Berberine enhances autophagic flux by activating the Nrf2 signaling pathway in bovine endometrial epithelial cells to resist LPS-induced apoptosis.

Berberine exerts many beneficial effects on lipopolysaccharide (LPS)-induced bovine endometrial epithelial cells (BEECs). Recently, we also found that berberine shows significant antiapoptotic and autophagy-promoting activities, but the underlying mechanism has not been elucidated. This research explored the association between the antiapoptotic and autophagy-promoting activities of berberine in LPS-treated BEECs. BEECs were first preconditioned with an inhibitor of autophagic flux (chloroquine [CQ]) for 1 h, treated with berberine for 2 h, and then incubated with LPS for 3 h. Cell apoptosis was assessed by flow cytometry, and autophagy activities were assessed by immunoblot analysis of LC3II and p62. The results indicated that the antiapoptotic activity of berberine was notably inhibited in LPS-treated BEECs after preconditioning with CQ for 1 h. Furthermore, to determine whether berberine promoted autophagy by activating the nuclear factor-erythroid 2 related factor 2 (Nrf2) signaling pathway, we assessed autophagy in LPS-treated BEECs after preconditioning with a signaling pathway inhibitor of Nrf2 (ML385). The results indicated that the enhanced autophagy activity induced by berberine was partially reversed in LPS-treated BEECs after the Nrf2 signaling pathway was disturbed by ML385. In conclusion, berberine enhances autophagic flux to allow resistance to LPS-induced apoptosis by activating the Nrf2 signaling pathway in BEECs. The present study may provide new insight into the antiapoptotic mechanism of berberine in LPS-induced BEECs.

Baicalin inhibits oxidative injures of mouse uterine tissue induced by acute heat stress through activating the Keap1/Nrf2 signaling pathway.

Heat stress effect the physiological functions of body, and reproductive system is one of the most sensitive. It's imperative to find out suitable measures to alleviate harmful effects of heat stress. Baicalin is well-known with its antioxidative property. To examine whether Baicalin could reduce oxidative injures of uterine tissue in heat-stressed mice. The mice were divided into four groups: control (Con), Baicalin (Bai), heat stress (H) and heat stress plus Baicalin (H + Bai). The oxidative damage of uterine tissue was detected by ELISA, H&E staining, tunnel assay and immunohistochemical staining. The protein/mRNA expressions of Keap1/Nrf2 related factors were detected by Western blot or QPCR. The results showed that mice heat-stressed at 41 °C for 2 h induced macroscopic changes, significantly increased MDA content and reduced activities of antioxidant enzymes including SOD, CAT and GSH-Px of the uterine tissue. Compared with Con group, heat stress up-regulated caspase-3 and caspase-9, enhanced the apoptosis of endometrial epithelial and glandular epithelial cells, improved the HO-1 mRNA/protein and NQO1 protein expressions, while down-regulated the mRNA/protein of Keap1. Compared with H group, antioxidant enzyme activities, Nrf2 protein and Nrf2, NQO1 and GCLC mRNA expressions were significantly increased in the H + Bai group. While the uterine epithelial cells apoptosis, MDA contents, caspase-3, caspase-9 and Keap1 protein and HO-1 mRNA expressions were decreased in the H + Bai group of mice compared with that in H group. Briefly, acute heat stress causes oxidative injures and apoptosis of mouse uterine tissue and Baicalin protects uterine tissue from the damages possibly through Keap1/Nrf2 signaling pathway.

Tanshinone IIa alleviates LPS-induced oxidative stress in dairy cow mammary epithelial cells by activating the Nrf2 signalling pathway.

