Q30 quality score (>90%). Raw data generated using Illumina sequencing were degraded using fastp (v0.20.0) to remove adapters and low-quality reads, and host genome fragments were removed using BWA-MEM (v0.7.17). A total of 153.43 Gb of high-quality sequence was obtained, with an average of 6.39 Gb per sample. Subsequently, Megahit (v1.1.2) was used to assemble high-quality sequences into contigs, yielding an average of 438,045.63 contigs per sample with an average N50 of 1,145.21 bp. The specific sample size and sequencing depth need to be supplemented according to the experimental design. Prodigal (v2.6.3) was further used to predict ORFs on the assembled contigs, ultimately obtaining 6.52 million ORFs with an average length of 520.82 bp. CD-HIT (v4.8.1) was used to cluster the ORFs, resulting in a goat hindgut microbial NR gene set with an average length of 517.49 bp and containing 6,308,850 non-redundant (NR) predicted genes. The non-redundant gene set was aligned with the NR database using DIAMOND software, alignment type: BLASTP (Version 2.2.28+), and the BLAST alignment parameter setting was an expected e-value of 1e. -5 ) and obtained species annotation results from the taxonomic information database corresponding to the NR library. Species abundance was then calculated using the sum of gene abundances corresponding to the species. The abundance of species in each sample was statistically analyzed at each taxonomic level. LefSe was used to analyze species with significant differences in abundance in the hindgut microbiota between the HS and US groups.

[0038] For untargeted metabolomics sequencing and analysis, fecal samples from sheep in the HS and US groups were thawed on ice, and 50 mg (±1 mg) of each sample was homogenized with 500 μL of ice-cold methanol / water (70%, v / v). The samples were vortexed for 3 minutes, sonicated in an ice-water bath for 10 minutes, and vortexed again for 1 minute. The samples were then centrifuged at 12,000 rpm for 10 minutes. After centrifugation, 200 μL of the supernatant was incubated at -20°C for 30 minutes and then centrifuged again at 12,000 rpm for 30 minutes at 4°C. 350 μL of the supernatant was then transferred to a vacuum concentrator and evaporated to dryness. The dry residue was reconstituted with 150 μL of 70% methanol / water, vortexed for 3 minutes, and sonicated in an ice-water bath for 10 minutes. Finally, the samples were centrifuged at 12,000 rpm for 3 minutes at 4°C, and 120 μL of the supernatant was analyzed by UHPLC-ESI-MS / MS. Ultra-high-performance liquid chromatography (UPLC) conditions were as follows: a Waters ACQUITY UPLC HSS T3 C18 column (1.8 μm, 2.1 mm × 100 mm); a mobile phase consisting of ultrapure water containing 0.1% formic acid and acetonitrile containing 0.1% formic acid; a column temperature of 40°C; a flow rate of 0.4 mL / min; and an injection volume of 2 μL. Mass spectrometry was acquired in both positive and negative ion modes. LC-MS / MS raw data were processed using the Analyst 1.6.3 software package. To expand the scope of metabolite identification, metabolite annotation was performed based on the self-built MWDB (METWARE database) and public databases (Metlin, HMDB, KEGG, and MoNA). Differential metabolites were screened using a threshold of VIP > 1 and P < 0.05 (Wilcoxon rank-sum test). Identified metabolites were mapped to KEGG pathways, and significantly enriched metabolic pathways were identified using a hypergeometric test.

[0039] Figure 2 A shows the PCoA analysis between the colonic microbiomes of sheep in the HS and US groups; Figure 2 B shows the results of Lefse analysis of colonic microorganisms in two groups of sheep; Figure 2 C is the difference in abundance of Prevotella copri (P. copri) in the colon contents of the two groups of sheep; Figure 2 D is the correlation analysis between P. copri and oxalate concentrations in sheep serum and feces; Figure 2 E is the circle map of the P. copri genome; Figure 2F shows the process by which P. copri metabolizes L-aspartate to produce nicotinic acid (NA), which is then converted into trigonelline (TRL) by nicotinamide N-methyltransferase (NNMT) expressed in the host intestine; Figure 2 G and H are the relative concentrations of NA and TRL in feces of the two groups of sheep determined by non-targeted metabolomics; Figure 2 I shows the NNMT gene expression levels in the colon tissues of two groups of sheep; Figure 2 J shows the results of in vitro culture of P. copri and the changes of NA in the culture medium over time; Figure 2 K and L show the correlation analysis between the relative concentrations of NA and TRL in sheep feces and the oxalate content in sheep urine and feces.

