Modulation of apoptosis and Inflammasome activation in chondrocytes: co-regulatory role of Chlorogenic acid.

BACKGROUND: The B-cell lymphoma 2 (Bcl-2) protein regulates programmed cell death throughout the disease conditions by upholding apoptotic pathways. However, the mechanism by which it's expressed in chondrocytes still needs to be studied in chondrocyte-related disorders. Additionally, exploring the potential therapeutic role of Chlorogenic acid (CGA) in confluence with Bcl-2 modulation is of significant interest. METHODS: In vivo and in vitro studies were performed according to our previous methodologies. The chondrocytes were cultured in specific growth media under standard conditions after expression verification of different microRNAs through high-throughput sequencing and verification of Bcl-2 involvement in tibial growth plates. The effect of Bcl-2 expression was investigated by transfecting chondrocytes with miR-460a, siRNA, and their negative controls alone or in combination with CGA. The RNA was extracted and subjected to a reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Western blot analysis and immunofluorescence assays were performed to visualize the intracellular localization of Bcl-2 and associated proteins related to apoptotic and inflammasome pathways. Moreover, apoptosis through flow cytometry was also performed to understand the modulation of concerning pathways. RESULTS: The suppression of Bcl-2 induced higher apoptosis and mitochondrial dysfunction, leading to IL-1ß maturation and affecting the inflammasome during chondrocyte proliferation. Conversely, overexpression attenuated the activation, as evidenced by reduced caspase activity and IL-1ß maturation. In parallel, CGA successfully reduced siRNA-induced apoptosis by decreasing Cytochrome C (Cyto C) release from the mitochondria to the cytoplasm, which in turn decreased Caspase-3 and Caspase-7 cleavage with Bcl-2-associated X protein (Bax). Furthermore, siBcl-2 transfection and CGA therapy increased chondrocyte proliferation and survival. The CGA also showed a promising approach to maintaining chondrocyte viability by inhibiting siRNA-induced apoptosis. CONCLUSIONS: Targeting Bcl-2-mediated regulation might be a possible treatment for chondrocyte-related conditions. Moreover, these results add knowledge of the complicated processes underlying chondrocyte function and the pathophysiology of related diseases, highlighting the significance of target specific therapies. Video Abstract.

Impact of Bacillus licheniformis from yaks following antibiotic therapy in mouse model.

Gut microorganism (GM) is an integral component of the host microbiome and health system. Abuse of antibiotics disrupts the equilibrium of the microbiome, affecting environmental pathogens and host-associated bacteria alike. However, relatively little research on Bacillus licheniformis alleviates the adverse effects of antibiotics. To test the effect of B. licheniformis as a probiotic supplement against the effects of antibiotics, cefalexin was applied, and the recovery from cefalexin-induced jejunal community disorder and intestinal barrier damage was investigated by pathology, real-time PCR (RT-PCR), and high-throughput sequencing (HTS). The result showed that A group (antibiotic treatment) significantly reduced body weight and decreased the length of jejunal intestinal villi and the villi to crypt (V/C) value, which also caused structural damage to the jejunal mucosa. Meanwhile, antibiotic treatment suppressed the mRNA expression of tight junction proteins ZO-1, claudin, occludin, and Ki67 and elevated MUC2 expression more than the other Groups (P < 0.05 and P < 0.01). However, T group (B. licheniformis supplements after antibiotic treatment) restored the expression of the above genes, and there was no statistically significant difference compared to the control group (P > 0.05). Moreover, the antibiotic treatment increased the relative abundance of 4 bacterial phyla affiliated with 16 bacterial genera in the jejunum community, including the dominant Firmicutes, Proteobacteria, and Cyanobacteria in the jejunum. B. licheniformis supplements after antibiotic treatment reduced the relative abundance of Bacteroidetes and Proteobacteria and increased the relative abundance of Firmicutes, Epsilonbacteraeota, Lactobacillus, and Candidatus Stoquefichus. This study uses mimic real-world exposure scenarios by considering the concentration and duration of exposure relevant to environmental antibiotic contamination levels. We described the post-antibiotic treatment with B. licheniformis could restore intestinal microbiome disorders and repair the intestinal barrier. KEY POINTS: • B. licheniformis post-antibiotics restore gut balance, repair barrier, and aid health • Antibiotics harm the gut barrier, alter structure, and raise disease risk • Long-term antibiotics affect the gut and increase disease susceptibility.

