Nanobodies with cross-neutralizing activity provide prominent therapeutic efficacy in mild and severe COVID-19 rodent models.

The weakened protective efficacy of COVID-19 vaccines and antibodies caused by SARS-CoV-2 variants presents a global health emergency, which underscores the urgent need for universal therapeutic antibody intervention for clinical patients. Here, we screened three alpacas-derived nanobodies (Nbs) with neutralizing activity from twenty RBD-specific Nbs. The three Nbs were fused with the Fc domain of human IgG, namely aVHH-11-Fc, aVHH-13-Fc and aVHH-14-Fc, which could specifically bind RBD protein and competitively inhibit the binding of ACE2 receptor to RBD. They effectively neutralized SARS-CoV-2 pseudoviruses D614G, Alpha, Beta, Gamma, Delta, and Omicron sub-lineages BA.1, BA.2, BA.4, and BA.5 and authentic SARS-CoV-2 prototype, Delta, and Omicron BA.1, BA.2 strains. In mice-adapted COVID-19 severe model, intranasal administration of aVHH-11-Fc, aVHH-13-Fc and aVHH-14-Fc effectively protected mice from lethal challenges and reduced viral loads in both the upper and lower respiratory tracts. In the COVID-19 mild model, aVHH-13-Fc, which represents the optimal neutralizing activity among the above three Nbs, effectively protected hamsters from the challenge of SARS-CoV-2 prototype, Delta, Omicron BA.1 and BA.2 by significantly reducing viral replication and pathological alterations in the lungs. In structural modeling of aVHH-13 and RBD, aVHH-13 binds to the receptor-binding motif region of RBD and interacts with some highly conserved epitopes. Taken together, our study illustrated that alpaca-derived Nbs offered a therapeutic countermeasure against SARS-CoV-2, including those Delta and Omicron variants which have evolved into global pandemic strains.

Ciliary Type III Adenylyl Cyclase in the VMH Is Crucial for High-Fat Diet-Induced Obesity Mediated by Autophagy.

Neuronal primary cilia are crucial for body weight maintenance. Type III adenylyl cyclase (AC3) is abundantly enriched in neuronal cilia, and mice with global AC3 ablation are obese. However, whether AC3 regulates body weight through its ciliary expression and the mechanism underlying this potential regulation are not clear. In this study, humanized AC3 knock-in mice that are resistant to high-fat diet (HFD)-induced obesity are generated, and increases in the number and length of cilia in the ventromedial hypothalamus (VMH) are shown. It is demonstrated that mice with specifically knocked down ciliary AC3 expression in the VMH show pronounced HFD-induced obesity. In addition, in vitro and in vivo analyses of the VMH show that ciliary AC3 regulates autophagy by binding an autophagy-related gene, gamma-aminobutyric acid A receptor-associated protein (GABARAP). Mice with GABARAP knockdown in the VMH exhibit exacerbated HFD-induced obesity. Overall, the findings may reveal a potential mechanism by which ciliary AC3 expression regulates body weight in the mouse VMH.

Mechanisms of Heshouwuyin in regulating apoptosis of testicular cells in aging rats through mitochondrial pathway.

BACKGROUND: Polygonum multiflorum has important effects on anti-aging and immunity enhancement. Many traditional Chinese medicine preparations based on Polygonum multiflorum are widely used for the clinical prevention and treatment of aging. However the mechanisms of these herb mixtures are often unknown. This study investigates the effect of Heshouwuyin, a Chinese herbal compound for invigorating the kidney, on the regulation of testicular cells apoptosis in aging rats. METHODS: In this study, 18-month-old Wistar rats served as a model of natural aging and 12-month-old rats served as a young control group. Heshouwuyin group 1 and group 2 were comprised 18-month-old rats given Heshouwuyin intragastrically for 60 days and 30 days respectively. Then testes of the young control group were isolated in the age of 12 months, the other three groups were in the age of 18 months. RESULTS: TUNEL assay showed that the rate of testicular cell apoptosis was obviously higher and Flow cytometry analysis showed that the rate of cell proliferation was significantly lower in the natural aging group than in the young control group and that intervention with Heshouwuyin could reverse this phenomenon. Therefore, we further applied microarray analysis to screen out differentially expressed genes regulated by Heshouwuyin and related to cell apoptosis. The expression of these genes was observed by quantitative fluorescence PCR, immunofluorescence staining, and western blot. The results showed that the expression of 14-3-3σ was significantly lower and that the expression of DR6, BAX, caspase-3 and Cytc were significantly higher in the natural aging group than in the young control group, but intervention with Heshouwuyin significantly reversed this phenomenon. Moreover, the curative efficacy of Heshouwuyin after 60 days was better than that of Heshouwuyin after 30 days. CONCLUSION: Our study suggests that Heshouwuyin has anti-aging effects on the testis by means of inhibiting the occurrence of apoptosis in spermatogenic cells, thus improving the spermatogenic function of the testis. This is mainly achieved by regulating the expression of key genes in the mitochondrial apoptosis pathway.

