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Acidic pH environment induces autophagy in osteoblasts

View Article: PubMed Central - PubMed

ABSTRACT

Osteoblasts (OBs) play an important role in bone fracture healing, yet the extreme adverse microenvironment in fracture sites has a negative impact on the survival of OBs. Therefore, it is important to study how OBs behave in the complex fracture microenvironment. Studies have shown that autophagy plays a pivotal role in maintaining cellular homeostasis and defending the cell against adverse microenvironments. In this study we found the induction of autophagy in OBs at femoral bone fracture sites, which may be a result of ischemia, oxidative stress and hypoxia within the local area. At fracture sites a low pH environment also developed. Until now it has been unclear whether the induction of autophagy in osteoblasts is triggered by the acidic pH environment. Therefore, we cultured OBs in vitro in media of different pH values, and found both autophagy and apoptosis increased in OBs in acidic conditions. However, when autophagy inhibitor chloroquine (CQ) was used, apoptosis increased significantly compared with that without CQ. Thus indicating that inhibition of autophagy may promote apoptosis in OBs in an acidic environment, which may provide a new therapeutic strategy to decrease cell apoptosis in OBs through the use of drugs that modulate the autophagic state.

No MeSH data available.


Acidic pH environment has a negative effect on the survival of OBs.OBs were cultured in media with different pH values for 12, 24, 48 h and cell viability was measured by MTT. Apoptosis was determined by flow cytometry. (A–D) MC3T3-E1 cells was cultured in media with pH6.4, 6.8 and 7.4 for 12, 24, 48 h, and cell viability was detected. *P < 0.05 vs.12 h with pH6.4 and 6.8, and *P < 0.05 vs.6 h with pH7.4. (E) Cell apoptosis increased in media with pH 6.8 and 6.4 at the time of 48 h including both early apoptosis and late apoptosis. (F) Analysis of apoptotic rate with pH 6.4, 6.8 and 7.4 at the time of 48 h. *P < 0.05 vs.pH 7.4. Values are the mean ± sd, *P < 0.05 in comparison to the control by one-way ANOVA.
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f2: Acidic pH environment has a negative effect on the survival of OBs.OBs were cultured in media with different pH values for 12, 24, 48 h and cell viability was measured by MTT. Apoptosis was determined by flow cytometry. (A–D) MC3T3-E1 cells was cultured in media with pH6.4, 6.8 and 7.4 for 12, 24, 48 h, and cell viability was detected. *P < 0.05 vs.12 h with pH6.4 and 6.8, and *P < 0.05 vs.6 h with pH7.4. (E) Cell apoptosis increased in media with pH 6.8 and 6.4 at the time of 48 h including both early apoptosis and late apoptosis. (F) Analysis of apoptotic rate with pH 6.4, 6.8 and 7.4 at the time of 48 h. *P < 0.05 vs.pH 7.4. Values are the mean ± sd, *P < 0.05 in comparison to the control by one-way ANOVA.

Mentions: Viability of osteoblastic cell line, MC3T3-E1, was markedly influenced by the acidity of the culture media in which they are grown. Both pH values of 6.4 and 6.8 induced a significant decrease in cell viability compared with that of pH 7.4 (P < 0.05). While all cells died when cultured in media at pH 6.0. Furthermore, a pH- and time-dependent decrease in cell viability was observed, with a significant difference in viability between pH value of 6.4 and 6.8 (Fig. 2A–D). Levels of apoptosis were determined by flow cytometry utilising Annexin-V and PI dual labelling. Cells in the Q2 and Q4 quadrants represent apoptotic cells. Increased rate of primarily early apoptosis (but also some late apoptosis) was detected in MC3T3-E1 cells in media with pH 6.8 and 6.4 after 48 h of exposure. Compared with cells cultured at pH 7.4, those at pH 6.4 and 6.8 exhibited statistically significantly increased levels of apoptosis (P < 0.05). (Fig. 2E,F). Comparison between the levels of apoptosis between the two acidic culture conditions showed increased apoptosis at pH6.4, however there is no statistically significant difference (P > 0.05).