Leucine improves thiram-induced tibial dyschondroplasia and gut microbiota dysbiosis in broilers.

Thiram, a commonly used agricultural insecticide and fungicide, has been found to cause tibial dyschondroplasia (TD) in broilers, leading to substantial economic losses in the poultry industry. In this study, we aimed to investigate the mechanism of action of leucine in mitigating thiram-induced TD and leucine effects on gut microbial diversity. Broiler chickens were randomly divided into five equal groups: control group (standard diet), thiram-induced group (thiram 80 mg/kg from day 3 to day 7), and different concentrations of leucine groups (0.3%, 0.6%, 0.9% leucine from day 8 to day 18). Performance indicator analysis and tibial parameter analysis showed that leucine positively affected thiram-induced TD broilers. Additionally, mRNA expressions and protein levels of HIF-1α/VEGFA and Ihh/PTHrP genes were determined via quantitative real-time polymerase chain reaction and western blot. The results showed that leucine recovered lameness disorder by downregulating the expression of HIF-1α, VEGFA, and PTHrP while upregulating the expression of Ihh. Moreover, the 16 S rRNA sequencing revealed that the leucine group demonstrated a decrease in the abundance of harmful bacteria compared to the TD group, with an enrichment of beneficial bacteria responsible for producing short-chain fatty acids, including Alistipes, Paludicola, CHKCI002, Lactobacillus, and Erysipelatoclostridium. In summary, the current study suggests that leucine could improve the symptoms of thiram-induced TD and maintain gut microbiota homeostasis.

Baicalin inhibits apoptosis and enhances chondrocyte proliferation in thiram-induced tibial dyschondroplasia in chickens by regulating Bcl-2/Caspase-9 and Sox-9/Collagen-II expressions.

Avian tibial dyschondroplasia (TD) is a skeletal disease affecting fast growing chickens, resulting in non-mineralized avascular cartilage. This metabolic disorder is characterized by lameness and reduced growth performance causing economic losses. The aim of this study was to investigate the protective effects of baicalin against TD caused by thiram exposure. A total of two hundred and forty (n = 240) one day-old broiler chickens were uniformly and randomly allocated into three different groups (n = 80) viz. control, TD, and baicalin groups. All chickens received standard feed, however, to induce TD, the TD and baicalin groups received thiram (tetramethylthiuram disulfide) at a rate of 50 mg/kg feed from days 4-7. The thiram induction in TD and baicalin groups resulted in lameness, high mortality, and enlarged growth-plate, poor production performance, reduction in ALP, GSH-Px, SOD, and T-AOC levels, and increased AST and ALT, and MDA levels. Furthermore, histopathological results showed less vascularization, and mRNA and protein expression levels of Sox-9, Col-II, and Bcl-2 showed significant downward trend, while caspase-9 displayed significant up-regulation in TD-affected chickens. After the TD induction, the baicalin group was orally administered with baicalin at a rate of 200 mg/kg from days 8-18. Baicalin administration increased the vascularization, and chondrocytes with intact nuclei, alleviated lameness, decreased GP size, increased productive capacity, and restored the liver antioxidant enzymes and serum biochemical levels. Furthermore, baicalin significantly up-regulated the gene and protein expressions of Sox-9, Col-II, and Bcl-2, and significantly down-regulated the expression of caspase-9 (p < 0.05). Therefore, the obtained results suggest that baicalin could be a possible choice in thiram toxicity alleviation by regulating apoptosis and chondrocyte proliferation in thiram-induced tibial dyschondroplasia.

The Effect of miR-140-5p with HDAC4 towards Growth and Differentiation Signaling of Chondrocytes in Thiram-Induced Tibial Dyschondroplasia.

