Glycyrrhizin Protects Submandibular Gland Against Radiation Damage by Enhancing Antioxidant Defense and Preserving Mitochondrial Homeostasis.

Aims: Radiotherapy inevitably causes radiation damage to the salivary glands (SGs) in patients with head and neck cancers (HNCs). Excessive reactive oxygen species (ROS) levels and imbalanced mitochondrial homeostasis are serious consequences of ionizing radiation in SGs; however, there are few mitochondria-targeting therapeutic approaches. Glycyrrhizin is the main extract of licorice root and exhibits antioxidant activity to relieve mitochondrial damage in certain oxidative stress conditions. Herein, the effects of glycyrrhizin on irradiated submandibular glands (SMGs) and the related mechanisms were investigated. Results: Glycyrrhizin reduced radiation damage in rat SMGs at both the cell and tissue levels, and promoted saliva secretion in irradiated SMGs. Glycyrrhizin significantly downregulated high-mobility group box-1 protein (HMGB1) and toll-like receptor 5 (TLR5). Moreover, glycyrrhizin significantly suppressed the increases in malondialdehyde and glutathione disulfide (GSSG) levels; elevated the activity of some critical antioxidants, including superoxide dismutase, catalase, glutathione peroxidase, and glutathione (GSH); and increased the GSH/GSSG ratio in irradiated cells. Importantly, glycyrrhizin effectively enhanced thioredoxin-2 levels and scavenged mitochondrial ROS, inhibited the decline in mitochondrial membrane potential, improved adenosine triphosphate synthesis, preserved the mitochondrial ultrastructure, activated the proliferator-activated receptor-gamma coactivator-1alpha (PGC-1α)/nuclear respiratory factor 1/2 (NRF1/2)/mitochondrial transcription factor A (TFAM) signaling pathway, and inhibited mitochondria-related apoptosis in irradiated SMG cells and tissues. Innovation: Radiotherapy causes radiation sialadenitis in HNC patients. Our data suggest that glycyrrhizin could be a mitochondria-targeted antioxidant for the prevention of radiation damage in SGs. Conclusion: These findings demonstrate that glycyrrhizin protects SMGs from radiation damage by downregulating HMGB1/TLR5 signaling, maintaining intracellular redox balance, eliminating mitochondrial ROS, preserving mitochondrial homeostasis, and inhibiting apoptosis.

The MocR family transcriptional regulator DnfR has multiple binding sites and regulates Dirammox gene transcription in Alcaligenes faecalis JQ135.

Microbial ammonia oxidation is vital to the nitrogen cycle. A biological process, called Dirammox (direct ammonia oxidation, NH3 →NH2 OH→N2 ), has been recently identified in Alcaligenes ammonioxydans and Alcaligenes faecalis. However, its transcriptional regulatory mechanism has not yet been fully elucidated. The present study characterized a new MocR-like transcription factor DnfR that is involved in the Dirammox process in A. faecalis strain JQ135. The entire dnf cluster was composed of 10 genes and transcribed as five transcriptional units, that is, dnfIH, dnfR, dnfG, dnfABCDE and dnfF. DnfR activates the transcription of dnfIH, dnfG and dnfABCDE genes, and represses its own transcription. The intact 1506-bp dnfR gene was required for activation of Dirammox. Electrophoretic mobility shift assays and DNase I footprinting analyses showed that DnfR has one binding site in the dnfH-dnfR intergenic region and two binding sites in the dnfG-dnfA intergenic region. Three binding sites of DnfR shared a 6-bp repeated conserved sequence 5'-GGTCTG-N17 -GGTCTG-3' which was essential for the transcription of downstream target genes. Cysteine and glutamate act as possible effectors of DnfR to activate the transcription of transcriptional units of dnfG and dnfABCDE, respectively. This study provided new insights in the transcriptional regulation mechanism of Dirammox by DnfR in A. faecalis JQ135.

Nicotinamide mitigates radiation injury in submandibular gland by protecting mitochondrial structure and functions.

