Abstract:

Objective

To investigate the effect and regulatory mechanism of chromosome structure maintenance protein 4 （SMC4） on hypoxemic human pulmonary artery smooth muscle（hPASMC）pyroptosis.

Method

hPASMCs were used as experimental subjects， and the cells were randomly divided into normoxic group， hypoxic group， hypoxic + NC group， and hypoxic + si-SMC4 group. Except for the normoxic group， which was cultured under 37 ℃ and 5% CO₂ conditions，the hypoxic group and hypoxic + si-SMC4 group were cultured for 24 hours under 3%O₂ and 5%CO₂ restrictions to establish a hypoxic model. Western blot was used to detect the expression levels of SMC4 and apoptosis related proteins such as interleukin-1β， GSDMD， GSDMD-N， and NOD like receptor protein 3 （NLRP3）. Lactate dehydrogenase （LDH） release assay was used to detect the release of LDH release in cell culture medium. Cell immunofluorescence DAPI/PI double staining to was used to detect the proportion of pyroptosis cells. Rats were divided into normoxic group and 10% O₂ low oxygen group. Blood pressure monitoring and HE staining were used to determine the success of the animal model. Tissue immunofluorescence was used to detect SMC4 tissue localization，and Western blot was used to detect differential expression of SMC4 in various tissues and organs.Independent sample t-test was used for comparison between the two groups； Multiple groups were analyzed using one- way ANOVA， and pairwise comparisons were processed using SNK-q test.

Result

Compared with the normoxic group， the expression level of SMC4 protein was increased in the lung tissue of rats in the hypoxic group （0.86 ± 0.06 vs 0.75 ± 0.05， P ＜ 0.05）， and SMC4 was localized in the smooth muscle layer of pulmonary vessels. Compared with the normoxic group， the hypoxic group showed increased pulmonary artery thickness [（30.33 ± 3.49 ） vs （13.52 ± 2.34） μm］，NEMO （0.77 ± 0.14 vs 0.31 ± 0.09）， NLRP3 （1.04 ± 0.15 vs 0.54 ± 0.22）， GSDMD （1.34 ± 0.32 vs 0.86 ± 0.34）， GSDMD-N （0.90 ± 0.11 vs 0.45 ± 0.02）， IL-1β （3.15 ± 0.56 vs 0.53 ± 0.06）expression， percentage of LDH release （71.69 ± 10.36 vs 19.94 ± 3.54）， and PI positive cells （4.81 ±0.64 vs 2.18 ± 0.31）， P ＜ 0.05. The low oxygen+si-SMC4 group can reverse the above effects.

Conclusion

SMC4 is upregulated in hypoxic pulmonary hypertension， activates the expression of cell apoptosis related proteins， and increases the release of LDH from hPASMCs， and increasing the proportion of PI positive cells. Knocking down SMC4 may reverse these changes， which may be related to NEMO regulation.

Key words: Pulmonary artery smooth muscle cells, SMC4, Cell pyroptosis, NLRP3