The Kidney Injury Induced By Short-term PM_2.5 Exposure And The Prophylactic Treatment Of Compound Essential Oils In Balb/c Mice

Purpose: PM2.5 is well known as a major environmental pollutant,with the aggravation of air pollution,PM2.5 has been paid much attention in recent years.However,most of the researches are limited to the influence of PM2.5 on respiratory system.In this study,we investigated the kidney injury on the basis of acute lung injury caused by short-term PM2.5 exposure.At the same time,we explored whether compound essential oil can interfere with this kind of damage so as to achieve the purpose of prophylactic treatment.Methods: 1.PM2.5 collection and PM2.5 suspension preparation: PM2.5 sample was collected in Langfang,China in 2013,which was collected in winter with a specific collector.Immersing the silica filter membrane with PM2.5 in ultra-pure water and ultrasonic for 2-3 hours.When the PM2.5 particles were separated from the filter membrane and suspended in pure water,freezed and dried to remove moisture.Configuration of a suspension with a concentration of 10 mg/ml with normal saline and PM2.5 particles.2.Animals and experiment design: 6-8 weeks Male BALB/c mice were kept in a conditioned room(24 ± 1 oC)and a 12/12 h light/dark cycle with free access to water and food.Experimental procedures were divided into two parts.In the first part,48 Balb/c mice were divided into two groups randomly(n = 24): control group;sterile saline(intratracheal instillation of 50 ?l)at day 0 and day 2.PM2.5 group;PM2.5 in sterile saline(intratracheal instillation of 50 ?l aqueous suspensions of 0.5 mg PM2.5 in sterile saline)at day 0 and day 2.In the second part,48 Balb/c mice were divided into two groups randomly(n = 24),as follows: PM2.5 + saline group;Static inhalation 200 ?l of sterile saline the day before PM2.5 exposure(intratracheal instillation of 50 ?l aqueous suspensions of 0.5 mg PM2.5 in sterile saline at day 0 and day 2).PM2.5 + CEOs group;Constant daily static inhalation 200 ?l of CEOs for 30 min per day until killed on the base of PM2.5 exposure which was described previously.Mice in every group were sacrificed respectively on days 3,7 and 14 after intratracheal instillation.3.Measurement of kidney injury biomarkers: Mice were killed on day 3,7 and 14 after PM2.5 exposure,and the kidney tissues and serum were obtained.The renal tissues were embedded in paraffin and stained with hematoxylin eosin(H&E)staining to observe whether there were pathological changes.The gene expression of KIM-1 in renal tissues was detected by q-PCR,cystatin C(Cys C)and uric acid(UA)in serum were detected by chemiluminescence.4.Oxidative stress indexes and inflammatory cytokines level: The MDA in mouse kidney tissues was detected by thiobarbituric acid(TBA)method.m RNA expressions of NOX4,SOD-1,HO-1,Nf-?B,TNF-? were measured by q-PCR.The protein level of NOX4,Nf-?B,TNF-? were detected by Western blot assay.5.The determination of renin angiotensin system(RAS): The protein secretion of ACE and AT1 R in renal tissues was detected by Western blot.Results: Part one: 1.Pathological examination: On the basis of known PM2.5 exposure can cause acute lung injury,HE staining of rat kidney revealed that there was no significant difference between PM2.5 exposure group and control group except for the loose structure and glomerular atrophy in individual samples.2.The level of kidney injury biomarkers increased: Compared with the control group,the gene expression of KIM-1 was increased in the PM2.5 exposure group,and the Cys C and UA in the serum increased at three time-points.3.Indicators of oxidative stress and inflammatory factors increased: Compared to control group,the MDA in kidney tissues increased after PM2.5 exposure.Both the gene expression and the protein level of NOX4 were higher than the control group,the gene level of HO-1 and SOD-1 increased and decreased respectively.The gene expression of Nf-?B,TNF-? were increased at all three time-points,however the protein level of Nf-?B,TNF-? only increased on day 7.4.The main indexes of RAS were activated: After PM2.5 exposure,the protein level of ACE and AT1 R were significantly higher than that in the control group.Part two: 1.The level of kidney injury biomarkers decreased: After administrated with CEOs,the gene expression of KIM-1 was decreased compare with PM2.5+saline group.The serum Cys C and UA were significantly decreased.2.Indicators of oxidative stress and inflammatory factors were inhibited: In PM2.5+CEOsgroup,MDA level,HO-1 and NOX4 gene expression were lower than PM2.5+saline group,the gene level of SOD-1 was higher than that of PM2.5+saline group.Both of gene expression and protein level of Nf-?B,TNF-? only decreased on day 7.3.The main indexes of RAS were suppressed: After the treatment with CEOs,the protein levels of ACE and AT1 R in renal tissues were suppressed significantly since day 3.Conclusion: Short-term PM2.5 exposure showed a certain damage to the kidney at the molecular level,but there was no substantial damage.PM2.5 induced persistent oxidative stress and inflammatory response by activating renin-angiotensin system.CEOs could inhibit the incidence of oxidative stress and inflammation by suppressing the renin angiotensin system.This study explored the kidney injury and the potential damage pathway induced by short-term PM2.5 exposure,and the preventive effect of CEOs on renal injury caused by PM2.5.