Submandibular Gland Pathogenesis Following SARS-CoV-2 Infection and Implications for Xerostomia.

Although SARS-CoV-2 induces mucin hypersecretion in the respiratory tract, hyposalivation/xerostomia has been reported by COVID-19 patients. We evaluate the submandibular gland (SMGs) pathogenesis in SARS-CoV-2-infected K18-hACE2 mice, focusing on the impact of infection on the mucin production and structural integrity of acini, ductal system, myoepithelial cells (MECs) and telocytes. The spike protein, the nucleocapsid protein, hACE2, actin, EGF, TNF-α and IL-1ß were detected by immunofluorescence, and the Egfr and Muc5b expression was evaluated. In the infected animals, significant acinar hypertrophy was observed in contrast to ductal atrophy. Nucleocapsid proteins and/or viral particles were detected in the SMG cells, mainly in the nuclear membrane-derived vesicles, confirming the nuclear role in the viral formation. The acinar cells showed intense TNF-α and IL-1ß immunoexpression, and the EGF-EGFR signaling increased, together with Muc5b upregulation. This finding explains mucin hypersecretion and acinar hypertrophy, which compress the ducts. Dying MECs and actin reduction were also observed, indicating failure of contraction and acinar support, favoring acinar hypertrophy. Viral assembly was found in the dying telocytes, pointing to these intercommunicating cells as viral transmitters in SMGs. Therefore, EGF-EGFR-induced mucin hypersecretion was triggered by SARS-CoV-2 in acinar cells, likely mediated by cytokines. The damage to telocytes and MECs may have favored the acinar hypertrophy, leading to ductal obstruction, explaining xerostomia in COVID-19 patients. Thus, acinar cells, telocytes and MECs may be viral targets, which favor replication and cell-to-cell viral transmission in the SMG, corroborating the high viral load in saliva of infected individuals.

Role of serotonin, estrogen, and TNF-α in the paroxetine-impaired steroidogenesis and testicular macrophages polarization.

BACKGROUND: Paroxetine, a selective serotonin reuptake inhibitor (SSRI) antidepressant, has caused male sexual dysfunction; however, the paroxetine mechanisms of action in testes are still unclear. OBJECTIVES: Paroxetine serotonergic effects in testes were evaluated, focusing on steroidogenesis and the correlation between macrophages population and possible TNF-α-derived oxidative stress. We also verified whether the changes are reversible following treatment interruption. MATERIALS AND METHODS: Adult rats received paroxetine (PG35 and PG65) or tap water (CG) for 35 days. PG65 was maintained without treatment for 30 more days. Intratesticular testosterone (IT), nitrite, and malondialdehyde concentrations were measured. To confirm serotonergic and estrogenic effects, Htr1b and Esr1 expressions were analyzed. The daily sperm production (DSP), frequency of abnormal seminiferous tubules (ST), SC number, ST area, and Leydig cells nuclear area (LCnu) were evaluated. TUNEL+ germ cells, M1 (CD68+ ), and M2 (Perls+ ) macrophages were quantified. 17ß-HSD7, CYP19A1, NDRG2, oxytocin, TNF-α, and iNOS were evaluated by immunoreactions. Oxytocin and NDRG2 protein levels as well as Tnfa mRNA expression were also analyzed. RESULTS: The Htr1b downregulation in testes confirmed the paroxetine serotonergic effect. The testicular sections showed abnormal ST frequency, ST atrophy and reduction of DSP, LCnu, SC number and Perls+ macrophages. TUNEL+ germ cells and LC were associated with strong NDRG2 immunoexpression. Paroxetine reduced IT levels and 17ß-HSD7 immunoexpression in parallel to increased CYP19A1, oxytocin, TNF-α and iNOS. Esr1 and Tnfa overexpression and increased number of CD68+ macrophages were also observed together with high nitrite and malondialdehyde levels. Most parameters were not recovered in PG65. CONCLUSIONS: Paroxetine serotonergic effect impairs LC steroidogenesis, via aromatization, increasing estrogen/testosterone ratio, which in turn upregulate NDRG2, promoting apoptosis, and impairing sperm production. Serotonin-estrogen pathways may be responsible for M2/M1 polarization, Tnfa upregulation, and induction of oxidative stress. The unrecovered testicular changes after treatment discontinuation are due to persistent paroxetine serotonin/estrogen effects.