OBJECTIVE: Mastitis is the most prevalent disease in dairy cows worldwide. Evidence has emerged that oxidative stress plays a crucial role in the development of mastitis. This study aimed to investigate the antioxidative effects of tanshinone IIa (Tan IIa) on LPS-induced oxidative stress in dairy cow mammary epithelial cells (CMECs). METHODS AND RESULTS: We examined the levels of ROS and MDA in LPS-treated CMECs after supplementation with Tan IIa using detection kits and found that Tan IIa significantly inhibited the upregulation of these factors. In addition, we also found that Tan IIa significantly reversed the decrease in mitochondrial membrane potential induced by LPS. Moreover, Tan IIa improved the activities of antioxidant enzymes, which were decreased by LPS. Finally, we examined the probable pathway in which Tan IIa exerted its antioxidant effects using qPCR and western blotting and found that Tan IIa significantly activated the Keap1/Nrf2 signalling pathway. CONCLUSION: These results suggest that Tan IIa might become a possible therapeutic agent for the treatment of dairy cow mastitis by weakening oxidative stress induced by LPS in CMECs.

Baicalin enhances the thermotolerance of mouse blastocysts by activating the ERK1/2 signaling pathway and preventing mitochondrial dysfunction.

Heat stress causes oxidative damage and induces excessive cell apoptosis and thus affects the development and/or even causes the death of preimplantation embryos. The effects of baicalin on the developmental competence of heat-stressed mouse embryos were investigated in this experiment. Two-cell embryos were cultured in the presence of baicalin and subjected to heat stress (42 °C for 1 h) at their blastocyst stage followed by continuous culture at 37 °C until examination. The results showed that heat stress (H group) increased reactive oxygen species (ROS) production, apoptosis and even embryo death, along with reductions in both mitochondrial activity and membrane potential (ΔΨm). Both heat stress (H group) and inhibition of the ERK1/2 signaling pathway (U group) led to significantly reduced expression levels of the genes c-fos, AP-1 and ERK2, and the phosphorylation of ERK1/2 and c-Fos, along with significantly increased c-Jun mRNA expression and phosphorylation levels. These negative effects of heat stress on the ERK1/2 signaling pathway were neutralized by baicalin treatment. To explore the signal transduction mechanism of baicalin in improving embryonic tolerance to heat stress, mitochondrial quality and apoptosis rate in the mouse blastocysts were also examined. Baicalin was found to up-regulate the expression of mtDNA and TFAM mRNA, increased mitochondria activity and ΔΨm, and improved the cellular mitochondria quality of mouse blastocysts undergoing heat stress. Moreover, baicalin decreased Bax transcript abundance in blastocyst, along with an increase in the blastocyst hatching rate, which were negatively affected by heat stress. Our findings suggest that baicalin improves the developmental capacity and quality of heat-stressed mouse embryos via a mechanism whereby mitochondrial quality is improved by activating the ERK1/2 signaling pathway and inducing anti-cellular apoptosis.

Tanshinone â ¡A inhibits the lipopolysaccharide-induced epithelial-mesenchymal transition and protects bovine endometrial epithelial cells from pyolysin-induced damage by modulating the NF-κB/Snail2 signaling pathway.

Mixed infection with Escherichia coli and Trueperella pyogenes (T. pyogenes) leads to purulent endometritis, but the underlying molecular mechanisms remain unclear. The aim of this study was to investigate the effect of tanshinone ⅡA (Tan ⅡA) on E. coli and T. pyogenes -induced purulent endometritis and explore the underlying mechanism. First, lipopolysaccharide (LPS) isolated from E. coli and bacteria-free filtrates (BFFs) isolated from T. pyogenes were used to induce a model of bovine endometrial epithelial cell (bEEC) damage in vitro. bEECs were pretreated with or without Tan ⅡA for 2 h, before LPS and BFFs were introduced to induce damage to investigate the protective effect of Tan IIA. Then, the cytolytic activity and inflammatory response in bEECs were examined using CCK-8, LDH and RT-qPCR assays. Furthermore, we confirmed the molecular mechanism by which Tan ⅡA reversed the damaged phenotypes in LPS- and BFFs-induced bEECs via the NF-κB/Snail2 pathway using qPCR and Western blotting. Tan ⅡA significantly decreased the cytolytic activity and inflammatory response in LPS- and BFFs-induced bEECs. In addition, Tan ⅡA reversed the dysregulation of E-cadherin, N-cadherin and vimentin. Moreover, Tan ⅡA significantly inhibited the activation of the NF-κB signaling pathway and decreased the expression level of Snail2, which is the main regulator of the epithelial-mesenchymal transition (EMT). In summary, Tan ⅡA inhibits the LPS-induced EMT and protects bEECs from pyolysin-induced damage by modulating the NF-κB/Snail2 signaling pathway.

