in the united states , foodborne pathogens cause 76 million illnesses , 325,000 hospitalizations , and 5,000 deaths annually . some of the main pathogens behind these sicknesses are gram - negative bacteria such as salmonella , escherichia , and campylobacter . infections with gram - negative bacteria may lead to endotoxemia caused by lipopolysaccharides ( lps ) which is a complex of glycolipids made up of two distinct regions . these are hydrophilic polysaccharide region ( composed of o antigen and core oligosaccharide ) and hydrophobic regions known as lipid a . lipid a is responsible for most of the lps induced biological effects . since lipid a is the innermost component of lps , it is closely adherent to the inner cell wall of bacteria and is generally not released ( and therefore not toxic ) until the death of the bacterial cell . lps is heat stable and not strongly immunogenic , so it can not be converted to a toxoid . histone modification is the central point for the control of cardiac growth and gene expression in response to acute and chronic stress stimuli . in the prevention and treatment of cardiac disease , histone modification and the signaling pathways manipulation is a major therapeutic step . histone acetyltransferases ( hats ) mediate acetylation of histone tails , loosening the interaction between dna and histones . acetylation of histones reduces their overall positive charge , thus decreasing their tight interactions with negatively charged dna . histone deacetylases ( hdacs ) can remove the acetyl groups on amino - terminus of histones , restore tight interactions between dna and histones , and deactivate the transcriptional pathway . therefore , hats ( histone acetyltransferases ) and hdacs ( histone deacetylases ) activity determines the transcriptional activation or repression . hat activity in cardiac muscle is determined by p300 that causes modification of chromatin and associated transcription factors and gene expression . studies done by yanazume et al . , 2003 , showed that agonist - induced cardiac hypertrophy accentuates p300 transcriptional activity and that this agonist - mediated cardiac hypertrophy is reversed by the blocking of p300-hat activity . curcumin is an inhibitor of p300-hat but very little is known about whether this regulatory effect is related to a protective role in cardiac dysfunction . curcumin which is derived from turmeric ( curcuma longa plant ) is a tropical plant that is native to southern and southeastern tropical asia . it is a perennial herb within the ginger family and is known for its yellow - orange color and for numerous therapeutic applications . curcumin is a polyphenolic compound and has been used in the treatment of many conditions , including cardiovascular diseases . this study was designed to determine the potential for curcumin to attenuate lps induced cardiac hypertrophy in rodents . curcumin c3 complex ( r ) was a kind gift from sabinsa corporation , hyderabad , india . the patented c3 complex ( r ) contains curcumin and its derivates demethoxycurcumin and bisdemethoxycurcumin , also known as curcuminoids . lps sc-3535 was obtained from santa cruz biotechnology as a white to yellow lyophilized powder . for the live and dead experiment , lps was used as 1.5  mg / mice ( dissolved in distilled water ) , and in curcumin attenuation of lps induced cardiac hypertrophy , lps was used as 60  mg / kg body weight dose ( dissolved in distilled water ) . histone h3 ( n-20 ) , was produced against a peptide mapping at the n - terminus of histone h3 of human origin . histone h4 ( n-18 ) , purified goat polyclonal antibody , was produced against a peptide mapping at the n - terminus of histone h4 of human origin . p300 ( c-20 ) was obtained from santa cruz biotechnology , santa cruz , ca . p300 ( c-20 ) , affinity purified rabbit polyclonal antibody , was raised against a peptide mapping at the c - terminus of p300 of human origin . acetyl - histone h3 ( lys9 ) antibody was obtained from cell signaling technology , danvers , ma . endogenous levels of histone h3 were detected by acetyl - histone h3 ( lys9 ) antibody only when acetylated at lysine 9 . acetyl - histone h4 ( lys12 ) antibody was obtained from cell signaling technology , danvers , ma . endogenous levels of histone h4 were detected by acetyl - histone h4 ( lys12 ) antibody only when acetylated at lys12 . acetyl - cbp ( lys1535)/p300 ( lys1499 ) antibody was obtained from cell signaling technology , danvers , ma . acetyl - cbp ( lys1535)/p300 ( lys1499 ) antibody detects endogenous levels of cbp or p300 only when acetylated at lysine 1535 or lysine 1499 , respectively . the animal care and use committee of tuskegee university approved the experimental protocol ( protocol number r0804 - 12 - 1 ) . eight breeding pairs of mice ( mus musculus ) strain 129s6/sveev , il-10 , were received from the gnotobiotic unit of the mutant mouse resource center , university of north carolina , chapel hill , nc ( iacuc approval number r076 - 10 - 2 ) . the mice were housed under specific pathogen - free ( spf ) conditions in the comparative medicine resource center ( cmrc ) , tuskegee university , and bred via