There is evidence to suggest that microRNA-140-5p (miR-140), which acts as a suppressor, is often elevated and has a role in various malignancies. Nevertheless, neither the function nor the mechanisms in chondrocytes linked with bone disorders, e.g., tibial dyschondroplasia (TD), have been satisfactorily established. The purpose of this study was to look into the role of microRNA-140-5p (miR-140) and its interaction with HDAC4 in chondrocytes, as well as the implications for tibial dyschondroplasia (TD), with a particular focus on the relationship between low miR-140 expression and poor pathologic characteristics, as well as its physiological effects on chondrocyte growth, differentiation, and chondrodysplasia. In this investigation, we discovered that TD had a reduced expression level of the miR-140. There was a correlation between low miR-140 expression, poor pathologic characteristics, and the short overall survival of chondrocytes. Our findings show an aberrant reduction in miR-140 expression, and HDAC4 overexpression caused disengagement in resting and proliferation zones. This further resulted in uncontrolled cell proliferation, differentiation, and chondrodysplasia. Mechanistically, HDAC4 inhibited the downstream transcription factors MEF2C and Runx2 and interacted with Col-Ⅱ, Col-X, and COMP. However, miR-140 binding to the 3'-UTR of HDAC4 resulted in the growth and differentiation of chondrocytes. Moreover, the expression of HDAC4 through LMK-235 was significantly decreased, and the expression was significantly increased under ITSA-1, referring to a positive feedback circuit of miR-140 and HDAC4 for endochondral bone ossification. Furthermore, as a prospective treatment, the flavonoids of Rhizoma drynariae (TFRD) therapy increased the expression of miR-140. Compared to the TD group, TFRD treatment increased the expression of growth-promoting and chondrocyte differentiation markers, implying that TFRD can promote chondrocyte proliferation and differentiation in the tibial growth plate. Hence, directing this circuit may represent a promising target for chondrocyte-related bone disorders and all associated pathological bone conditions.

Complete genome analysis of Lactobacillus fermentum YLF016 and its probiotic characteristics.

Lactobacillus fermentum (L. fermentum) YLF016 is a well-characterized probiotic with several favorable characteristics. This study aimed to analyze the probiotic characteristics of L. fermentum and uncover the genes implicated in its potential probiotic ability on the base of its genomics features. The complete genome of L. fermentum YLF016 was found to have a circular chromosome of 2,094,354 bp, and 51.46% G + C content without any plasmid. Its chromosome contained 2,130 predicted protein-encoding genes, 58 tRNA, and 15 rRNA-encoding genes. Also, it was found to have many other probiotic properties, such as a high survival rate in the gastrointestinal tract with strong adherence to intestinal cells, antibacterial activity against pathogens, and antioxidant activity. Moreover, the genome sequence analysis demonstrated specific genes coding for carbon metabolism pathway, genetic adaption, stress resistance, and adhesive ability. Further analysis revealed its non-hemolytic activity and its non-functional ability of virulence factors. In conclusion, L. fermentum YLF016 possesses many valuable probiotic properties that refer to its potential probiotic ability.

Thiram induces myocardial oxidative damage and apoptosis in broilers via interfering their cardiac metabolism.

Thiram is a dithiocarbamate pesticide extensively used as a fungicide to preserve crops and seeds. Long-term exposure to thiram causes potential harm to the health of human beings and animals. So far, most of the researches on thiram focused on erythrocyte toxicity, immune system, kidney damage, and tibial dyschondroplasia; however, there is less data on cardiac toxicity. In this study, we examined cardiac histopathology, inflammatory factors, oxidative stress indicators, and apoptosis markers in the heart of broilers that were exposed to thiram. According to our findings, the continuous exposure to thiram caused pathological changes and abnormal function of myocardial tissues with increased level of inducible nitric oxide synthase (iNOS), inflammatory factors (IL-6, IL-8, TNF-α and NF-κB), and decreased level of anti-inflammatory factor (IL-10). In addition, thiram significantly upregulated the protein expression of cleaved-caspase 3, cleaved-PARP, and caused cardiomyocyte apoptosis. Meanwhile, the expression of heat shock proteins (HSP60, HSP70, HSP90) markedly decreased in the thiram-treated groups. An excessive accumulation of peroxidation products (MDA, H2O2), a decrease in T-AOC, and antioxidant activity enzymes (T-SOD, GST and GPX) were also noticed, all of which led to oxidative stress and activation of Nrf2 signal pathway by up-regulating key target genes (HO-1 and SODs). Thiram-induced metabolites were further identified via non-targeted metabonomic analysis. Correlation analysis revealed eighteen differentially expressed metabolites, closely related to cardiac injury. Importantly, thiram primarily affected the taurine and hypotaurine metabolism, pyrimidine metabolism as well as glycerol metabolism. Collectively, our study suggests that thiram could cause cardiotoxicity by interfering with taurine and hypotaurine metabolism, pyrimidine metabolism, and glycerolipid metabolism, which further induce oxidative stress via triggering Nrf2 signal pathway. This study may provide new evidence for the molecular mechanism of cardiotoxicity caused by thiram and resonate the alarm for animals and workers who have been exposed to thiram for a long time.

