[Inhibition of connexin 43-mediated hemichannel activity promotes odontoblast differentiation of human dental pulp cells induced by lipopolysaccharide].

PURPOSE: To investigate the role and mechanism of connexin 43（Cx43）in odontoblast differentiation of human dental pulp cells (hDPCs) induced by lipopolysaccharide (LPS). METHODS: The maxillary first molar injury model of SD rats was established. The expression pattern of Cx43 in dental pulp repair after injury was detected by immunofluorescence(IF) staining. hDPCs was respectively stimulated with 0, 1, 10, 100 and 1 000 ng/mL LPS for 6 h to screen the optimal concentration, and then the expression of Cx43 was inhibited and overexpressed in hDPCs. Quantitative real-time PCR(qRT-PCR) and Western blot(WB) were used to detect the expression of Cx43 and dentin sialophosphoprotein (DSPP), dental matrix protein-1 (DMP-1), osterix (Osx) and extracellular signal-regulated kinase (ERK) activity. Furthermore, hDPCs were treated with specific Cx43 channel inhibitors to investigate the effect of Cx43-mediated channel activity in odontoblast differentiation of hDPCs, and to explore the role and mechanism of Cx43 in regulating odontoblast differentiation of hDPCs induced by LPS. Statistical analysis was performed with SPSS 26.0 software package. RESULTS: IF results showed that Cx43 was mainly expressed in the odontoblast layer in healthy dental pulp tissues. At 3-24 h after tooth injury, the expression of Cx43 decreased and then gradually increased to the normal level; from 3 days to 2 weeks after injury, the expression of Cx43 tended to be down-regulated which was in the odontoblast layer and pulp proper. The expression of DSPP mRNA was significantly up-regulated in the hDPCs stimulated with 10 ng/mL LPS for 6 h(P＜0.01). Inhibition of Cx43 significantly up-regulated the expression of DSPP, DMP-1 and Osx mRNA induced by LPS in hDPCs(P＜0.05), while overexpression of Cx43 obviously inhibited the expression of factors related to LPS-induced odontoblast differentiation(P＜0.01) and the fluorescence intensity of DSPP. 10 ng/mL LPS activated ERK signal in hDPCs, and overexpression of Cx43 significantly attenuated the activity of ERK signal induced by LPS(P＜0.01). Inhibition of Cx43-mediated hemichannel (HC) promoted mRNA expression of factors related to odontoblast differentiation in hDPCs and the activity of ERK signal induced by LPS(P＜0.05), while blocking Cx43-mediated gap junction channel (GJC) inhibited odontoblast differentiation. CONCLUSIONS: Cx43 participates in the regulation of dental pulp repair after injury, and its expression shows a downward trend as a whole. Inhibition of Cx43 or blocking of HC promotes LPS-induced ERK signal activity and odontoblast differentiation of hDPCs.

Connexin43 reduces LPS-induced inflammation in hDPCs through TLR4-NF-κB pathway via hemichannels.

OBJECTIVES: Connexin43 (Cx43) is involved in the inflammation of many tissue types. Dental caries is infectious disease resulting from mineralized tissue dissolution by a specific bacterial population, causing pulp inflammation. However, Cx43's role in dental pulp remains unclear. Here, we investigated the function of Cx43 during pulp inflammation. MATERIALS AND METHODS: We constructed a dentin injury model in Sprague-Dawley rats to investigate changes in Cx43 expression during pulp inflammation. Cx43 was inhibited in human dental pulp cells (hDPCs) that had been stimulated with lipopolysaccharide (LPS) to investigate the effect of Cx43 on inflammatory response. Promotion of TLR4-NF-κB pathway activity and special Cx43 channel inhibitors were used to clarify the function of Cx43 in hDPCs. RESULTS: Dentin injury led to low-level inflammation in dental pulp. Following dentin injury, Cx43 expression initially decreased before gradually recovering to normal levels. Cx43 inhibition reduced LPS-induced expression of inflammatory cytokines and NF-κB pathway activity. Promotion of NF-κB pathway activity counteracted the effect of Cx43 in hDPCs. Furthermore, inhibition of Cx43 hemichannels reduced LPS-induced inflammatory cytokine expression. CONCLUSIONS: Cx43 is involved in inflammation of dental pulp, while its inhibition reduced LPS-induced inflammation in hDPCs through NF-κB pathway via blockage of hemichannels.

[High titer ethanol production from an atmospheric glycerol autocatalytic organosolv pretreated wheat straw].

The expensive production of bioethanol is because it has not yet reached the 'THREE-HIGH' (High-titer, high-conversion and high-productivity) technical levels of starchy ethanol production. To cope with it, it is necessary to implement a high-gravity mash bioethanol production (HMBP), in which sugar hydrolysates are thick and fermentation-inhibitive compounds are negligible. In this work, HMBP from an atmospheric glycerol autocatalytic organosolv pretreated wheat straw was carried out with different fermentation strategies. Under an optimized condition (15% substrate concentration, 10 g/L (NH4)2SO4, 30 FPU/g dry matter, 10% (V/V) inoculum ratio), HMBP was at 31.2 g/L with a shaking simultaneous saccharification and fermentation (SSF) at 37 degrees C for 72 h, and achieved with a conversion of 73% and a productivity of 0.43 g/(L x h). Further by a semi-SFF with pre-hydrolysis time of 24 h, HMBP reached 33.7 g/L, the conversion and productivity of which was 79% and 0.47 g/(L x h), respectively. During the SSF and semi-SSF, more than 90% of the cellulose in both substrates were hydrolyzed into fermentable sugars. Finally, a fed-batch semi-SFF was developed with an initial substrate concentration of 15%, in which dried substrate (= the weight of the initial substrate) was divided into three portions and added into the conical flask once each 8 h during the first 24 h. HMBP achieved at 51.2 g/L for 72 h with a high productivity of 0.71 g/(L x h) while a low cellulose conversion of 62%. Interestingly, the fermentation inhibitive compound was mainly acetic acid, less than 3.0 g/L, and there were no other inhibitors detected, commonly furfural and hydroxymethyl furfural existing in the slurry. The data indicate that the lignocellulosic substrate subjected to the atmospheric glycerol autocatalytic organosolv pretreatment is very applicable for HMBP. The fed-batch semi-SFF is effective and desirable to realize an HMBP.