BACKGROUND: Radiation damage to salivary gland is inevitable in head and neck cancer patients receiving radiotherapy. Safe and effective treatments for protecting salivary glands from radiation are still unavailable. Mitochondrial damage is a critical mechanism in irradiated salivary gland; however, treatment targeting mitochondria has not received much attention. Nicotinamide is a key component of the mitochondrial metabolism. Here, we investigated the effects and underlying mechanisms of nicotinamide on protecting irradiated submandibular gland. METHODS: Submandibular gland cells and tissues were randomly divided into four groups: control, nicotinamide alone, radiation alone, and radiation with nicotinamide pretreatment. Cell viability was detected by PrestoBlue cell viability reagent. Histopathological alterations were observed with HE staining. Pilocarpine-stimulated saliva was measured from Wharton's duct. Cell apoptosis was determined by flow cytometry and terminal deoxynucleotidyl transferase dUTP nick-end labeling assay. Nicotinamide phosphoribosyl transferase was examined with immunofluorescence. The levels of nicotinamide adenine dinucleotide, mitochondrial membrane potential, and adenosine triphosphate were measured with the relevant kits. The mitochondrial ultrastructure was observed under transmission electron microscopy. RESULTS: Nicotinamide significantly mitigated radiation damage both in vitro and in vivo. Also, nicotinamide improved saliva secretion and reduced radiation-induced apoptosis in irradiated submandibular glands. Moreover, nicotinamide improved nicotinamide phosphoribosyl transferase and the levels of nicotinamide adenine dinucleotide/adenosine triphosphate and mitochondrial membrane potential, all of which were decreased by radiation in submandibular gland cells. Importantly, nicotinamide protected the mitochondrial ultrastructure from radiation. CONCLUSION: These findings demonstrate that nicotinamide alleviates radiation damage in submandibular gland by replenishing nicotinamide adenine dinucleotide and maintaining mitochondrial function and ultrastructure, suggesting that nicotinamide could be used as a prospective radioprotectant for preventing radiation sialadenitis.

Salidroside Ameliorates Radiation Damage by Reducing Mitochondrial Oxidative Stress in the Submandibular Gland.

Radiotherapy for patients with head and neck cancer inevitably causes radiation damage to salivary glands (SGs). Overproduction of reactive oxygen species (ROS) leads to mitochondrial damage and is critical in the pathophysiology of SG radiation damage. However, mitochondrial-targeted treatment is unavailable. Herein, both in vitro and in vivo models of radiation-damaged rat submandibular glands (SMGs) were used to investigate the potential role of salidroside in protecting irradiated SGs. Cell morphology was observed with an inverted phase-contrast microscope. Malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT), glutathione (GSH), mitochondrial ROS, mitochondrial membrane potential (MMP), and ATP were measured using relevant kits. The mitochondrial ultrastructure was observed under transmission electron microscopy. Cell apoptosis was determined by Western blot and TUNEL assays. Saliva was measured from Wharton's duct. We found that salidroside protected SMG cells and tissues against radiation and improved the secretion function. Moreover, salidroside enhanced the antioxidant defense by decreasing MDA, increasing SOD, CAT, and GSH, and scavenging mitochondrial ROS. Furthermore, salidroside rescued the mitochondrial ultrastructure, preserved MMP and ATP, suppressed cytosolic cytochrome c and cleaved caspase 3 expression, and inhibited cell apoptosis. Together, these findings first identify salidroside as a mitochondrial-targeted antioxidant for preventing SG radiation damage.

Genetic Foundations of Direct Ammonia Oxidation (Dirammox) to N2 and MocR-Like Transcriptional Regulator DnfR in Alcaligenes faecalis Strain JQ135.

Ammonia oxidation is an important process in both the natural nitrogen cycle and nitrogen removal from engineered ecosystems. Recently, a new ammonia oxidation pathway termed Dirammox (direct ammonia oxidation, NH3→NH2OH→N2) has been identified in Alcaligenes ammonioxydans. However, whether Dirammox is present in other microbes, as well as its genetic regulation, remains unknown. In this study, it was found that the metabolically versatile bacterium Alcaligenes faecalis strain JQ135 could efficiently convert ammonia into N2 via NH2OH under aerobic conditions. Genetic deletion and complementation results suggest that dnfABC is responsible for the ammonia oxidation to N2 in this strain. Strain JQ135 also employs aerobic denitrification, mainly producing N2O and trace amounts of N2, with nitrite as the sole nitrogen source. Deletion of the nirK and nosZ genes, which are essential for denitrification, did not impair the capability of JQ135 to oxidize ammonia to N2 (i.e., Dirammox is independent of denitrification). Furthermore, it was also demonstrated that pod (which encodes pyruvic oxime dioxygenase) was not involved in Dirammox and that AFA_16745 (which was previously annotated as ammonia monooxygenase and is widespread in heterotrophic bacteria) was not an ammonia monooxygenase. The MocR-family transcriptional regulator DnfR was characterized as an activator of the dnfABC operon with the binding motif 5'-TGGTCTGT-3' in the promoter region. A bioinformatic survey showed that homologs of dnf genes are widely distributed in heterotrophic bacteria. In conclusion, this work demonstrates that, besides A. ammonioxydans, Dirammox occurs in other bacteria and is regulated by the MocR-family transcriptional regulator DnfR. IMPORTANCE Microbial ammonia oxidation is a key and rate-limiting step of the nitrogen cycle. Three previously known ammonia oxidation pathways (i.e., nitrification, anaerobic ammonia oxidation [Anammox], and complete ammonia oxidation [Comammox]) are mediated by autotrophic microbes. However, the genetic foundations of ammonia oxidation by heterotrophic microorganisms have not been investigated in depth. Recently, a previously unknown pathway, termed direct ammonia oxidation to N2 (Dirammox), has been identified in the heterotrophic bacterium Alcaligenes ammonioxydans HO-1. This paper shows that, in the metabolically versatile bacterium Alcaligenes faecalis JQ135, the Dirammox pathway is mediated by dnf genes, which are independent of the denitrification pathway. A bioinformatic survey suggests that homologs of dnf genes are widely distributed in bacteria. These findings enhance the understanding of the molecular mechanisms of heterotrophic ammonia oxidation to N2.