Sodium butyrate ameliorates thiram-induced tibial dyschondroplasia and gut microbial dysbiosis in broiler chickens.

Thiram is a dithiocarbamate pesticide widely used in agriculture as a fungicide for storing grains to prevent fungal diseases. However, its residues have threatened the safety of human beings and the stability of the ecosystem by causing different disease conditions, e.g., tibial dyschondroplasia (TD), which results in a substantial economic loss for the poultry industry. So, the research on TD has a great concern for the industry and the overall GDP of a country. In current study, we investigated whether different concentrations (300, 500, and 700 mg/kg) of sodium butyrate alleviated TD induced under acute thiram exposure by regulating osteogenic gene expression, promoting chondrocyte differentiation, and altering the gut microbial community. According to the findings, sodium butyrate restored clinical symptoms in broilers, improved growth performance, bone density, angiogenesis, and chondrocyte morphology and arrangement. It could activate the signal transduction of the Wnt/ß-catenin pathway, regulate the expression of GSK-3ß and ß-catenin, and further promote the production of osteogenic transcription factors Runx2 and OPN for restoration of lameness. In addition, the 16S rRNA sequencing revealed a significantly different community composition among the groups. The TD group increased the abundance of the harmful bacteria Proteobacteria, Subdoligranulum, and Erysipelatoclostridium. The sodium butyrate enriched many beneficial bacteria, such as Bacteroidetes, Verrucomicrobia, Faecalibacterium, Barnesiella, Rikenella, and Butyricicoccus, etc., especially at the concentration of 500 mg/kg. The mentioned concentration significantly limited the intestinal disorders under thiram exposure, and restored bone metabolism.

Characterization of fungal microbial diversity in healthy and diarrheal Tibetan piglets.

BACKGROUND: Diarrhea is an important ailment limiting the production of the Tibetan pig industry. Dynamic balance of the intestinal microbiota is important for the physiology of the animal. The objective of this work was to study fungal diversity in the feces of early weaning Tibetan piglets in different health conditions. RESULTS: In the present study, we performed high-throughput sequencing to characterize the fungal microbial diversity in healthy, diarrheal and treated Tibetan piglets at the Tibet Autonomous Region of the People's Republic of China. The four alpha diversity indices (Chao1, ACE, Shannon and Simpson) revealed no significant differences in the richness across the different groups (P > 0.05). In all samples, the predominant fungal phyla were Ascomycota, Basidiomycota and Rozellomycota. Moreover, the healthy piglets showed a higher abundance of Ascomycota than the treated ones with a decreased level of Basidiomycota. One phylum (Rozellomycota) showed higher abundance in the diarrheal piglets than in the treated. At genus level, compared with that to the healthy group, the proportion of Derxomyces and Lecanicillium decreased, whereas that of Cortinarius and Kazachstania increased in the diarrheal group. The relative abundances of Derxomyces, Phyllozyma and Hydnum were higher in treated piglets than in the diarrheal ones. CONCLUSIONS: A decreased relative abundance of beneficial fungi (e.g. Derxomyces and Lecanicillium) may cause diarrhea in the early-weaned Tibetan piglets. Addition of probiotics into the feed may prevent diarrhea at this stage. This study presented the fungal diversity in healthy, diarrheal and treated early-weaned Tibetan piglets.