Berberine attenuates the inflammatory response by activating the Keap1/Nrf2 signaling pathway in bovine endometrial epithelial cells.

Endometritis is a common inflammatory disease that disturbs the rapid development of dairy farming. In the present study, we investigated the anti-inflammatory effects of berberine on the LPS-induced inflammatory response in bovine endometrial epithelial cells (bEECs) and the participation of the Keap1/Nrf2 signaling pathway in this process. Berberine treatment significantly reduced the LPS-induced expression levels of CRP, IL-1ß, IL-6, and TNF-α in bEECs. The Nrf2 signaling pathway in these cells was also activated by berberine. We further evaluated the effects of Nrf2 activators and inhibitors on the downregulation of proinflammatory cytokines. The activator of Nrf2 significantly inhibited the production of these proinflammatory cytokines that was induced by LPS. However, an inhibitor of Nrf2 only partially inhibited the anti-inflammatory effects of berberine on the LPS-induced inflammatory response in bEECs. In conclusion, our findings suggest that berberine exerts anti-inflammatory effects partially by activating the Keap1/Nrf2 signaling pathway.

Aucubin ameliorates the LPS-induced inflammatory response in bovine endometrial epithelial cells by inhibiting NF-κB and activating the Keap1/Nrf2 signalling pathway.

Cows are susceptible to pathogenic bacterial infection after pregnancy, leading to inflammation of the endometrium. Aucubin (AU) has been proven to exhibit highly effective anti-inflammatory activity, but its ability to protect against endometritis in dairy cows remains unclear. Therefore, the goal of the present study was to evaluate the protective effect of AU on the LPS-induced inflammatory response of bovine endometrial epithelial cells (BEECs). After pre-treating BEECs with AU (10, 20 and 50 µM) for 6 hr, the cells were stimulated with LPS for 3 hr. Subsequently, BEECs apoptosis was analysed by flow cytometry, the expression of pro-inflammatory cytokine mRNA was detected by qRT-PCR, and changes in NF-κB and Keap1/Nrf2 signalling were analysed by western blotting and immunofluorescence analyses. The results showed that AU can reduce TNF-α, IL-1ß, IL-6, COX-2 and iNOS mRNA expression in BEECs and reduce cell apoptosis. Furthermore, AU significantly reduced the level of NF-κB p65 and IκB phosphorylation and inhibited the nuclear translocation of NF-κB p65. AU also activated the Keap1/Nrf2 pathway, promoting the nuclear transfer of Nrf2 and increasing Keap1, Nrf2, HO-1 and NQO1 mRNA and protein levels. Taken together, these results indicate that AU ameliorates the LPS-induced inflammatory response by inhibiting NF-κB and activating the Keap1/Nrf2 signalling pathway, which has a protective effect on BEECs.

Tanshinone IIA exhibits anti-inflammatory and antioxidative effects in LPS-stimulated bovine endometrial epithelial cells by activating the Nrf2 signaling pathway.

Endometritis is a major disease in productive bovines, and is also caused by conditional pathogens after delivery. The integrity and activity of bovine endometrial epithelial cells (bEECs) determine the development of endometritis. Tanshinone IIA, a compound purified from Salvia miltiorrhiza bunge, has been reported to have anti-inflammatory effects. The aim of this study was to investigate the anti-inflammatory effects of tanshinone IIA in the LPS-induced inflammatory response of bEECs. The results showed that tanshinone IIA inhibited the mRNA expression levels of COX-2 and iNOS, and reduced the expression levels of IL-1ß, TNF-α, IL-6 and IL-8 induced by LPS. In addition, we found that tanshinone IIA inhibited the level of MDA, but increased the activities of CAT and SOD. To evaluate the anti-inflammatory mechanism of tanshinone IIA, we examined the activation of Nrf2. The results showed that the Nrf2 signaling pathway was significantly activated by tanshinone IIA. In conclusion, these results showed that tanshinone IIA exhibited anti-inflammatory and antioxidative effects by activating the Nrf2 signaling pathway.