Phenylephrine alleviates 131I damage in submandibular gland through promoting endogenous stem cell regeneration via lissencephaly-1 upregulation.

Thyroid cancer is the most common endocrine malignancy. 131I ablation therapy is an effective treatment for patients with differentiated thyroid cancer (DTC) but frequently causes radiation damage in salivary glands (SGs). Stem cell-based regenerative therapy has been found to reduce radiation sialadenitis. We hypothesize that microtubule motor-regulating protein lissencephaly-1 (LIS1) may be a key stem cell regulator responsible for its efficacy and that upregulating LIS1 would decrease131I-induced radiation sialadenitis. Here, we report that LIS1 was reduced by 131I in submandibular glands (SMGs) of rats, using both proteomic analysis and Western blot approach. Moreover, the levels of LIS1-Sca-1 and LIS1-SOX2 were downregulated by 131I together with the decrease of LIS1. In contrast, phenylephrine pretreatment enhanced LIS1 and improved the co-expressions and co-localizations of LIS1-Sca-1 and LIS1-SOX2 in 131I-irradiated SMGs. Since Sca-1 and SOX2 are the established stem cell biomarkers in salivary gland, our findings demonstrate that LIS1 may be a potential target for regulating stem cell maintenance in irradiated SGs. Importantly, phenylephrine may have the ability to promote endogenous stem cell regeneration in SMGs via upregulating the LIS1/Sca-1 and LIS1/SOX2 signaling pathways, suggesting that phenylephrine application before 131I ablation therapy may provide a practical and effective way to prevent radiation sialadenitis for DTC patients.

Phenylephrine Alleviates 131I Radiation Damage in Submandibular Gland Through Maintaining Mitochondrial Homeostasis.

PURPOSE: The impairment of the salivary glands is a permanent side effect of 131I ablation therapy for patients with differentiated thyroid cancer. Effective and safe treatments for protecting the salivary glands against 131I are currently not available. Mitochondria are susceptible to ionizing radiation, but alterations after 131I exposure are unknown. Here, we investigated the mechanisms of 131I damage in submandibular glands (SMGs) and evaluated the cytoprotective effect of phenylephrine (PE) against mitochondrial radiation damage. METHODS AND MATERIALS: Rats were randomly divided into 4 groups: control, PE alone, 131I alone, and 131I with PE pretreatment. The mitochondrial structure of SMGs was observed under transmission electron microscopy. Apoptosis was detected using terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling. Cytochrome c, cleaved-caspase 3, SIRT1, NAMPT, and PGC-1α protein levels were determined with Western blot and immunohistochemistry. Levels of mitochondrial membrane potential, nicotinamide adenine dinucleotide (NAD), and adenosine triphosphate (ATP) were measured with relevant kits. RESULTS: After exposing rat SMGs to 131I, the mitochondrial membrane structures were destroyed, the mitochondrial membrane potential decreased, the release of cytochrome c increased, and cleaved-caspase 3 and cell apoptosis were activated. Moreover, the expression of SIRT1, NAMPT, and PGC-1α was downregulated, and the levels of NAD and ATP decreased. In contrast, PE alleviated the 131I-induced mitochondrial damages and upregulated the expression of SIRT1/NAMPT/PGC-1α and the levels of NAD and ATP. CONCLUSIONS: These findings demonstrate that 131I impairs the salivary glands via the downregulation of SIRT1/NAMPT/PGC-1α signal pathways, which disturbs mitochondrial homeostasis. PE alleviated the 131I damage in SMGs at the mitochondrial level, suggesting that PE could be used as a potential radioprotector for patients with differentiated thyroid cancer with radiation sialadenitis.

