Effect Of Reduced Paralemmin-3 Expression On LPS-Induced Acute Lung Injury In Rat And Research On Its Protective Mechanism

Objective The study was performed to investigate the effect of reduced paralemmin-3(PALM3) expression on LPS-induced ALI in vivo and vitro by constructing recombinant adenoviral vector of short hairpin RNA(sh RNA) against rat PAML3. Meanwhile, to illuminate the protective mechanisms of reduced PALM3 expression in ALI, the co-immunoprecipitation was used to identify whether the PALM3 interacting with the adaptor molecules in LPS-TLR4 signaling such as My D88, TICAM-2, IRAK-1, TRAF-6.Methods1. Construction and identification of recombinant adenovirus expressing short Hairpin RNA of rat PALM3According to the principle of the RNA interfering sequence design, three different sequences were chosen as the target sequences. Then, the short hairpin complementary strand targeting the consensus sequences of PALM3 were designed and inserted into the shuttle plasmid p GV120-EGFP. The p GV120-PALM3-sh RNA-EGFP was identified by PCR and DNA sequencing. Recombinant viruses were generated by using 293 packaging cells co-transfected with the shuttle plasmid and the framing plasmid(p BHGlox) via the Ad MaxTMsystem. The recombinant viruses were named as Ad.PALM3-sh RNA-1, Ad.PALM3-sh RNA-2, Ad.PALM3-sh RNA-3, respectively. And, the titers of recombinant adenoviruses were determined by means of 50% tissue culture infectious dose. The rat alveolar epithelial cell Ⅱ(AEⅡ cell) was separated from the rat lung tissue by using the enzyme digestion procedure. The methods of differential velocity centrifugation and immune adherence were used to purify the AE Ⅱ cell.Then, the pulmonary surfactant-associated protein C(SP-C) was used as the specific marker to identify the AEⅡ cell. The three different recombinant adenoviruses were used to infect the AE Ⅱ cells, respectively. By observing EGFP expression with fluorescence microscopy, the optimal multiplicity of infection(multiplicity of infection,MOI) was determinated. The PALM3 protein expression was detected by Western Blot to compare the differences of transfection efficiency.2. Effect of downregulation of PALM3 on LPS-induced acute lung injury in ratAccording to the previous results, Ad.PALM3-sh RNA-1 was selected for subsequent experiment. Wistar rats were lightly anesthetized by ether inhalation, and inoculated intranasally with 100μL of PBS(Control group), Ad.PALM3-sh RNA(3×108PFU in 100 μL)(Ad.PALM3-sh RNA group) or Ad.EGFP(3×108PFU in 100 μL)(Ad.EGFP group). Forty-eight hours later, LPS at 10 mg/kg(dissolved in saline) was injected into the peritoneal cavity of conscious mice to establish ALI rat model. The rats were sacrificed at 0, 3, 6, 12, 24, and 48 h after LPS challenge. The expression of PLAM3 was determined by RT-PCR and Western Blot. Meanwhile, the differences of pulmonary histological changes and 7-day survival rate among three groups were compared. The levels of TNF-α, IL-1β, IL-6 and MPO in BALF and serum were detected by ELISA. NF-κB p65 DNA binding activity in lung tissue was also assessed on isolated nuclear extracts by ELISA.3. Downregulation of PALM3 expression inhibits the inflammatory reaction induced by LPS in AEII cellsAccording to the previous results, Ad.PALM3-sh RNA-1 was selected for subsequent experiment. The AE Ⅱ cells were divided into three groups(Ad.PALM3-sh RNA group, Ad.EGFP group and Control group). The AEII cells were transfected by Ad.PALM3-sh RNA, Ad.EGFP or PBS, respectively. LPS was given to the cell culture medium at 48 h after transfection, the culture supernatant and cells were collected at 0, 3, 6, 12, 24 and 48 h after LPS-stimulation. The expression levels of PALM3 in AEⅡ cells were determined by Western Blot and PCR. The levels of TNF-α,IL-1β and IL-6 in culture supernatant were detected by enzyme-linked immunosorbent assay(ELISA). Otherwise, the activity of NF-κB was also measured by ELISA.4. To identify whether PALM3 interacting with the adaptor molecules in LPS-TLR4signalingThe AEⅡ cells were obtained by using previously described procedure and were divided into two groups(PBS group and LPS group). LPS(10 μg/m L) was given to the culture supernatant in LPS group. However, in PBS group, an equal volume of PBS was given. At 24 h after administration, the total protein was extracted from the AE Ⅱcells. The protein expressions of PALM3, My D88, TICAM-2, IRAK-1 and TRAF-6 in two groups were detected by Western Blot. And, the co-immunoprecipitation analysis was used to verify whether PALM3 interacting with the adaptor molecules in LPS-TLR4 signaling such as My D88, TICAM-2, IRAK-1 and TRAF-6.Results1. The PCR and DNA sequencing results demonstrated that the shuttle plasmid p GV120-PALM3-sh RNA-EGFP was successfully constructed. Cytopathic effect and fluorescence detection indicated that the adenovirus was successfully packaged after co-transfection. The titers of Ad.PALM3-sh RNA-1, Ad.PALM3-sh RNA-2 and Ad.PALM3-sh RNA-3 were 3×1010pfu/ml, 2×1010pfu/ml and 3×1010pfu/ml,respectively, which met the requirement of subsequent experiments. The AEⅡ cells were obtain from rat lung tissue successfully and identified by the SP-C of cellular immunofluorescence. The expression of EGFP was observed at 24 h after transfection.And, at 48 h after transfection, the expression of EGFP was the strongest. Otherwise,the definite optimum MOI used in vitro experiment was 200. Compared with Ad.EGFP group and Control group, PALM3 protein expression levels in Ad.PALM3-sh RNA-1group was lowest.2. PALM3 was constitutively expressed in normal rat lung tissues. The PALM3 protein and m RNA levels were enhanced in a time dependent manner in LPS-induced ALI rat models. Compared with Ad.EGFP and Control group, PALM3 m RNA and protein expression were down-regulated in Ad.PALM3-sh RNA group(P<0.05). Reduced expression of PLAM3 significantly decreased the levels of TNF-α, IL-1β, IL-6 and MPO,inhibited the activation of NF-κB, attenuated pulmonary pathological changes and neutrophil infiltration, and improved the outcome of ALI(P < 0.05).3. After LPS stimulation, the PALM3 protein and m RNA levels were enhanced in a time dependent manner. Compared with those in Ad.EGFP group and Control group,PALM3 m RNA and protein expression were depressed in Ad.PALM3-sh RNA group(P<0.05). Downregulated the expression of PLAM3 significantly decreased the levels of TNF-α, IL-1β and IL-6, inhibited the activation of NF-κB and suppressed the LPS-induced inflammatory response in AEⅡ cells.4. Whether LPS was given or not, the expression of PALM3, My D88, TICAM-2, IRAK-1and TRAF-6 were detected by Western Blot. The results of co-immunoprecipitation have demonstrated that PALM3 interacted with IRAK-1 in native status, but interacted with TIACM-2 and TRAF-6 in a ligand dependent manner.Conclusion1. Downregulation of PALM3 expression could inhibit the inflammatory reaction induced by LPS in AEII cells and has a protective effect on LPS-induced ALI in rat.2. PALM3 might be one of the adaptor molecules in LPS-TLR4 signaling. The protective effect of reduced PALM3 expression on ALI might be ascribed to blocking the transduction cascade of LPS-TLR4 signaling.