Taurine is an effective therapy against thiram induced tibial dyschondroplasia via HIF-1α/VEGFA and ß-catenin/ GSK-3ß pathways in broilers.

Thiram causes tibial dyschondroplasia in broilers, leading to a significant economic loss in the poultry industry. Our study explored the effects of taurine in thiram induced tibial dyschondroplasia (TD) through in vivo and in vitro approches. In in vivo study, thiram resulted in lameness disorder, low production parameters ALP, ACP, and a high level of NOS. While, the taurine exhibited promising effect by reducing lameness, increasing ALP, ACP levels, and significantly lowering NOS level with the restoration of the growth plate. In in vitro study, thiram caused distortion and disintegration of chondrocytes. The CCK-8 technique revealed the lower cell activity in TD as compared with the treatment group. Even, the treatment and taurine groups had higher cell activity than control group. Also, the chondrocyte morphology progressively reverted to normal after taurine treatment. It might effectively decreased the symptoms of TD in broilers and their production performance. Further research found that the taurine effectively improved chondrocytes' cell viability and recovered lameness disorder by regulation of HIF-1α, VEGFA, and Wnt/ß-catenin signaling pathways. In summary, these results indicate that taurine has a protective effect on thiram-induced broilers and it can enhance the growth activity by directly affecting the development of chondrocytes and blood vessels.

Cluster of differentiation 147 (CD147) expression is linked with thiram induced chondrocyte's apoptosis via Bcl-2/Bax/Caspase-3 signalling in tibial growth plate under chlorogenic acid repercussion.

Tibial dyschondroplasia (TD) is a metabolic disease of young poultry that affects bone andcartilage's growth. It mostly occurs in broilers due to thiram toxicity in the feed. In this disease, tibial cartilage is not yet ripe for ossification, but it also results in lameness, death, and moral convictions of commercial poultry due to numerous apoptotic changes on cell level. These changes serve a cardinal role in this situation. Many potential problems indicate that chlorogenic acid (CGA) performs an extensive role in controlling apoptosis's perception. However, the actual role of CGA in TD affected chondrocytes in-vitro is still unidentified. The current study investigates the imperceptible insight of CGA on chondrocyte's apoptosis via B-cell lymphoma 2 (Bcl-2), Bcl-2 associated x-protein (Bax), and Caspase-3 with CD147 signalling. The expression of these markers was investigated by Immunofluorescence, western blot analysis, and reverse transcription-quantitative polymerase chain (RT-qPCR). Chondrocytes from the growth plate of tibia were isolated, cultured, and processed. A sub-lethal thiram (2.5 µg/mL) was used to induce cytotoxicity and then treated with an optimum dose (40 µg/ mL) of CGA. According to the results, thiram distorted chondrocyte cells with enhanced apoptotic rate. But, in case of CGA, high expression of CD147 enhanced cell viability of chondrocytes, accompanied by downregulation of Bax/Caspase-3 signalling with the upregulation of Bcl-2. The first possibility has ruled out in the present study by the observation that the cells apoptosis marker, Caspase-3 showed a significant change in CD147 overexpressing cells. Conversely, immunodepletion of CD147 with enhanced cleavage of Caspase-3, indicating the activation of apoptosis in chondrocytes cells. Therefore, these findings suggest a novel insight about CD147 in thiram induced TD about the regulation of Bcl-2/Bax/Caspase-3 apoptosis-signalling axis.

Isolation, characterization, and interaction of lignin-degrading bacteria from rumen of buffalo (Bubalus bubalis).

The purpose of this study was to isolate lignin-degrading bacteria from buffalo rumen and to explore their interactions further. Using lignin as the carbon source, three bacteria, B-04 (Ochrobactrum pseudintermedium), B-11 (Klebsiella pneumoniae), and B-45 (Bacillus sonorensis), which have shown lignin degradation potential, were successfully isolated and identified from the rumen fluid of buffalo by colony morphology, 16S ribosomal RNA gene sequencing, and biochemical and physiological analyses. The degradation rates of lignin were determined, and the maximum values were 4.86%, 11.1%, and 7.68% for B-04, B-11, and B-45, respectively. The maximum laccase activities were 0.65, 0.93, and 1.15 U/ml, while the maximum lignin peroxidase activities were 5.72, 8.29, and 18.69 U/ml, respectively. Pairwise interaction studies showed inhibitory interaction between B-04 and B-45, inhibitory interaction between B-04 and B-11, and symbiotic interaction between B-11 and B-45. This is the first report on the lignin degradation ability of bacteria isolated from the buffalo's rumen, which provides a new understanding for revealing the mechanism of roughage tolerance of buffalo.