Chlorogenic acid ameliorates mice clinical endometritis by activating Keap1/Nrf2 and inhibiting NFκB signalling pathway.

OBJECTIVES: Clinical endometritis is a common reproductive disorder in mammals that seriously endangers animal health and causes economic losses worldwide. This study aims to use lipopolysaccharide and Trueperella pyogenes exotoxin as modelling reagents (LC) to perfuse the mouse uterus in order to establish a model of clinical endometritis and to investigate the anti-inflammatory and antioxidant effects of chlorogenic acid (CGA). METHODS: In this study, five LC uterine perfusions were selected to model clinical endometritis. The anti-inflammatory and antioxidant effects of CGA were clarified. Through HE staining, proinflammatory cytokines, blood testing, NFκB and Keap1/Nrf2 signalling pathways and other index changes to explore the protection mechanism of CGA. KEY FINDINGS: After CGA treatment, the appearance, inflammatory damage and blood indicators of the mouse uterus returned to normal. Simultaneously, CGA could inhibit the activation of NFκB and reduce the release of inflammatory cytokines; CGA could also activate Keap1/Nrf2, promote the dissociation of Keap1 and Nrf2 and significantly increase the expression of the downstream genes HO-1 and NQO1. CONCLUSIONS: The above results together explain that five LC uterine perfusions can be used to establish a mouse model of clinical endometritis. CGA can treat clinical endometritis by activating Keap1/Nrf2 and inhibiting the NFκB signalling pathway.

Andrographolide attenuates inflammatory response induced by LPS via activating Nrf2 signaling pathway in bovine endometrial epithelial cells.

Endometritis is one of the main causes of bovine infertility, which causes serious economic losses to the industry. The endometrium is the first line of defense against invading microbial pathogens in the uterus. Andrographolide is the primary active component of A. paniculate, and has been shown to have anti-inflammatory and antioxidant effects. However, its effects on the LPS-induced signaling pathway in bovine endometrial epithelial cells (bEECs) have not been reported yet. The aim of this study was to investigate the anti-inflammatory effects and mechanism of andrographolide in the LPS-induced inflammatory response of bEECs. We found that andrographolide strongly reduced LPS-induced NO and iNOS expression. The production of cytokines that were upregulated by LPS was significantly suppressed. To investigate the anti-inflammatory mechanism of andrographolide, we examined the activation of Nrf2. The results shown that andrographolide inhibited the expression of Keap1 but increased the expression of Nrf2. The expression levels of target genes of Nrf2 including Ho-1 and Nqo-1 were increased by andrographolide. Taken together, these results suggest that andrographolide may serve as a candidate to protect against the LPS-induced inflammatory response by inducing Nrf2 activation.

Determination of uterine bacterial community in postpartum dairy cows with metritis based on 16S rDNA sequencing.