Inhibition of nicotinamide phosphoribosyltransferase and depletion of nicotinamide adenine dinucleotide contribute to arsenic trioxide suppression of oral squamous cell carcinoma.

Emerging evidence suggests that increased nicotinamide phosphoribosyltransferase (NAMPT) expression is associated with the development and prognosis of many cancers, but it remains unknown regarding its role in oral squamous cell carcinoma (OSCC). In the present study, the results from tissue microarray showed that NAMPT was overexpressed in OSCC patients and its expression level was directly correlated with differential grades of cancer. Interestingly, treatment of OSCC cells with chemotherapy agent arsenic trioxide (ATO) decreased the levels of NAMPT protein and increased cellular death in an ATO dose- and time-dependent manner. Most importantly, combination of low concentration ATO with FK866 (a NAMPT inhibitor) exerted enhanced inhibitive effect on NAMPT protein and mRNA expressions, leading to synergistic cytotoxicity on cancer cells through increasing cell apoptosis and depleting intracellular nicotinamide adenine dinucleotide levels. These findings demonstrate the crucial role of NAMPT in the prognosis of OSCC and reveal inhibition of NAMPT as a novel mechanism of ATO in suppressing cancer cell growth. Our results suggest that ATO can significantly enhance therapeutic efficacy of NAMPT inhibitor, and combined treatment may be a novel and effective therapeutic strategy for OSCC patients.

Underlying protective mechanism of α1-adrenoceptor activation against irradiation-induced damage in rat submandibular gland.

OBJECTIVES: Damage to salivary gland after radiotherapy for head and neck malignant tumours can lead to irreversible oral complaints, which severely impair quality of life. The protective effect of α1-adrenoceptor activation on the salivary glands after irradiation has previously been demonstrated. However, the exact mechanism remains unclear. In this study, we investigated the underlying cytoprotective mechanism of α1-adrenoceptor activation in rat submandibular glands after irradiation. STUDY DESIGN: Rats were locally irradiated using a linear accelerator in the head and neck region with a dose of 20Gy. After irradiation, phenylephrine (5mg/kg) was injected intraperitoneally for 7 successive days and the submandibular glands were then collected. The antiapoptotic effect of phenylephrine on the gland was examined by TUNEL, the proliferative cellular nuclei antigen (PCNA) was determined by immunohistochemistry, and the activation of c-Jun N-terminal kinase (JNK) was detected by Western blot. RESULTS: The irradiation only group showed severe atrophy, increased apoptosis, enhanced cell proliferation, and the phosphorylation of JNK was markedly increased by 26.89% (P<0.05), compared to the control. The phenylephrine-treated group, however, showed remarkably alleviated atrophy, decreased apoptosis, and further increased cell proliferation, and the phosphorylation of JNK was markedly decreased by 36.00% (P<0.05), compared to the irradiation only group. CONCLUSIONS: The data showed that the underlying protective mechanism of α1-adrenoceptor activation in irradiated gland might be related to improved cell proliferation, inhibited cell apoptosis, and depressed activation of JNK. It could be helpful in protecting salivary glands against irradiation damage.

Mechanism of the protective effect of phenylephrine pretreatment against irradiation-induced damage in the submandibular gland.

Irradiation is a fundamental treatment modality for head and neck malignancies. However, a significant drawback of irradiation treatment is the irreversible damage to salivary glands in the radiation field. Although the protective effect of phenylephrine pretreatment on salivary glands following irradiation has previously been demonstrated, the exact mechanism remains unclear. In this study, we investigated the cytoprotective mechanisms of phenylephrine pretreatment in rat submandibular glands following irradiation. Rats were locally irradiated using a linear accelerator in the head and neck region with a single dose of 20 Gy. Phenylephrine (5 mg/kg) was injected intraperitoneally 30 min prior to irradiation and the submandibular glands were collected on day 7 after irradiation. In comparison with the control group, the irradiation-only group demonstrated severe atrophy, enhanced cell proliferation and increased apoptosis. The phenylephrine-pretreated group, however, demonstrated markedly alleviated atrophy, further increased cell proliferation and decreased apoptosis compared with the irradiation-only group. The data indicated that the cytoprotective mechanisms of phenylephrine pretreatment in the submandibular gland following irradiation may be related to improved cell proliferation and inhibition of cell apoptosis.