Probiotic potential and safety assessment of Lactobacillus isolated from yaks.

Current problem of antibiotic resistance and the high incidence of bacterial diseases has brought huge losses to the yak breeding industry in Tibet. Therefore, the purpose of this study was to isolate Lactobacillus with safety and beneficial probiotic potential for the prophylaxis of intestinal diseases in yaks. After 16S rDNA sequence, four strains i.e. Lactobacillus sakei (named L4), Enterococcus hirae (named E5), Pediococcus acidilactici (named P7), Weissella confusa (named W8) were isolated from feces of yaks. The results of tolerance to acid, bile salt, enzyme and temperature showed that P7 was highly tolerant to acid, bile salt and digestive enzyme, while E5 was more resistant to temperature. The antibacterial assay showed L4 had a strong inhibitory effect against Staphylococcus aureus (BNCC186335), and E5, P7, W8 had effective antibacterial ability against Escherichia coli (C83902). In addition, L4, E5, P7 and W8 mainly produced organic acids and bacteriocin production to inhibit common intestinal pathogens. The results of antibiotic susceptibility assay indicated that L4, E5, P7 and W8 were highly sensitive to most clinically used antibiotics and didn't contain the VanA and VanB genes on the basis of PCR amplification, and L4, E5, P7 and W8 didn't exhibit hemolytic activity. The animal toxicity experiment results showed that no obvious pathological change was found in intestinal tissue sections, and L4, E5 and W8 strains also promoted the growth performance of mice, consequently, the L4, E5, P7 and W8 had no toxic effect on mice. In conclusion, lactobacillus isolated from feces of yaks not only have potential probiotics and strong antibacterial ability in vitro, but also are safe. Therefore, they have the potential to reduce the occurrence of bacterial diseases as new feed additives.

Prevalence and molecular characterization of Cryptosporidium spp. in yaks (Bos grunniens) in Naqu, China.

The intestinal protozoan specie, Cryptosporidium causes serious diarrheal syndrome in humans and animals worldwide. However, limited knowledge is known about the infection caused by this specie in yaks in Naqu. About 950 serum and 150 fecal samples were collected and assayed by using commercial ELISA kits and nPCR detection methods to find the prevalence and molecular characterization of Cryptosporidium spp. in yaks. Results found that 103 out of 950 (10.8%) serums were uncovered against C. parvum antibodies. In different regions, the prevalence of C. parvum in yaks were in a range from 9.1% to 16.7%, with obvious difference among the three areas (P < 0.001). In male and female yaks, the prevalence of C. parvum was found to be 7.2% and 13.3% respectively (P < 0.001); and a significant difference (P < 0.001) with a range of 9.8%-18.2% was observed among different age groups. Out of 150 fecal samples, only 2 (1.3%) positive samples were detected via nPCR. The positive samples were sequenced and identified to be C. bovis. The two isolates were clustered to cattle and yak clade separately. Our results highlight the prevalence and epidemiological status of Cryptosporidium spp. in yaks which may contribute towards the prevention and control of this zoonotic disease in Naqu, China.

Molecular Characterization of Giardia duodenalis and Enterocytozoon bieneusi Isolated from Tibetan Sheep and Tibetan Goats Under Natural Grazing Conditions in Tibet.