Metritis is a frequently occurring diseases in postpartum cows and is one of the important reasons for the infertility of dairy cows, accounting for 20-30% of dairy cow diseases and has serious implications for the dairy industry. It has been reported in the literature that the bacterial balance of genital tracts is directly related to the maintenance of physiological function and the development of various diseases of the reproductive system. By analyzing the changes in abundance and diversity of bacteria in the cow uterus from 1 to 35 days postpartum, the objective was to reveal the mechanism of metritis in cows and provide the basis for diagnosis, treatment and prevention of metritis in postpartum dairy cows. Uterine contents were taken from six cows (three healthy and three with metritis) on 1, 7, 14, 21 and 35 days after parturition. DNA genomes extracted from the samples were primed with 515F5'-GTGCCAGCMGCCGCGG-3' and 907R5'-CCGTCAATTCMTTRAGTTT-3' for PCR amplification of the V4+V5 regions of the 16S rDNA genes and construction of a gene library. The sequence of the bacterial structure of the cow uterine contents was analyzed using 16S rDNA high-throughput sequencing technology. A total of 30 samples were tested by PCR, and 29 samples qualified. The results of cluster analysis showed that except for one sample, the number of OTUs in the healthy cows was above 200, while in the cows with metritis, except for three samples, OTUs were below 200. The Chao1 and Shannon indices showed that the abundance of bacteria in the cow uterus was lower than that of healthy cows. Analysis of the relative abundance of bacteria in the cow uterus showed that there were six phyla present, including Bacteroidetes, Firmicutes, Fusobacteria, Proteobacteria, Actinobacteria and Tenericutes. There were 10 dominant genera in healthy cows, including Bacteroides, Clostridium sensu stricto 1, Escherichia-Shigella, Fusobacterium, Halomonas, Helcococcus, Porphyromonas, Prevotella 6, Rikenellaceae RC9 gut group and Streptococcus. There were nine dominant genera in cows with metritis, including Bacteroides, Caviibacter, Clostridium sensu stricto 1, Falsiporphyromonas, Fusobacterium, Halomonas, Helcococcus, Porphyromonas and Prevotella 7. Phylogenetic tree analysis showed that uterine contents from 29 samples could be separated into two clusters. Eleven samples from the cows with metritis were clustered with one sample from the healthy group, and 13 samples from the healthy cows were clustered together with four samples from the metritis group. Principal co-ordinate analysis showed that the points representing healthy cows and those representing the metritis group were concentrated in two distinct regions, which shows that there were significant differences in the structure evolution between healthy cows and cows with metritis. The above results indicate that bacterial diversity declines with time postpartum in healthy cows and is lower in cows with metritis, with characteristic changes in the relative abundances, including increases in Bacteroidetes and Fusobacteria, decreases in Firmicutes and Proteobacteria, increases in Porphyromonas, Bacteroides and Fusobacterium, and a decrease in Clostridium sensu stricto 1.

Baicalin protects mouse testis from injury induced by heat stress.

Heat stress has been documented to reduce reproductive performance of female animals through injury to germ cells, with few studies available in male animals. The objectives of this study were to evaluate protective effects of baicalin on testicular tissue damage of mice subjected to heat stress and its related mechanisms. In this experiment, A total of forty mice were divided into four groups, including control group (C), baicalin group (B), heat stressed group (H) and heat stress with baicalin treatment (H + B) group. Morphological changes, activities of antioxidant enzymes and apoptosis-related parameters in the mice testes tissue were monitored. The results showed that the process of spermatogenesis in mice testis was impaired and the cellular apoptosis increased due to acute heat stress at 41 °C. Interestingly, the tissue damage was alleviated with the significant (P < 0.05) increase in the activities of SOD, CAT and GSH-Px enzymes, decrease (P < 0.05) in MDA content and number of cellular apoptosis recorded in mice of H + B group compared with those in mice from H group. In addition, the Fas, FasL and P-JNK protein expressions were significantly (P < 0.05) increased; and apaf-1, caspase-3, -9 were slightly expressed in the H group, while there was no difference in Bcl-2 expression, compared with C, B and H + B groups. The above results clearly indicate that heat stress induces macroscopic/apoptotic and oxidative changes in the testicular tissue of mice; these changes are alleviated by Baicalin through increasing anti-oxidative enzyme activities and possibly through blocking Fas/FasL pathway.

Characterization of the cervical bacterial community in dairy cows with metritis and during different physiological phases.