Possible mechanism underlying the effect of Heshouwuyin, a tonifying kidney herb, on sperm quality in aging rats.

BACKGROUND: Herb mixtures are used as alternatives to hormone therapy in China for the treatment of partial androgen deficiency in aging men. However, the compositions of these herb mixtures are complex and their mechanisms are often unknown. This study investigates the effect of Heshouwuyin, a Chinese herbal compound for invigorating the kidney, on the control of testosterone secretion and sperm function. METHODS: Aged Wistar rats were administered with Heshouwuyin. A Shouwu pill group and young group were used as controls. RESULTS: Morphology, chemiluminescence, fluorescence immunohistochemistry, and western blot showed that the epididymal sperm of naturally aged rats had intact plasma membranes. They also had abnormal mitochondrial function and DNA integrity, a significant decline in serum testosterone levels, and significant pathological changes in the structure of testicular tissues. Heshouwuyin significantly improved sperm function and serum testosterone levels, and improved testicular morphology. Moreover, the curative efficacy of Heshouwuyin after 60 days was better than that of Heshouwuyin after 30 days and the Shouwu pill group. CONCLUSION: Heshouwuyin exerts an important role in controlling testosterone secretion and sperm function.

A study on isolation of chemical constituents from Sophora flavescens Ait. and their anti-glioma effects.

BACKGROUND: Sophora flavescens Ait. is a traditional Chinese medicine with a long history in China. It is mainly used in the treatment of heat dysentery and similar ailments in the clinical. The objective of this paper was to isolate, purify and identify alkaloids from Sophora flavescens Ait. and to explore their inhibitory effects on C6 glioma cells. MATERIALS AND METHODS: Column chromatography, extraction and NMR spectroscopy were used to structurally identify the isolated compounds. MTT assay and flow cytometry were used to detect the inhibitory effect of matrine on C6 cells. RESULTS: Three compounds were isolated from Sophora flavescens Ait., namely matrine, oxymatrine and lupeol. Different concentrations of matrine solution all had inhibitory effects on growth of C6 cell lines, which showed apparent dose-effect relationship. Compared with the control group, proportion of G0/G1 phase cells increased in each matrine concentration group to a maximum of 79.8%; proportion of S phase cells reduced, and proportion of G2/M phase cells declined slightly to a minimum of 6.3%, suggesting that after the action of matrine proliferation of C6 cells was significantly inhibited and the cells were arrested in the G1 phase. CONCLUSION: We concluded that Sophora flavescens Ait. has an inhibitory effect on C6 cell proliferation.

Effects of lignans extracted from Eucommia ulmoides and aldose reductase inhibitor epalrestat on hypertensive vascular remodeling.

AIM OF THE STUDY: To investigate the effects of lignans extracted from Eucommia ulmoides and epalrestat on vascular remodeling in spontaneously hypertensive rats. MATERIALS AND METHODS: Ten-week-old male spontaneously hypertensive rats were randomly divided into 3 groups (12 rats each group), and treated orally with 100 mg/kg/d of captopril (an angiotensin-converting enzyme inhibitor), 100 mg/kg/d of epalrestat (an aldose reductase inhibitor) and 300 mg/kg/d of lignans by gavage daily for 16 weeks, respectively. Sex-, age-, and number-matched spontaneously hypertensive rats and normotensive Wistar Kyoto rats, were treated with distilled water (vehicle) as controls. The rats were weighed weekly. Mean arterial blood pressure and heart rate were measured periodically by non-invasive blood pressure monitoring. They were sacrificed at the end of experiment (26-week-old). Superior mesenteric artery and aorta were isolated for determination of histomorphometry and the expression of aldose reductase by immunohistochemistry. RESULTS: Captopril and lignans, but not epalrestat, decreased mean arterial blood pressure in spontaneously hypertensive rats. Vascular remodeling was improved in all three treated groups by histomorphometry. CONCLUSIONS: Both lignans and epalrestat reversed hypertensive vascular remodeling. Aldose reductase played a vital role in the pathologic process of hypertensive vascular remodeling rather than elevation of blood pressure. These data suggested that aldose reductase could be a new therapeutic target for the treatment of cardiovascular diseases.

Effect of Myrtol standardized on mucus hypersecretion and clearance of Pseudomonas aeruginosa in a rat model of chronic obstructive pulmonary disease.