[0040] Results showed significant separation of colonic microorganisms between the HS and US groups. Prevotella, represented by P. copri, was significantly elevated in the colons of healthy sheep (P < 0.01). Its abundance was significantly negatively correlated with serum oxalate concentration (|R| = 0.4162, P < 0.05), but positively correlated with fecal oxalate concentration (|R| = 0.753, P < 0.0001). The genomic map of the P. copri strain revealed that it participates in both nucleotide and coenzyme transport and metabolism pathways, metabolizing L-aspartate to NA, which is then converted to TRL by NNMT expressed in the host intestine. Untargeted metabolomics results also demonstrated significantly higher relative concentrations of NA and TRL in feces of HS-treated sheep compared with US-treated sheep (P < 0.01). NNMT gene expression levels in colonic tissue of sheep with urolithiasis were significantly lower than those in healthy sheep (P < 0.01). Further in vitro culture of the P. copri strain and measurement of NA concentration in the culture medium confirmed the strain's ability to synthesize NA. Furthermore, the relative concentrations of NA and TRL in sheep feces were significantly negatively correlated with oxalate concentration in sheep urine (P < 0.05) and positively correlated with fecal oxalate concentration (P < 0.05). These results suggest that TRL, produced from NA metabolism, plays an important role in the prevention and treatment of urinary stones in animals.

[0041] Example 3 Trigonelline inhibits the occurrence of urinary stones by promoting the transport of oxalate to the intestine

[0042] The main sources of oxalate in animals are exogenous dietary intake and endogenous production in the liver, which is then excreted through the circulatory system to the kidneys, bladder, or intestines. To reveal the regulation of intestinal oxalate transport by trigonelline, we measured the protein expression levels of intestinal oxalate transporters SLC26A6 and SLC26A3, the expression level of lactate dehydrogenase (LDH) in the liver, and the oxalate concentration in feces. We further explored this using Transwell chambers, plating MCEC cells into Transwell chambers, treating them with siRNA targeting the SLC26A6 gene, and adding TRL to treat the cells. After the cells were confluent, a 10 μM sodium oxalate solution was added to the lower layer of liquid. No sodium oxalate was added to the upper chamber liquid. The oxalate concentration in the upper chamber of the Transwell was measured at the time of liquid change and 1 hour after the liquid change.

[0043] Figure 3 A shows traces of oxalate metabolism in animals. Figure 3 B is the flow chart of cell experiment. Figure 3 C shows the oxalate concentration in the upper chamber of the Transwell. Figure 3 D is the LDH expression level in the liver of sheep in the HS group and US group. Figure 3 E is the summary expression levels of SLC26A6 and SLC26A3 in the intestine of sheep in the HS group and US group. Figure 3 F shows the difference in oxalate concentration in feces of sheep in the HS and US groups.

[0044] In the animal intestine, SLC26A6 promotes the elimination of oxalate from the intestine, while SLC26A3 promotes the absorption of oxalate from the intestine into the blood. The results showed that inhibiting SLC26A6 gene expression in animal intestinal cells reduced the transport of oxalate by cells, and supplementation with TRL relieved the inhibition of oxalate transport. There was no significant difference in the expression of LDH, which can promote the conversion of glyoxylate to oxalate, in the livers of sheep in the HS and US groups. However, the expression level of SLC26A6 protein in the colon of sheep in the HS group was upregulated, while the expression level of SLC26A3 was decreased. At the same time, the oxalate concentration in the feces of sheep in this group was significantly reduced. These results indicate that TRL can inhibit the occurrence of urinary stones in animals by promoting the transport of oxalate into the intestine and inhibiting its absorption in the intestine.

[0045] Example 4 Therapeutic Effect of Trigonelline on Glyoxylic Acid-Induced Kidney Stones in Mice

[0046] The mouse model was further used to investigate the therapeutic effect of trigonelline on mice with glyoxylic acid-induced kidney stones. Fifty 8-week-old C57BL / 6J male mice were randomly divided into the following five groups: (1) NC group: combined antibiotics were added to drinking water for 7 days and a normal diet was maintained; (2) CON group: combined antibiotics were added to drinking water for 7 days and a glyoxylic acid solution of 75 mg / kg / day was injected intraperitoneally starting on the 8th day and a normal diet was maintained; (3) PC group: combined antibiotics were added to drinking water for 7 days and a glyoxylic acid solution of 75 mg / kg / day was injected intraperitoneally starting on the 8th day and a normal diet was maintained and a gavage of 10 mg / kg / day was administered daily. 8 (4) HKPC group: Combined antibiotics were added to drinking water for 7 days, and 75 mg / kg / day of glyoxylic acid solution was injected intraperitoneally starting on the 8th day. The patients were fed a normal diet and given 10% heat-killed sputum every day. 8 CFU / mLP.copri bacterial liquid; (5) TRL group: Combined antibiotics were added to drinking water for 7 days, and 75 mg / kg / day of glyoxylic acid solution was injected intraperitoneally starting on the 8th day. The animals were fed a normal diet and received 50 mg / kg of TRL by oral gavage daily.