brother  :  sister mating to produce the mice used in this study . subsequently , 48 of the resulting mice were randomly assigned to one of four treatment groups . the adult 129svev mice ( 68 weeks old ) used in the current study were assigned to 4 groups ( 3 males and 3 females in each group ) : ( a ) control , ( b ) curcumin : 100  g / kg of body weight , ( c ) lps : 60  mg / kg , and ( d ) lps ( 60  mg / kg ) + curcumin ( 100  g / kg of body weight ) . then , following a 24 acclimation period , curcumin and curcumin + lps treated groups received intraperitoneal ( i.p . ) injections of curcumin ( 100  g / kg of body weight ) . one day after the pretreatment with 100  g / kg curcumin , the lps and curcumin + lps groups received lps : 60  mg / kg i.p . all mice in the 12  hrs subgroups were sacrificed with co2 12  hrs after lps injection . all mice in the 24  hrs subgroups were sacrificed with co2 , 24  hrs after lps injection . hearts and lungs of the sacrificed mice were dissected and weighed to compare heart weight / brain weight ( mg / g ) and lung weight / brain weight ( mg / g ) ratios in lps and curcumin treated mice . the collected organs were then preserved in 10% regular formalin in 50  ml tubes . immunohistochemistry is the process of detecting antigens in cells of a tissue section by exploiting the principle of antigen - antibody binding specificity . the sections of murine tissue were placed on the heated water bath and picked up , placed on the slides , and placed in a rack . after all slides had been cut and placed in a rack they were put into a 60 degrees celsius oven over night . the slides were deparaffinized by going through 3 changes of xylene 2 minutes each , followed by 3 changes of 100% alcohol at 1 minute and one change of 95% alcohol at 1 minute . slides were washed in running tap water for 1 minute and stored in distilled water slides . each group was normally distributed , and the variances were equal in the two groups . the test statistic , ts , was calculated using a formula that has the difference between the means in the numerator . the denominator was the standard error of the difference in the means , which got smaller as the sample variances decrease or the sample sizes increase . thus ts got larger as the means got farther apart , the variances got smaller , or the sample sizes increased . mice in the lps treated group showed significant cardiac hypertrophy as measured by heart weight / brain weight ( hw / bw ) ratio . figure 1 illustrates that the lps treated group showed significant cardiomegaly . in 12  hrs subgroup , the p = .04 denotes a significant difference between the control and lps , and in the 24  hrs subgroup , the p = .0026 denotes a significant difference between the control and lps treated groups . mice , in the lps + curcumin treated group , showed attenuation of cardiac hypertrophy as measured by heart weight / brain weight ( hw / bw ) ratio ( figure 1 ) . in 12  hrs subgroup , the p = .005 denotes a significant difference between the lps and lps + curcumin , and in 24  hrs subgroup , the p = .04 denotes a significant difference between the lps and lps + curcumin treated groups . no significant changes were observed in the mice treated with either curcumin or control group ( figure 1 ) . in figure 2 , the heart collected from lps treated mice group showed significant cardiomegaly . dna wound around eight core histone proteins ( two each of h2a , h2b , h3 , and h4 ) form the nucleosome which is the primary building block of chromatin . the amino - terminal tails of core histones pass through various posttranslational modifications , including acetylation , phosphorylation , methylation , and ubiquitination . these modifications happen in response to various stimuli and serve to open the transcriptional machinery , increase the accessibility of chromatin to transcription factors , and ultimately result gene expression . acetylation of h3 has a central role in histone deposition and chromatin assembly in some organisms . hats acetylate histone proteins , relax chromatin , and expose prohypertrophic genes for activation by cardiogenic transcription factors . inhibition of histone acetylation has a central role for the antihypertrophic activity of curcumin . in order to evaluate the mechanism of action of curcumin against lps induced cardiac hypertrophy , we determined the condition of histone h3 acetylation by using acetylation specific antibodies . in this study , curcumin repressed the lps induced acetylation of histone-3 in mice ( figure 4 ) . as a control , the paraffin blocks were exposed to histone h3 antibody , and endogenous levels of histone h3 were detected by h3 antibody ( figure 3 ) . as expected , lps induced a significant increase in histone acetylation that was blocked and sustained by curcumin ( figure 4 ) . histone proteins were detected by histone h3 antibody in control ( figure 3(a ) ) , curcumin ( figure 3(b ) ) , lps ( figure 3(c ) ) , and curcumin + lps ( figure 3(d ) ) groups . but the cardiac cells in the lps treated group showed more staining in the cytoplasm indicating hypertrophy . figure 4 illustrates the degree of histone acetylation in cardiac