Research On Therapeutic Effect Of Patchouli Alcohol On Hypertension-induced Renal Injury And Its Molecular Mechanisms

Objective:To investigate the therapeutic effect of patchouli alcohol?PA?on kidney damage in spontaneously hypertensive rat?SHR?and its possible molecular mechanisms,and to further provide theoretic basis for new drug research anddevelopment of PAMethods:Eight-weeks old SHRs?n=60,half male and half female?were randomly divided into six groups,including SHR model group?equal volume of saline?,solvent group?equal volume of extra virgin olive oil?,PA high(80mg·Kg^-1·d^-1),mediate(40 mg·Kg^-1·d^-1)and low dose(20 mg·Kg^-1·d^-1)group,and positive group(captopril,2.5 mg·Kg^-1·d^-1).In addition,10 Wistar-kyoto?WKY?rats of the same ages were set as the normal control group?half male and half female,equal volume of saline?.After adaption for 1 week,all rats were orally gavaged with corresponding drug for consecutive 8 weeks.General conditions,body weight and tail arterial systolic blood pressure?SBP?by non-invasive tail-cuff were monitored at 1week before administration and at every week after administration.After the last administration,the plasma from whole blood collected through abdominal aorta was obtained by centrifugation,and the left and right kidney were harvested and weighed to calculate its left and right kidney weight index,respectively.The levels of plasmic creatinine?Cr?and blood urea nitrogen?BUN?were measured with the method of sarcosine oxidase and urease,respectively.The contents of renin,angiotension??Ang??,transforming growth factor?₁?TGF-?₁?,plasminogen activator inhibitor 1?PAI-1?in plasma and renal tissues,and the contents of short-chain fatty acids?SCFAs?in plasma were determined by enzyme-linked immunosorbent assay?ELISA?.Renal sections were prepared to observe alternation of renal histopathology referring to renal tubular injury,collagenous fibers and glomerulosclerosis by H&E and Masson staining,respectively,and to examine the expressions of collagen??Col??,collagen??Col??,and fibronection?FN?proteins by immunohistochemistry assay.The expressions of?smooth muscle actin??-SMA?,matrix metalloproteinase 9?MMP-9?,tissue inhibitor of metalloproteinase 1?TIMP-1?,ras,raf-1,and extracellular signal-regulated kinase 1 and 2(ERK_1/2)mRNA in renal tissues were detected by real-time PCR.The expressions of?-SMA,ras,raf-1,ERK_1/2,pERK_1/2proteins in kidney were detected by Western blot.And the 16SrRNA platform was employed for the examination on richness,diversity and structure of gut flora.Results:Part 1 Study referring to effect of PA on blood pressure of SHRCompared to WKY group,it showed a poor states,including bad psychosis,reduced movements,not shiny hair,dry stool,and less urine in the SHR group;the growth rate of body mass declined,especially at the second,third,and eighth week after administration?p<0.01?;the SBP were significantly raised in a fluctuant manner?p<0.01?with the remarkable increase of plasmic renin,Ang?,TGF-?₁ and PAI-1levels?p<0.01 or p<0.05?;and there were no statistical difference in the levels of renin,Ang?,TGF-?₁ and PAI-1 of renal tissues between WKY and SHR group.After treatment with different doses of PA,finding that mediate(40 mg·Kg^-1)and low(20 mg·Kg^-1)dose of PA could significantly improve general condition,but treatment male SHR instead of the female with high(80 mg·Kg^-1)dose of PA resulted in a series of toxic effects,i.e.shaggy hair,soy urine,tan-colour skin,exudation of grease,and scabs.Mediate dose of PA significantly reduced the growth rate of body mass at the second week after administration?p<0.01?,but increased the growth rate of body mass at the third week after administration?p<0.01?;low dose of PA could reduce growth rate of body mass,especially at the first and eighth week after administration?p<0.05 or p<0.01?;high,mediate and low dose of PA significantly lowered the SBP at the sixth to eighth week,the seventh to eighth week and the eighth week after administration?p<0.05 or p<0.01?,respectively,and the onset time other than depressurizing range PA produced was in an concentration-dependent manner;All doses of PA significantly reduced renin levels in plasma and renal tissues?p<0.01 in all?;high dose of PA significantly reduced plasmic Ang?levels?p<0.01?;high and mediate doses of PA significant decreased PAI-1 levels in renal tissues?p<0.05 or p<0.01?;mediate and low doses of PA significantly reduced TGF-?₁?p<0.05 or p<0.01?and PAI-1 levels?p<0.05 in both?in renal tissues;besides,PA had no obvious effect on the levels of Ang?in renal tissues?p>0.05?.Part 2 Study referring to effect of PA on renal fibrosis of SHRCompared to the WKY group,the SHR group showed a remarkable increase in left and right kidney weight indices?p<0.01 in both?,plasmic Cr and BUN levels?p<0.01 in both?,Col??p<0.01?,Col??p<0.05?,FN?p<0.01?,and raf-1 protein?p<0.05?,and?