In the present study, fecal samples from a total of 620 Tibetan sheep and 260 Tibetan goats from six counties in Tibet were examined by nested PCR. The results showed that the overall infection rates of Giardia duodenalis and Enterocytozoon bieneusi were 0.8% (5/620) and 15% (93/620), respectively, in Tibetan sheep, and 0% (0/260) and 9.6% (25/260), respectively, in Tibetan goats. Based on sequence analysis of the SSU rRNA, tpi, bg, and gdh genes of G. duodenalis, only assemblage E was identified. Based on sequence analysis of the ribosomal internal transcriptional spacer (ITS) region of E. bieneusi, a total of 12 genotypes (three novel and nine known) were detected, and these clustered into two separate phylogenetic groups. Genotypes CHG19, EbpA, EbpC, H, PigEBITS5, and CTS3 clustered into Group 1 with high zoonotic potential, while genotypes BEB6, CHC8, CHG1, I, CTS1, and CTS2 fell within the host-specific Group 2. Ten genotypes were detected in Tibetan sheep, and two genotypes were found in Tibetan goats. The current study indicated that E. bieneusi infections are widespread among these livestock, and Tibetan goats may play an important role as a reservoir of zoonotic E. bieneusi genotypes.

LPS-induced inflammation disorders bone modeling and remodeling by inhibiting angiogenesis and disordering osteogenesis in chickens.

Inflammation plays a negative role in the growth and development of bone. However, the underlining mechanisms of inflammation caused abnormal bone development and even bone disease are still poorly understood, especially in chickens. In this study, we explored the influence of inflammation on bone formation in broilers for the first time by using lipopolysaccharide (LPS) to establish systemic inflammatory models in chickens with tibia as the research object. The measurements of production and tibial parameters showed an inefficient production performance and lower growth rate in LPS group. We also found a large amount of platelets, inflammatory cells in chickens' blood and higher levels of inflammatory factors in serum after LPS injection, meanwhile, increase in thrombus, chondrocyte nucleolysis, and osteoclasts and a reduction in blood vessels were observed in growth plate through histological observation. The qPCR analysis showed that the mRNA expression levels of NF-κB, TLR4, TF, TPO, and its receptor C-MPL enhanced, while VEGFA was inhibited in LPS group. In addition, in OPG/RANKL system, OPG was decreased while RANKL enhanced. It was also observed that the mRNA levels of MMP-9 and its inducing factor CD147 enhanced in LPS group. The western blot results were basically in consistent with mRNA test. Thus, we infer that inflammation can inhibit bone modeling and remodeling by affecting angiogenesis and osteogenesis, and result in negative effect on bone formation furtherly.

Puerarin enhance vascular proliferation and halt apoptosis in thiram-induced avian tibial dyschondroplasia by regulating HIF-1α, TIMP-3 and BCL-2 expressions.

Tetramethyl thiuram disulfide (thiram) is a dithiocarbamate pesticide used for crop protection and storage. But, it's widespread utilization is associated with deleterious growth plate cartilage disorder in broilers termed as avian tibial dyschondroplasia (TD). TD results in non-mineralized and less vascularized proximal tibial growth plate cartilage causing lameness and poor growth performance. This study investigated the therapeutic potential of puerarin against thiram toxicity in TD affected chickens. One-day-old broiler chickens (n = 240) were alienated into three equal groups i.e. control, TD and puerarin (n = 80) and were offered standard feed. Additionally, TD and puerarin groups were offered thiram at 50 mg/kg of feed from 4 to 7 days for TD induction followed by puerarin therapy at 120 mg/kg to puerarin group only from 8 to 18 days for TD treatment. Thiram feeding to TD and puerarin group chickens caused lameness, mortality, and increased the aspartate aminotransferase (AST), alanine aminotransferase (ALT), malondialdehyde (MDA) levels and growth plate (GP) size and upregulated HIF-1α expression. Besides, the production parameters, alkaline phosphatase (ALP), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) levels and the expressions of TIMP-3 and BCL-2 were decreased (p < 0.05). Puerarin alleviated lameness, enhanced angiogenesis and growth performance and serum and antioxidant enzymes, decreased apoptosis and recuperated GP width by significantly downregulating HIF-1α and upregulating the TIMP-3 and BCL-2 mRNA and protein expressions in puerarin group chickens (p < 0.05). In conclusion, the toxic effects associated with thiram can be mitigated using puerarin.

Effect of total flavonoids of Rhizoma Drynariae in thiram induced cytotoxicity of chondrocyte via BMP-2/Runx2 and IHH/PTHrP expressions.