[0047] The combined antibiotics used in the experiment consisted of 1 g / L streptomycin, 1 g / L ampicillin, 1 g / L gentamicin, and 0.5 g / L vancomycin, which were added to the mice's drinking water for 7 days. The glyoxylic acid used in the experiment was injected intraperitoneally for 14 days. The P. copri, heat-inactivated P. copri, and TRL used in the experiment were administered orally for 14 days.

[0048] The weight of mice was recorded every day during the experiment; urine and feces of mice were collected before the end of the experiment; kidney, colon, and liver tissues of mice were collected for subsequent determination of protein and gene expression and tissue morphology observation; and serum of mice was collected for determination of serum biochemical indicators.

[0049] Figure 4 A shows the experimental process; Figure 4 B shows the changes in mouse body weight during the experiment; Figure 4 C is the difference in the weight of collected mouse kidneys; Figure 4 D, E, F, and G are the differences in the concentrations of urea nitrogen, creatinine, calcium ions, and phosphorus ions in mouse serum; Figure 4 H is the H&E staining result of mouse kidney section; Figure 4 I, J are the oxalate concentrations in mouse feces and serum; Figure 4 K is the NNMT gene expression level in mouse colon tissue; Figure 4 L shows the expression levels of SOD2, NOX2, SPP1, CD44, and GAPDH proteins in mouse kidney tissue.

[0050] The results showed that P. copri and TRL reversed the weight loss induced by intraperitoneal injection of glyoxylic acid, significantly reduced kidney weight (P<0.05), significantly decreased serum urea nitrogen, creatinine, and calcium and phosphate concentrations in mice (P<0.05), and significantly reduced calcium oxalate crystal deposition in the renal tubules of mice. Furthermore, TRL and P. copri treatment significantly reduced serum oxalate concentrations (P<0.05), increased fecal oxalate concentrations, and colonic NNMT gene expression (P<0.05). Furthermore, TRL and P. copri treatment increased SOD2 protein expression in kidney tissue and decreased NOX2, SPP1, and CD44 protein expression. These results suggest that trigonelline can significantly ameliorate the progression of glyoxylic acid-induced nephrolithiasis.

[0051] Example 5 Trigonelline regulates kidney protein expression and inhibits calcium oxalate crystal deposition

[0052] To reveal the mechanism by which trigonelline inhibits urinary stone formation, we performed proteomics analysis on the mouse kidneys described in Example 4 to analyze the regulatory effects of trigonelline on renal protein expression. Protein lysis buffer was added to mouse kidney tissue, lysed on ice for 30 minutes, and centrifuged at 12,000 g for 30 minutes at 4°C. The supernatant was then assayed for protein concentration using a BCA protein assay kit. Take 100 μg of protein sample, supplement with lysis buffer, add triethylammonium bicarbonate buffer (TEAB) with a final concentration of 100 mM; add TCEP with a final concentration of 10 mM and react at 37 ° C for 60 min; add iodoacetamide with a final concentration of 40 mM and react at room temperature in the dark for 40 min; add pre-cooled acetone to each tube (acetone: sample v: v = 6: 1) and precipitate at -20 ° C for 4 h; centrifuge at 10000g for 20 min, take the precipitate and fully dissolve the sample with 100 μL 100 mM TEAB; add Trypsin at a mass ratio of 1:50 (enzyme: protein) and enzymatically hydrolyze at 37 ° C overnight; desalt and quantify the peptides. After trypsin digestion, the peptides were vacuum-pumped and re-dissolved with 0.1% trifluoroacetic acid (TFA). The peptides were desalted with HLB and vacuum-concentrated. The peptides were quantified using a Thermo Fisher Scientific peptide quantification kit.

