Huangqi Baihe Granules alleviate hypobaric hypoxia-induced acute lung injury in rats by suppressing oxidative stress and the TLR4/NF-κB/NLRP3 inflammatory pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Huangqi Baihe Granules (HQBHG) are a modified formulation based on the traditional recipe "Huangqi Baihe porridge" and the Dunhuang medical prescription "Cistanche Cistanche Soup." The Herbal medicine moistens the lungs and tones the kidneys in addition to replenishing Qi and feeding Yin, making it an ideal choice for enhancing adaptability to high-altitude hypoxic environments. AIM OF THE STUDY: The purpose of this study was to examine a potential molecular mechanism for the treatment and prevention of hypoxic acute lung injury (ALI) in rats using Huangqi Baihe Granules. MATERIALS AND METHODS: The HCP-III laboratory animal low-pressure simulation chamber was utilized to simulate high-altitude environmental exposure and establish an ALI model in rats. The severity of lung damage was evaluated using a battery of tests that included spirometry, a wet/dry lung ratio, H&E staining, and transmission electron microscopy. Using immunofluorescence, the amount of reactive oxygen species (ROS) in lung tissue was determined. Superoxide dismutase (SOD), glutathione (GSH), malondialdehyde (MDA), and myeloperoxidase (MPO) levels in lung tissue were determined using this kit. Serum levels of proinflammatory cytokines, including tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and interleukin-1 beta (IL-1 beta), and antiinflammatory cytokines like interleukin-10 (IL-10) were measured using an enzyme-linked immunosorbent assay kit. Gene expression changes in lung tissue were identified using transcriptomics, and the relative expression of proteins and mRNA involved in the toll-like receptor 4 (TLR4)/nuclear factor-kappa B (NF-κB p65)/Nod-like receptor protein 3 (NLRP3) pathway were determined using western blotting and quantitative real-time PCR. RESULTS: HQBHG was shown to enhance lung function considerably, decrease the wet/dry ratio of the lungs, attenuate lung tissue damage, suppress ROS and MDA formation, and increase SOD activity and GSH expression. The research also demonstrated that HQBHG inhibited the activation of the TLR4/NF-κB p65/NLPR3 signaling pathway in lung tissue, reducing the release of downstream pro-inflammatory cytokines. CONCLUSIONS: HQBHG exhibits potential therapeutic effects against ALI induced by altitude hypoxia through suppressing oxidative stress and inflammatory response. This suggests it may be a novel drug for treating and preventing ALI.

Free fatty acids promote degranulation of azurophil granules in neutrophils by inducing production of NADPH oxidase-derived reactive oxygen species in cows with subclinical ketosis.

Subclinical ketosis (SCK) in dairy cows, a common metabolic disorder during the peripartal period, is accompanied by systemic inflammation. Excessive release of azurophil granule (AG) contents during degranulation of polymorphonuclear neutrophils (PMN) could contribute to systemic inflammation in SCK cows. Although the increase in blood free fatty acids (FFA) in SCK cows may promote AG degranulation from PMN, the underlying mechanisms are unclear. Thirty multiparous cows (within 3 wk postpartum) with similar lactation numbers (median = 3, range = 2-4) and days in milk (median = 6, range = 3-15) were classified based on serum ß-hydroxybutyrate (BHB) level as control (n = 15, BHB < 0.6 mM) or SCK (n = 15, 1.2 mM < BHB < 3.0 mM). Cows with SCK had greater levels of serum haptoglobin, serum amyloid A, IL-1ß, IL-6, IL-8 and tumor necrosis factor-α. These proinflammatory factors had strong positive correlations with myeloperoxidase (MPO), a marker protein of PMN AG, whose content was greater in the serum of SCK cows. Both the number of AG and the protein abundance of MPO were lower in PMN isolated from SCK cows. Additionally, we found a greater ratio of blood CH138A+/CD63high cells and greater mean fluorescence intensity of CD63 on the PMN membrane, further confirming the greater degree of AG degranulation in cows with SCK. In vitro FFA dose response (0, 0.3, 0.6, 1.2, and 2.4 mM for 4 h) and time course (0, 0.5, 1, 2, and 4 h with 0.6 mM) experiments were performed on PMN isolated from control cows. The increase in MPO content in extracellular supernatant resulting from those experiments led to the selection of 0.6 mM FFA for 1 h duration as conditions for subsequent studies. After FFA treatment, release of intracellular MPO was increased along with increased levels of CD63 mean fluorescence intensity on the PMN membrane, confirming that FFA promoted degranulation of AG. In addition, FFA treatment increased reactive oxygen species (ROS) production by PMN, an effect that was attenuated by incubation with diphenyleneiodonium chloride (DPI), a NADPH oxidase-derived ROS inhibitor. The mitochondrial-derived ROS inhibitor carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP) did not affect ROS in response to FFA treatment. Treatment with FFA increased p47 phosphorylation and mRNA abundance of NCF1, NCF2, and CYBB in PMN. Furthermore, DPI, but not FCCP, dampened the degranulation of PMN AG induced by FFA in vitro. These data suggested that the degranulation of AG in PMN induced by FFA was mediated by NADPH oxidase-derived ROS. As verified ex vivo, PMN from SCK cows had greater levels of ROS, phosphorylation of p47, and mRNA abundance of NCF1, NCF2, and CYBB. Overall, the present study revealed that high blood concentrations of FFA in SCK cows induce the production of NADPH oxidase-derived ROS, thereby promoting degranulation of AG in PMN. The stimulatory effect of FFA on the release of AG content during degranulation, especially MPO, provides a new insight into the systemic inflammation experienced by peripartal cows with SCK.