Tibial Dyschondroplasia (TD) is a prevailing skeletal disorder that mainly affects rapidly growing avian species. It results in reduced bone strength, lameness and an increase risk of fragility fractures. Total flavonoids of Rhizoma drynariae (TFRD) have been used as an effective treatment of different bone diseases in humans. The current in vitro study was conducted to explore the therapeutic effect of TFRD on thiram-induced cytotoxicity in avian growth plate cells via bone morphogenetic protein-2/runt related transcription factor-2 (BMP-2/Runx2) and Indian hedgehog/Parathyroid hormone-related peptide (IHH/PTHrP) expressions. Chondrocytes were isolated, cultured and refined from chicken's tibial growth plates in a special medium. Then chondrocytes were treated with sublethal thiram having less concentration (2.5 µg/mL) to induce cytotoxicity of chondrocyte, and then treated with providential doses (100 µg/mL) of TFRD. Thiram caused distorted morphology of chondrocytes, nuclei appeared disintegration or lysed along with decreased expressions of BMP-2/Runx2 and IHH/PTHrP. TFRD administration not only enhanced the viability of chondrocytes by itself, but also well restored the damage caused by thiram on growth plate chondrocytes by significantly up-regulating the expressions of BMP-2/Runx2 and IHH/PTHrP. Therefore, this study provides a novel insight into the further treatment of TD and other skeletal ailments and lays the foundation for prevention and treatment.

Characterization of the microbial community structure in intestinal segments of yak (Bos grunniens).

Yak (Bos grunniens), a ruminant, has a complex gastrointestinal microbial ecosystem, which is essential for host nutrition and health. However, not much is known about gut microbial communities of yak. This study was conducted to characterize the gut microbial diversity and composition of small intestinal and cecal contents of yaks through high-throughput sequencing targeting V3-V4 hypervariable region of 16S rRNA gene. A total of 916,934 high-quality sequences were obtained and 224 core operational taxonomic units (OTUs) shared all samples. The result showed that the microbial community in the small intestine was different from cecum sample. In all samples, the majority of bacterial phyla were Firmicutes, Bacteroidetes and Proteobacteria. A large proportion of anaerobes in the families Peptostreptococcaceae, Prevotellaceae, Flavobacteriaceae, Lachnospiraceae, and Succinivibrionaceae were present in the various intestinal segments. The relative abundance of Ruminococcaceae, Bacteroidaceae and Muribaculaceae were significantly higher in cecum than in other segments of intestines. At the genus level, Bacteroides was the most predominant genus in cecum. The results indicated that yak have abundant and diverse gut microbial community. In conclusion, this study characterized the profiles of microbial communities across intestinal segments and provide better insight into microbial population structure and diversity of yak.

Influence of dietary supplementation with Bacillus velezensis on intestinal microbial diversity of mice.

Yaks are an aboriginal breed of the Qinghai-Tibet plateau (3000 m), which are highly adaptable to cold and hypoxic environments. It is noticed that hypoxia and hypothermia can induce changes in intestinal microbial structure in animals. Increasing evidences suggested that probiotics supplementation can improve the balance of gut microbiota of animals. However, so far, very few studies have emphasized on the probiotics isolated from yaks in the Qinghai-Tibet Plateau. Therefore, a potential probiotic strain Bacillus velezensis was isolated from yaks. In the present study, a total of 18 Kunming mice (15-18 g) were equally distributed into two groups; control and probiotic treated groups (1 × 109 CFU/day). During the experimental period, all the mice from both groups were given standard normal diet ad libitum. At the end of the experiment, mice were euthanized and the intestines (duodenum, jejunum, ileum, and cecum) were removed for high-throughput sequencing. The results demonstrated that Bacillus velezensis supplementation showed beneficial effects on the gut microbiota of mice. Specifically, Bacillus velezensis supplementation increased the population of Lactobacillus and Ruminococcus in the duodenum, and Candidatus Arthromitus in the jejunum. Additionally, Acinetobacter in the duodenum and Helicobacter in the cecum were decreased after feeding Bacillus velezensis. Altogether, these findings suggested that Bacillus velezensis isolated from Tibetan yaks can improve gut microbiota of mice.