[0053] For DIA proteomics sequencing and data analysis, samples were digested with trypsin and treated with HLB desalting columns. Equal amounts of peptides were dissolved in mass spectrometry buffer and eluted using a Vanquish Neo ultra-high performance liquid chromatography system combined with a uPAC high-throughput column (75 μm × 5.5 cm). Gradient elution (180 SPD) was performed with 2% acetonitrile aqueous solution containing 0.1% formic acid (mobile phase A) and 80% acetonitrile aqueous solution (mobile phase B). The peptides were analyzed by Orbitrap Astral mass spectrometer in DIA mode (positive ion mode, ion source voltage 1.5 kV; primary mass spectrometry scan range 380–980 m / z, secondary mass spectrometry 150–2000 m / z). The raw data were obtained by Spectronaut. TM 18 software was used for database search analysis, with strict screening criteria set: protein / peptide false discovery rate (FDR) ≤ 1%, peptide confidence ≥ 99%, exclusion of shared peptides and modified peptides, and quantification using a six-peptide / protein (three characteristic ions / peptide) peak area integration strategy. Significant P values ​​were determined using SPSS (v27), and the fold difference (FC) criterion was also included: proteins with P < 0.05 and FC > 1.2 were considered upregulated, while proteins with P < 0.05 and FC < 0.83 were considered downregulated. Protein sequences were aligned with various protein databases using DIAMOND (v2.1.8.162) to identify the biological functions of each differentially expressed protein and annotation of the pathway to which it belongs.

[0054] Figure 5 A is the volcano plot of protein differences between TRL group and CON group; Figure 5 B shows the predicted binding conformation of TRL and ZFX protein; Figure 5 C and D are Veen plots of enriched proteins in each group; Figure 5 E and F are Figure 5 Heatmap analysis results of 35 and 17 proteins in the enrichment center in C and D; Figure 5 G and H are the protein expression levels of mouse renal tubular epithelial cells (TCMK-1) treated with TRL, calcium oxalate, and Si-ZFX in vitro; Figure 5 I is the dual-luciferase reporter results of transcription factor binding to Cdc34, Cabs1, Flt1, MYO9A, and SPP1 promoters; Figure 5 J is the adhesion of calcium oxalate to TCMK-1 cells in vitro.

[0055] Proteomic analysis revealed significant differences in renal tissue protein expression between the TRL and CON groups after treatment. TRL significantly upregulated ZFX protein expression. Molecular structure-based binding prediction using the CB-DOCK2 website revealed multiple binding sites between the TRL molecule and the ZFX protein. ZFX is a transcription factor that can bind to the promoter regions of multiple target genes to promote or repress gene expression. Further analysis of proteins that were downregulated in both the TRL and PC groups and upregulated in the CON group, as well as proteins that were upregulated in both the TRL and PC groups and downregulated in the CON group, identified proteins downregulated in both the TR1 and PC groups: cell division cycle protein 34 (Cdc34), osteopontin (SPP1), and vascular endothelial growth factor receptor 1 (Flt1), as well as proteins upregulated: calmodulin (Cabs1), myosin 9A (Myo9A), and the transcription factor ZFX protein. In vitro validation using TCMK-1 cells revealed that TRL regulates ZFX expression, which in turn regulates the expression of downstream proteins, including Cdc34, SPP1, Flt1, Cabs1, and Myo9A. Dual-luciferase reporter assays demonstrated that ZFX binds to the promoter regions of the Cdc34, SPP1, Flt1, Cabs1, and Myo9A genes, promoting the expression of Cabs1 and Myo9A proteins and inhibiting the expression of Cdc34, SPP1, and Flt1 proteins. Further in vitro calcium oxalate crystal adhesion assays demonstrated that TRL directly inhibits the adhesion of calcium oxalate crystals to cells. These results suggest that TRL, by binding to the renal transcription factor ZFX protein and regulating downstream protein expression, can inhibit abnormal cell proliferation, restore vascular permeability, accelerate calcium ion transport, and promote barrier recovery.

[0056] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. The use of trigonelline in the preparation of medicines for preventing or treating animal urinary stones.

2. The use according to claim 1, characterized in that The animal is a mammal, a ruminant or a rodent.

3. The use according to claim 2, characterized in that The ruminant is a cow or a sheep; the mammal is a cat or a dog; and the rodent is a mouse or a rabbit.

4. The use according to claim 1, characterized in that The urinary stones are metabolic stones, oxidative damage stones or drug-induced stones.

5. The use according to claim 1 or 4, characterized in that The type of urinary stones is calcium oxalate stones.

6. A pharmaceutical composition for preventing and treating urinary stones in animals, characterized in that: Contains trigonelline and excipients.

7. The pharmaceutical composition according to claim 6, characterized in that The adjuvant is citrate, antioxidant or diuretic.

8. The pharmaceutical composition according to claim 6, characterized in that The mass ratio of the trigonelline to the adjuvant is 1:0.1-2.

9. The pharmaceutical composition according to claim 7, characterized in that The citrate is potassium citrate; the antioxidant is α-lipoic acid; and the diuretic is hydrochlorothiazide.

10. Application of trigonelline in the preparation of functional food for preventing animal urinary stones.

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