For the development of disease prevention and intervention strategies, a better understanding of the dynamics and interactions within cervical bacterial communities in both healthy cows and cows with metritis is required. Understanding the complexity and ecology of microorganisms in the vagina of dairy cows with metritis and during different physiological phases is critical for developing strategies to balance microorganism content. To gain deeper insight into fluctuations within the cervical microbiota, swab samples were collected from 40 Holstein dairy cows, and16S rDNA amplicon sequencing was used to analyze cervical bacterial diversity. Meanwhile, vaginal bacterial composition was analyzed during different physiological phases, including the formative (CF), gestational (CG), and postpartum (CP) stages, and in cows with metritis (CM). The results revealed a complex profile with extensive differences in the cervical bacterial composition. A total of 678,043clean 16S rDNA V4-V6 reads were gained, and 1877 Operational Taxonomic Units (OTUs) were observed after calculation. At both the phylum and genus levels, the top 10 bacteria by percentage were the same when comparing the CF, CG, and CP groups of cows, with some variation in abundance. At the phylum level, the cervical microbial community in the CF, CG, and CP groups included mainly Firmicutes, which accounted for 39.3%, 48.3%, and 49.6% of the total microbial composition of each group, respectively. However, the cervical bacterial community in the CM group consisted of mostly Bacteroidetes, which accounted for 72.6% of the total microbial composition. The second major bacterial community in the CF and CG groups of cows was Proteobacteria, which accounted for 28.3%and 30.1% of the total microbial compositions of these groups, respectively, while the second major bacterial community in the CP group was Bacteroidetes (23.5%). However, in the CM group, the second major bacterial community was Fusobacteria, which accounted for18.0% of the total microbial composition. At the genus level, the cervical bacterial community in the CM group of cows was dominated by Porphyromonas(44.4%) and Fusobacterium(12.1%), while Porphyromonas accounted for only 1.3%, 1.1%, and 1.4% of the total microbial compositions of the CF, CG, and CP groups, respectively. Likewise, Fusobacterium accounted for 2.3%, 0.7%, and 4.7% of the total microbial compositions of the CF, CG, and CP groups, respectively. The results demonstrate that cervical bacterial diversity decreases in cows with metritis and that the predominant bacterial genera are Porphyromonas and Fusobacterium. Cervical bacterial diversity was rich in all observed physiological phases, and the predominant bacterial phylum was Firmicutes. Pregnancy had little effect on the cervical bacterial community; however, there were increases in the abundances of pathogenic species in postpartum cows. Cervical bacterial diversity decreased in cows with metritis, however, due to the highly dynamic and complex course of metritis, the relationship between cervical bacterial diversity and metritis requires further investigation.

Antitumor effects of baicalin on ovarian cancer cells through induction of cell apoptosis and inhibition of cell migration in vitro.

Baicalin, an active flavone isolated from Scutellaria baicalensis Georgi, has been demonstrated to induce various beneficial biochemical effects such as anti­inflammatory, anti­viral, and antitumor effects. However, the antitumor mechanism of baicalin is not well understood. In the present study, baicalin was demonstrated to inhibit the viability and migration of a widely used ovarian cancer cell line, A2780, in a dose­dependent manner. MTT assays revealed that cell viability significantly decreased in ovarian cancer cells treated with baicalin compared with untreated cells, without effect on normal ovarian cells. Flow cytometric analysis indicated that baicalin suppressed cell proliferation by inducing apoptosis. The underlying mechanisms involved were indicated to be downregulation of the anti­apoptotic protein B­cell lymphoma 2 apoptosis regulator and activation of caspase­3 and ­9. In addition, wound healing and transwell assays revealed that cell migratory potential and expression of matrix metallopeptidase (MMP)­2 and MMP­9 were significantly inhibited when cells were exposed to baicalin, compared with untreated cells. The present study therefore suggested that baicalin has the potential to be used in novel anti­cancer therapeutic formulations for treatment of ovarian cancer.

Cytotoxic and chemosensitization effects of Scutellarin from traditional Chinese herb Scutellaria altissima L. in human prostate cancer cells.