This study aimed to investigate the effect of Myrtol standardized (GeloMyrtol forte) in the treatment of chronic obstructive pulmonary disease (COPD) in an animal model. A total of 93 experimental rats were randomly divided into 6 groups: control (n = 6), exposure to cigarette smoke (CS, n = 6), CS plus Myrtol standardized treatment (CS + M, n = 6), Pseudomonas aeruginosa (PA) infection (PA, n = 25), CS + PA (n = 25), and CS + PA + M (n = 25). For all 62 CS rats, they were exposed to cigarette smoke for a period of 12 weeks. During this time period the 31 CS + M rats (CS + M; CS + PA + M) received 300 mg/kg/day Myrtol standardized intragastrically always 30 min prior to smoke exposure. For CS + PA and CS + PA + M rats, intratracheal PA inoculation was performed after the 12 weeks of smoke exposure. All intratracheal PA inoculations were followed by a post-infection examination at 6, 12, 24, 48 and 72 h in each 5 rats. All study animals were euthanized and their lungs were excised; the left lung was homogenized for determination of bacterial load and measurements of TNF-alpha and IL-6, the right lungs were preserved for histo- and immunohistochemical examinations (e. g. MUC5AC). The lungs from CS rats were pathologically similar to those of COPD patients with the characteristics of goblet cell metaplasia and MUC5AC hypersecretion. CS animals had a significantly greater number of MUC5AC positive cells in the bronchial epithelial cells, and significantly increased expression levels of TNF-alpha and IL-6 after PA infection. However, the administration of Myrtol standardized significantly (p = 0.002) attenuated MUC5AC hypersecretion, measured as integrity optical density (IOD), in CS + M rats (45.98 +/- 6.25) as compared to CS alone (65.55 +/- 11.18) rats. The same applies at different time points between CS + PA rats (65.15 +/- 11.94, 75.88 +/- 7.42, 81.2 +/- 6.49, 75.14 +/- 6.85 and 67.32 +/- 10.61, respectively) and CS + PA + M rats (47.08 +/- 4.78, 54.22 +/- 6.59, 65.4 +/- 6.12, 59.98 +/- 4.96 and 48.43 +/- 7.29, respectively). Similar effects were found in the production of IL-6 and TNF-alpha in the CS + PA + M lungs. Similarly the bacterial load of 10,980 +/- 4,253 CFU in CS + PA + M was significantly lower compared to 42,400 +/- 3,296 CFU in CS + PA lungs after 72 h PA infection. In conclusion, this experimental study demonstrates a significant therapeutic effect of Myrtol standardized in treating common pathological conditions, such as airway mucus hypersecretion and defect of mucociliary functions in COPD.

Zicam-induced damage to mouse and human nasal tissue.

Intranasal medications are used to treat various nasal disorders. However, their effects on olfaction remain unknown. Zicam (zinc gluconate; Matrixx Initiatives, Inc), a homeopathic substance marketed to alleviate cold symptoms, has been implicated in olfactory dysfunction. Here, we investigated Zicam and several common intranasal agents for their effects on olfactory function. Zicam was the only substance that showed significant cytotoxicity in both mouse and human nasal tissue. Specifically, Zicam-treated mice had disrupted sensitivity of olfactory sensory neurons to odorant stimulation and were unable to detect novel odorants in behavioral testing. These findings were long-term as no recovery of function was observed after two months. Finally, human nasal explants treated with Zicam displayed significantly elevated extracellular lactate dehydrogenase levels compared to saline-treated controls, suggesting severe necrosis that was confirmed on histology. Our results demonstrate that Zicam use could irreversibly damage mouse and human nasal tissue and may lead to significant smell dysfunction.

Adult type 3 adenylyl cyclase-deficient mice are obese.

BACKGROUND: A recent study of obesity in Swedish men found that polymorphisms in the type 3 adenylyl cyclase (AC3) are associated with obesity, suggesting the interesting possibility that AC3 may play a role in weight control. Therefore, we examined the weight of AC3 mice over an extended period of time. METHODOLOGY/PRINCIPAL FINDINGS: We discovered that AC3(-/-) mice become obese as they age. Adult male AC3(-/-) mice are about 40% heavier than wild type male mice while female AC3(-/-) are 70% heavier. The additional weight of AC3(-/-) mice is due to increased fat mass and larger adipocytes. Before the onset of obesity, young AC3(-/-) mice exhibit reduced physical activity, increased food consumption, and leptin insensitivity. Surprisingly, the obesity of AC3(-/-) mice is not due to a loss of AC3 from white adipose and a decrease in lipolysis. CONCLUSIONS/SIGNIFICANCE: We conclude that mice lacking AC3 exhibit obesity that is apparently caused by low locomotor activity, hyperphagia, and leptin insensitivity. The presence of AC3 in primary cilia of neurons of the hypothalamus suggests that cAMP signals generated by AC3 in the hypothalamus may play a critical role in regulation of body weight.