Acidic pH environment induces autophagy in osteoblasts
Acidic pH environment has a negative effect on the survival of OBs.OBs were cultured in media with different pH values for 12, 24, 48 h and cell viability was measured by MTT. Apoptosis was determined by flow cytometry. (A–D) MC3T3-E1 cells was cultured in media with pH6.4, 6.8 and 7.4 for 12, 24, 48 h, and cell viability was detected. *P < 0.05 vs.12 h with pH6.4 and 6.8, and *P < 0.05 vs.6 h with pH7.4. (E) Cell apoptosis increased in media with pH 6.8 and 6.4 at the time of 48 h including both early apoptosis and late apoptosis. (F) Analysis of apoptotic rate with pH 6.4, 6.8 and 7.4 at the time of 48 h. *P < 0.05 vs.pH 7.4. Values are the mean ± sd, *P < 0.05 in comparison to the control by one-way ANOVA.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5382697&req=5

f2: Acidic pH environment has a negative effect on the survival of OBs.OBs were cultured in media with different pH values for 12, 24, 48 h and cell viability was measured by MTT. Apoptosis was determined by flow cytometry. (A–D) MC3T3-E1 cells was cultured in media with pH6.4, 6.8 and 7.4 for 12, 24, 48 h, and cell viability was detected. *P < 0.05 vs.12 h with pH6.4 and 6.8, and *P < 0.05 vs.6 h with pH7.4. (E) Cell apoptosis increased in media with pH 6.8 and 6.4 at the time of 48 h including both early apoptosis and late apoptosis. (F) Analysis of apoptotic rate with pH 6.4, 6.8 and 7.4 at the time of 48 h. *P < 0.05 vs.pH 7.4. Values are the mean ± sd, *P < 0.05 in comparison to the control by one-way ANOVA.
Mentions: Viability of osteoblastic cell line, MC3T3-E1, was markedly influenced by the acidity of the culture media in which they are grown. Both pH values of 6.4 and 6.8 induced a significant decrease in cell viability compared with that of pH 7.4 (P < 0.05). While all cells died when cultured in media at pH 6.0. Furthermore, a pH- and time-dependent decrease in cell viability was observed, with a significant difference in viability between pH value of 6.4 and 6.8 (Fig. 2A–D). Levels of apoptosis were determined by flow cytometry utilising Annexin-V and PI dual labelling. Cells in the Q2 and Q4 quadrants represent apoptotic cells. Increased rate of primarily early apoptosis (but also some late apoptosis) was detected in MC3T3-E1 cells in media with pH 6.8 and 6.4 after 48 h of exposure. Compared with cells cultured at pH 7.4, those at pH 6.4 and 6.8 exhibited statistically significantly increased levels of apoptosis (P < 0.05). (Fig. 2E,F). Comparison between the levels of apoptosis between the two acidic culture conditions showed increased apoptosis at pH6.4, however there is no statistically significant difference (P > 0.05).

View Article: PubMed Central - PubMed

ABSTRACT

Osteoblasts (OBs) play an important role in bone fracture healing, yet the extreme adverse microenvironment in fracture sites has a negative impact on the survival of OBs. Therefore, it is important to study how OBs behave in the complex fracture microenvironment. Studies have shown that autophagy plays a pivotal role in maintaining cellular homeostasis and defending the cell against adverse microenvironments. In this study we found the induction of autophagy in OBs at femoral bone fracture sites, which may be a result of ischemia, oxidative stress and hypoxia within the local area. At fracture sites a low pH environment also developed. Until now it has been unclear whether the induction of autophagy in osteoblasts is triggered by the acidic pH environment. Therefore, we cultured OBs in vitro in media of different pH values, and found both autophagy and apoptosis increased in OBs in acidic conditions. However, when autophagy inhibitor chloroquine (CQ) was used, apoptosis increased significantly compared with that without CQ. Thus indicating that inhibition of autophagy may promote apoptosis in OBs in an acidic environment, which may provide a new therapeutic strategy to decrease cell apoptosis in OBs through the use of drugs that modulate the autophagic state.

No MeSH data available.