Enhanced mitochondrial dysfunction and oxidative stress in the mammary gland of cows with clinical ketosis.

Ketosis is a common metabolic disorder in high-producing dairy cows during the peripartal period. Negative energy balance leads to increased circulating levels of nonesterified fatty acids (NEFA) and ß-hydroxybutyrate (BHB), consequently increasing the risk of ketosis. It is well-known that NEFA and BHB can induce lipotoxicity and oxidative stress in bovine tissues/organs including the liver and adipose tissue. Although the mammary gland is one important site for NEFA and BHB metabolism, whether an overload in their concentrations within mammary cells causes oxidative stress during ketosis remains unclear. Thus, the present study compared oxidative stress status and mitochondrial function in mammary tissues harvested by biopsy from healthy (n = 15) and clinically ketotic (n = 15) dairy cows within 2 to 3 wk postpartum. Compared with healthy cows, ketotic cows had depressed daily milk yield (median: 28.92 vs. 21.56 kg) and dry matter intake (median: 22.36 vs. 19.92 kg/d), accompanied by elevated plasma NEFA (median: 0.32 vs. 1.26 mM), BHB (median: 0.52 vs. 3.69 mM), and lower plasma glucose (median: 4.55 vs. 2.13 mM). As detected by a commercial kit, a greater level of reactive oxygen species in mammary epithelial cells of ketotic cows, and greater oxidant indices including hydrogen peroxide and malondialdehyde coupled with lower antioxidant indices including glutathione peroxidase, catalase, and superoxide dismutase activities as detected by the respective biochemical kits in the homogenate of mammary tissue of ketotic cows indicated increased oxidative stress status. Lower citrate synthase activity and ATP production as detected by the respective commercial kits coupled with lower mRNA and protein abundance of mitochondrial respiratory chain oxidative phosphorylation complexes I-V (CO I-V) in ketotic cows suggested an impairment of mitochondrial function. This was supported by lower mRNA and protein abundance of nucleus-derived mitochondrial function regulators including peroxisome proliferator activated receptor gamma coactivator 1 α, mitofusin 2, nuclear respiratory factor 1, and mitochondrial transcription factor A. Lower mitochondrial membrane potential evaluated via the tetraethylbenzimidazolylcarbocyanine iodide (JC-1) labeling method and swollen mitochondria in mammary epithelial cells of ketotic cows suggested the existence of mitochondrial damage. Overall, the present study revealed extensive mitochondrial dysfunction and oxidative stress in the mammary gland of clinically ketotic cows. As such, data suggest that reduced milk yield in cows with ketosis is partly due to enhanced oxidative stress along with mitochondrial dysregulation in the mammary gland.

Assessment of intraocular pressure measured by Reichert Ocular Response Analyzer, Goldmann Applanation Tonometry, and Dynamic Contour Tonometry in healthy individuals.