Scutellaria altissima L. is a common traditional Chinese medicine used to treat inflammation in some countries. Scutellarin, an active major flavone glycoside isolated from the traditional Chinese medicine Scutellaria altissima L., has been shown to offer various beneficial biochemical effects on cerebrovascular diseases and inflammation. However, the antiproliferative effects of Scutellarin in prostate cancer and the underlying mechanism are not fully elucidated. In the present study, we aimed to ascertain whether Scutellarin inhibits cancer cell growth and to further explore the molecular mechanism. Scutellarin enhanced the sensitivity of prostate cancer cells to cisplatin. MTT assays revealed that cell viability was significantly decreased in the prostate cancer cells treated with Scutellarin. Flow cytometric analysis indicated that Scutellarin suppressed cell proliferation by promoting G2/M arrest and inducing apoptosis. We employed western blotting to delineate the underlying mechanisms involved in the G2/M arrest and apoptosis. Comet assay and γH2AX immunocytochemistry were used to detect levels of DNA damage in PC3 cells exposed to Scutellarin and/or cisplatin. Our data revealed that Scutellarin significantly induced prostate cancer cell apoptosis by activating the caspase cascade. An increase in the Bax/Bcl-2 ratio, depolarization of mitochondrial membrane potential and cell cycle arrest at G2/M phase were accompanied by the apoptosis induction. Additionally, Scutellarin altered the protein expression of cell cycle and apoptosis regulatory genes by downregulating Cdc2, cyclin B1 and Bcl-2 and upregulating caspase-3, caspase-9 and Bax in prostate cancer cells. Furthermore, Scutellarin sensitized PC3 cells to cisplastin treatment in a dose-dependent manner. Taken together, our data confirmed the cytotoxicity of Scutellarin against prostate cancer PC3 cells and provide new findings in regards to Scutellarin sensitizing prostate cancer cells to chemotherapy. Our findings suggest that Scutellarin has potential to be used as a novel antineoplastic therapeutic candidate for prostate cancer patients.

Puerarin ameliorates heat stress-induced oxidative damage and apoptosis in bovine Sertoli cells by suppressing ROS production and upregulating Hsp72 expression.

Puerarin, a bioactive isoflavone glucoside extracted from radix Puerariae, has been proven to possess many biological activities. However, the role of puerarin in protecting bovine Sertoli cells (bSCs) under heat stress conditions remains to be clarified. The present study aimed to explore the possible protective mechanism of puerarin for primary cultured bSCs subjected to heat stress. Bovine Sertoli cells were treated with 15 µM of puerarin before they were exposed to 42 °C for 1 hour. The dose of puerarin (15 µM) was determined on the basis of cell viability. The results showed that puerarin treatment suppressed the production of reactive oxygen species and decreased the oxidative damage of the bSCs subjected to heat stress, as indicated by changes in superoxide dismutase, catalase, and glutathione peroxidase activities and malondialdehyde content. Moreover, puerarin treatment also suppressed the initiation of mitochondria-dependent apoptotic pathway, as revealed by changes in Bax to Bcl-2 ratio, mitochondrial membrane potential, cytochrome C release, caspase-3 activation, and apoptotic rate compared with the heat stress group. In addition, puerarin treatment increased Hsp72 expression in the bSCs with no apparent cellular cytotoxicity compared with the control group. Furthermore, increased Hsp72 was detected in the heat stress plus puerarin group compared with the heat stress group. In conclusion, puerarin attenuates heat stress-induced oxidative damage and apoptosis of bSCs by suppressing reactive oxygen species production and upregulating Hsp72 expression.

Baicalin attenuates lipopolysaccharide induced inflammation and apoptosis of cow mammary epithelial cells by regulating NF-κB and HSP72.

Baicalin is the main ingredient of traditional Chinese herbal medicine, Scutellaria baicalensis, which has been widely used clinically as an anti-inflammatory agent. However, molecular mechanism of action of this drug is not yet clear. In the present study, the protective mechanism of baicalin against lipopolysaccharide (LPS) induced inflammatory injury in cow mammary epithelial cells (CMECs) was explored. For this purpose, in vitro cultured CMECs were treated with baicalin (10µg/mL) and LPS (10µg/mL) for 24 and 12h, respectively, and the cell viability was measured by using cell counting kit-8 (CCK-8). The results revealed that LPS induced inflammatory responses, as p-p65/p65 and p-IκBα/IκBα ratios and TNF-α and IL-1ß production was increased in the CMECs. Both Bcl-2/Bax ratio and cell viability were decreased and caspase-3 cleaved following LPS treatment, indicating apoptosis of CMECs. Moreover, both LPS and baicalin increased HSP72 expression of the CMECs. However, cellular inflammatory responses and apoptosis were significantly reduced in baicalin treated CMECs. In conclusion, baicalin ameliorated inflammation and apoptosis of the CMECs induced by LPS via inhibiting NF-κB activation and up regulation of HSP72.