-SMA?p<0.01?and ERK_1/2/2 mRNA?p<0.05?expressions in renal tissues,and had no statistical alterations in MMP-9,TIMP-1,ras as well as raf-1mRNA and?-SMA,ras,ERK_1/2/2 as well as pERK_1/2 proteins expressions?p>0.05?.In addition,it also showed a notably pathological changes including renal tubules damage,glorulosclerosis,renal tubulointerstitial fibrosis,infiltration of inflammatory cells etc.,with renal tubules score and fibrotic index?p<0.01 in both?.After treatment with different doses of PA for consecutive 8 weeks,observing that low dose of PA remarkably abated left kidney weight index?p<0.05?,while mediate and low doses of PA remarkably abate right kidney weight index?p<0.05 or p<0.01?;high dose of PA remarkably reduced plasmic BUN levels?p<0.05?and raised plasmic Cr levels?P<0.05?;all dose of PA remarkably abated renal tubules injury score?p<0.05 or p<0.01?;mediate and low doses of PA remarkably abated renal fibrotic index?p<0.01in both?;all doses of PA remarkably dwindled Col?expressions in renal tissues?p<0.05 or p<0.01?;mediate and low doses of PA remarkably dwindled FN?p<0.05 or p<0.01?,?-SMA?P<0.05?as well as raf-1 proteins expressins?p<0.05 or p<0.01?in renal tissues;mediate dose of PA exhibited a remarkable elevation in MMP-9 mRNA expressions?p<0.05?,and reduction in?-SMA mRNA?p<0.05?and TIMP-1 mRNA expressions?p<0.05?of renal tissues;low dose of PA remarkably dwindled ras?p<0.05?,raf-1?p<0.05?and ERK_1/2 mRNA?p<0.05?,and Col??p<0.01?,ERK_1/2?p<0.05?as well as pERK_1/2 proteins expressions?p<0.05?in renal tissues;additionally,all doses of PA had no evidently effect on the ras proteins expressions in renal tissues?p>0.05?.Part 3 Study referring to effect of PA on gut flora of SHRCompared to WKY group,SHR group showed a reduced trend in abundance and diversity of gut flora?p>0.05 in both?;in phylum levels,it presented a marked reduction in Bacteroidetes richness?p<0.05?and increase in the ratio of Firmicutes to Bacteroidetes?p<0.05?,and slight reduction in Firmicutes as well as Proteobacteria richness?p>0.05 in both?and increase in Verrucomicrobia?p>0.05?;the results on genus clsssification using LEfSe analysis revealed that Lachnospiraceae UCG 005and Alloprevotell abundance markedly declined?p<0.01 in both?,and Coprococcusand and Peptoclostridium abundance markedly raised?p<0.01 in both?;the levels of SCFAs in plasma also presented a reduced trend,but the difference had no statistical significance?p>0.05?.After challenge with different doses of PA,finding that high dose of PA could markedly elevate the Chao 1?p<0.05?and Faith's PD index?p<0.05?;Shannon index showed a marked elevation in PA high and low dose groups?p<0.05 in both?and reduction in PA mediate dose group?p<0.05?;high dose of PA could markedly increase Bacteroidetes?p<0.05?as well as Proteobacteria richness?p<0.05?and decrease the ratio of F/B?p<0.05?;mediate dose of PA markedly increased Verrucomicrobia richness?p<0.05?and decreased Firmicutes richness?p<0.05?;high and mediate doses of PA markedly elevated Lachnospiraceae UCG 005 abundance?p<0.05 or p<0.01?and lowered Peptoclostridium abundance?p<0.01?;all doses of PA markedly raised Alloprevotella abundance?p<0.05 or p<0.01?;low dose of PA markedly raised Coprococcus abundance?p<0.01?;furthermore,all doses of PA could markedly elevated plasmic SCFAs levels?p<0.05or p<0.01?.Conclusions:1 PA had a potential in treating hypertension-induce renal injury.However,we found that high dose of PA(80 mg·Kg^-1)in the period of long-term administration resulted in a toxic effects and aggravated renal damage in SHR with kidney injury,suggesting that it was cautious to use high dose of PA like 80 mg·Kg^-1.2 PA had an evident effect on moderately lowering hypertension and its mechanisms maybe were related to inhibition of RAS and decrease of TGF-?₁ as well as PAI-1 levels.3 PA could markedly alleviate pathological alternation of renal tissues and protect kidneys of SHR,which was associated with the proliferation and activation of myofibroblast?MFB?,decreased synthesis of matrix protein,degradation of extracellular matrix?ECM?,and the regulation of ras/raf-1/ERK_1/2 signaling pathway,beside that inhibition of over-activated RAS and of over-elevated TGF-?₁ as well as PAI-1 levels.4 On the basis of the results that PA could produce the alternation of richness,diversity,and structure of Gut flora and promote the synthesis of SCFAs in SHR accompanying with kidney damage,it was speculated that gut flora was likely to participate in the effect of PA on treating hypertension-induced renal injury.But the further studies were needed to illuminate its molecular mechanisms.