AIM: To investigate the accuracy of intraocular pressure (IOP) as measured by a Reichert Ocular Response Analyzer (ORA), as well as the relationship between central corneal thickness (CCT) and IOP as measured by ORA, Goldmann applanation tonometry (GAT), and dynamic contour tonometry (DCT). METHODS: A total of 158 healthy individuals (296 eyes) were chosen randomly for measurement of IOP. After CCT was measured using A-ultrasound (A-US), IOP was measured by ORA, GAT, and DCT devices in a randomized order. The IOP values acquired using each of the three tonometries were compared, and the relationship between CCT and IOP values were analyzed separately. Two IOP values, Goldmann-correlated IOP value (IOPg) and corneal-compensated intraocular pressure (IOPcc), were got using ORA. Three groups were defined according to CCT: 1) thin cornea (CCT<520µm); 2) normal-thickness cornea (CCT: 520-580µm); and 3) thick cornea (CCT>580µm) groups. RESULTS: In normal subjects, IOP measurements were 14.95±2.99mmHg with ORA (IOPg), 15.21±2.77mmHg with ORA (IOPcc), 15.22±2.77mmHg with GAT, and 15.49±2.56mmHg with DCT. Mean differences were 0.01±2.29mmHg between IOPcc and GAT (P>0.05) and 0.28±2.20mmHg between IOPcc and DCT (P>0.05). There was a greater correlation between IOPcc and DCT (r=0.946, P=0.000) than that between IOPcc and GAT (r=0.845, P=0.000). DCT had a significant correlation with GAT (r=0.854, P=0.000). GAT was moderately correlated with CCT (r=0.296, P<0.001), while IOPcc showed a weak but significant correlation with CCT (r=-0.155, P=0.007). There was a strong negative correlation between CCT and the difference between IOPcc and GAT(r=-0.803, P=0.000), with every 10µm increase in CCT resulting in an increase in this difference of 0.35mmHg. The thick cornea group (CCT>580µm) showed the least significant correlation between IOPcc and GAT (r=0.859, P=0.000); while the thin cornea group (CCT<520µm) had the most significant correlation between IOPcc and GAT (r=0.926, P=0.000). The correlated differences between IOPcc and DCT were not significant in any of the three groups (P>0.05). CONCLUSION: Measurement of IOP by ORA has high repeatability and is largely consistent with GAT measurements. Moreover, the ORA measurements are affected only to a small extent by CCT, and are likely to be much closer to the real IOP value than GAT.

Intraocular pressure instrument reading comparisons after LASIK.

PURPOSE: To evaluate the agreement between intraocular pressure (IOP) readings measured by the Ocular Response Analyzer (ORA) and corrected Goldmann applanation tonometry (cGAT), derived from the "gold standard" for the clinical measurement of IOP, in eyes of subjects who have undergone laser in situ keratomileusis (LASIK). The effects of corneal biomechanical properties on IOP were also evaluated. METHODS: This was a retrospective cross-sectional study. The IOP of 148 eyes of 148 patients who have undergone LASIK was measured by the ORA and GAT one after another. cGAT was calculated according to a correction formula for IOP after LASIK. Central corneal thicknesses and corneal curvature (K value) were also measured. Corneal hysteresis and corneal resistance factor (CRF) were measured by the ORA. RESULTS: For the postoperative subjects, whose ages ranged from 18 to 37 years, the average IOP readings for GAT, cGAT, IOPg, IOPcc were 10.09 ± 2.43, 13.34 ± 2.52, 9.79 ± 2.52, 13.91 ± 2.26 mm Hg, respectively. The mean difference between IOPcc and cGAT was 0.63 ± 1.31 mm Hg (p<0.05). The upper and lower limits of agreement between the IOPcc and cGAT were +3.2 and -1.9 mm Hg. There was a significant correlation between cGAT and CRF; however, no significant correlation could be found between cGAT and corneal hysteresis. CONCLUSIONS: After being corrected for central corneal thinkness, curvature of the corneal anterior surface and reduced biomechanical bending strength of the cornea, the IOP reading obtained by GAT is in fair agreement with that obtained by IOPcc, which may be helpful for IOP readings in subjects after LASIK. CRF might be a factor that affects accurate IOP readings.