Baicalin increases developmental competence of mouse embryos in vitro by inhibiting cellular apoptosis and modulating HSP70 and DNMT expression.

Scutellaria baicalensis has been effectively used in Chinese traditional medicine to prevent miscarriages. However, little information is available on its mechanism of action. This study is designed specifically to reveal how baicalin, the main effective ingredient of S. baicalensis, improves developmental competence of embryos in vitro, using the mouse as a model. Mouse pronuclear embryos were cultured in KSOM medium supplemented with (0, 2, 4 and 8 µg/ml) baicalin. The results demonstrated that in vitro culture conditions significantly decreased the blastocyst developmental rate and blastocyst quality, possibly due to increased cellular stress and apoptosis. Baicalin (4 µg/ml) significantly increased 2- and 4-cell cleavage rates, morula developmental rate, and blastocyst developmental rate and cell number of in vitro-cultured mouse embryos. Moreover, baicalin increased the expression of Gja1, Cdh1, Bcl-2, and Dnmt3a genes, decreased the expression of Dnmt1 gene, and decreased cellular stress and apoptosis as it decreased the expression of HSP70, CASP3, and BAX and increased BCL-2 expression in blastocysts cultured in vitro. In conclusion, baicalin improves developmental competence of in vitro-cultured mouse embryos through inhibition of cellular apoptosis and HSP70 expression, and improvement of DNA methylation.

TIIA attenuates LPS-induced mouse endometritis by suppressing the NF-κB signaling pathway.

Endometritis is one of the main diseases that harms the dairy cow industry. Tanshinone IIA (TIIA), a fat-soluble alkaloid isolated from Salviae miltiorrhizae, has been reported to have potent anti-inflammatory properties. However, the anti-inflammatory effects of TIIA on a mouse model of lipopolysaccharide (LPS)-induced endometritis remain to be elucidated. The purpose of the present study was to investigate the effects of TIIA on LPS-induced mouse endometritis. TIIA was intraperitoneally injected 1 h before and 12 h after perfusion of LPS into the uterus. A histological examination was then performed, and the concentrations of myeloperoxidase (MPO) and nitric oxide (NO) in the uterine tissue were determined. The levels of tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß) in a homogenate of the uterus were detected by enzyme-linked immunosorbent assay. The extent of phosphorylation of IκBα and p65 was detected by Western blotting. TIIA markedly reduced the infiltration of neutrophils, suppressed MPO activity and the concentration of NO, and attenuated the expression of TNF-α and IL-1ß. Furthermore, TIIA inhibited the phosphorylation of the nuclear factor-kappa B (NF-κB) p65 subunit and the degradation of its inhibitor IκBα. All the results suggest that TIIA has strong anti-inflammatory effects on LPS-induced mouse endometritis.

Lactoferrin suppresses lipopolysaccharide-induced endometritis in mice via down-regulation of the NF-κB pathway.

Lactoferrin (LF) is one of the most abundant proteins found in milk, and it has been reported to have anti-inflammatory properties. However, the anti-inflammatory effects of LF on lipopolysaccharide (LPS)-induced endometritis and the underlying molecular mechanisms remain to be elucidated. In this study, we evaluated the effects of LF on LPS-induced endometritis in mice. The endometritis model was established by the perfusion of mice with LPS. LF was administered by intraperitoneal injection 1h before and 12h after LPS induction. Our results demonstrated that LF significantly attenuated the histopathological changes in the uterus, reduced the activity of myeloperoxidase (MPO) and the levels of nitric oxide (NO), and inhibited the activation of NF-κB and the expression of tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß) in a dose-dependent manner. The results suggest that LF has an anti-inflammatory effect on LPS-induced endometritis in mice. Therefore, LF may be a potential therapeutic agent for the treatment of endometritis.