Venlafaxine increases aromatization, reduces apical V-ATPase in clear cells and induces increased number of mast cells and smooth muscle cells death in rat cauda epididymis.

Depressive disorders (DD) have affected millions of people worldwide. Venlafaxine, antidepressant of the class of serotonin and norepinephrine reuptake inhibitors, has been prescribed for the treatment of DD. In rat testes, venlafaxine induces testosterone (T) aromatization and increases estrogen levels. Aromatase is a key enzyme for the formation of estrogen in the epididymis, an essential organ for male fertility. We investigated the impact of serotonergic/noradrenergic venlafaxine effect on the epididymal cauda region, focusing on aromatase, V-ATPase and EGF epithelial immunoexpression, smooth muscle (SM) integrity and mast cells number (MCN). Male rats were distributed into control (CG; n = 10) and venlafaxine (VFG, n = 10) groups. VFG received 30 mg/kg b.w. of venlafaxine for 35 days. The epididymal cauda was processed for light and transmission electron microscopy (TEM). The expression of connexin 43 (Cx43) and estrogen alpha (Esr1), adrenergic (Adra1a) and serotonergic (Htr1b) receptors were analyzed. Clear cells (CCs) area, SM thickness, viable spermatozoa (VS) and MCN were evaluated. Apoptosis was confirmed by TUNEL and TEM. The following immunoreactions were performed: T, aromatase, T/aromatase co-localization, V-ATPase, EGF, Cx43 and PCNA. The increased Adra1a and reduced Htr1b expressions confirmed the noradrenergic and serotonergic venlafaxine effects, respectively, corroborating the increased MCN, apoptosis and atrophy of SM. In VFG, the epithelial EGF increased, explaining Cx43 overexpression and basal cells mitotic activity. T aromatization and Esr1 downregulation indicate high estrogen levels, explaining CCs hypertrophy and changes in the V-ATPase localization, corroborating VS reduction. Thus, in addition to serotonergic/noradrenergic effects, T/estrogen imbalance, induced by venlafaxine, impairs epididymal structure and function.

Cimetidine-induced androgenic failure causes cell death and changes in actin, EGF and V-ATPase immunoexpression in rat submandibular glands.

Submandibular gland (SMG) is responsive to androgens via androgen receptor (AR). We verified whether cimetidine induces androgenic dysfunction in SMG, and evaluated the structural integrity, cell death and immunoexpression of actin, EGF and V-ATPase in androgen-deficient SMG. Male rats received cimetidine (CMTG) and control animals (CG) received saline. Granular convoluted tubules (GCTs) diameter and number of acinar cell nuclei were evaluated. TUNEL and immunofluorescence reactions for detection of AR, testosterone, actin, EGF and V-ATPase were quantitatively analysed. In CG, testosterone immunolabelling was detected in acinar and ductal cells cytoplasm. AR-immunolabelled nuclei were observed in acinar cells whereas ductal cells showed AR-immunostained cytoplasm, indicating a non-genomic AR action. In CMTG, the weak testosterone and AR immunoexpression confirmed cimetidine-induced androgenic failure. A high cell death index was correlated with decreased number of acinar cells, GCTs diameter and EGF immunoexpression under androgenic dysfunction. Actin immunofluorescence decreased in the SMG cells, but an increased and diffuse cytoplasmic V-ATPase immunolabelling was observed in striated ducts, suggesting a disruption in the actin-dependent V-ATPase recycling due to androgenic failure. Our findings reinforce the androgenic role in the maintenance of SMG histophysiology, and point to a potential clinical use of cimetidine against androgen-dependent glandular tumour cells.