tissue occurring in each treatment group . as can be seen in figure 4(c ) , lps induced a significant increase in histone acetylation as indicated by the intense staining . the histone proteins were mainly acetylated in the n - terminal end of the histone protein on lysine residues . in the curcumin + lps treatment group ( figure 4(d ) ) , there was a much lesser degree of nuclear staining with staining being similar to the control group , indicating that curcumin inhibited the histone acetylation activity of lps . these findings indicate that acetylation of histone tails by histone specific antibodies , mediated by histone acetyltransferases , is involved in activation of gene expression in the myocardium . conversely , repression of gene expression can be induced by histone deacetylation , which is mediated by histone deacetylases ( hdacs ) . modulation of chromatin structure has a central role in the regulation of transcription in eukaryotes . in eukaryotes , histone h4 acetylation at the position of lysine 16 ( h4-k16ac ) is a reversible posttranslational chromatin modification . the incorporation of this modified histone into the nucleosome inhibits the formation of compact 30 fibers and inhibits the ability of chromatin to form cross - fiber interactions . acetylation of histone tails , mediated by histone acetyltransferases , is involved in activation of gene expression in the myocardium . conversely , repression of gene expression can be induced by histone deacetylation , which is mediated by histone deacetylases ( hdacs ) . histone h4 acetylation at the position of lysine 16 ( h4-k16ac ) is a posttranslational chromatin modification . in addition to histone h3 we also measured histone h4 acetylation by measuring the [ h ] acetate incorporation into histones using h4 acetylation specific antibody . figure 5 illustrates the level of histone proteins as detected by histone h4 antibody in cardiac tissue from control ( figure 5(a ) ) , curcumin ( figure 5(b ) ) , lps ( figure 5(c ) ) , and curcumin + lps ( figure 5(d ) ) treatment groups . as can be seen in the figure , there was little variation in histone h4 basal expression levels among any of the treatment groups . as can be seen in figure 6(c ) , the lps treated group showed a significant increase in histone [ h ] acetylation . control and curcumin alone treatment groups , figures 6(a ) and 6(b ) , respectively , showed little difference in the staining of the acetylated histone h4 . similar to histone h3 acetylation , lps induced h4 acetylation was also blocked by curcumin treatment ( figure 6(d ) ) . this data strongly indicates that curcumin inhibits the acetylation of histones induced by lps . hats ( histone acetyl - transferase ) acetylate histone proteins , relax chromatin , and expose genes for activation by cardiogenic transcription factors . two highly related and widely expressed molecules , camp regulated enhancer binding protein ( cbp ) and p300 , have emerged as important cofactors for a broad number of transcription factors . cbp was originally discovered based on its ability and to interact with the camp response element binding protein , whereas p300 was isolated as a cellular target of the adenoviral oncoprotein e1a . sequence analysis of cbp and p300 has shown substantial homology that these proteins are transcriptional coactivators . p300 , high molecular weight transcriptional factor , has three cysteine and histidine - rich regions . p300 knockout mice showed the defects of cardiac muscle differentiation and trabeculation , indicating the importance of p300 for early cardiac morphogenesis and heart development . curcumin ( diferuloylmethane ) , a major curcumanoid in the spice turmeric , is a specific inhibitor of the p300/creb - binding protein ( cbp ) . to further check the mechanism of action of curcumin , we analyzed p300 protein acetylation levels . curcumin inhibited the lipopolysaccharide - induced acetylation resulting in an increase in the binding of p300 to the gene promoter ( figure 7(c ) ) . as was seen with histone acetylation , p300 acetylation was also inhibited by curcumin ( figure 7(d ) ) . the results of the present study indicate that inhibition of histone acetylation is a key mechanism for the anti - cardiac hypertrophy activity of curcumin and that p300-hat serves as its molecular target . p300 may maintain basal function in the normal heart but it also promotes cardiac hypertrophy under lps infusion . we can conclude from our study that curcumin attenuated lps induced cardiac hypertrophy in vivo . curcumin plays its anti - cardiac hypertrophy role by the involvement of chromatin remodeling , especially histone acetylation . acetylation of lysine residues upon a single histone tail ( hyperacetylation ) would produce a stronger effect . histone acetyltransferases have a critical role to acetylate histone proteins , relax chromatin , and expose cardiac hypertrophy genes for activation by cardiogenic transcription factors . hat activity in muscle is determined by p300 that causes modification of chromatin and associated transcription factors and gene expression . lps induced cardiac hypertrophy accentuates p300 transcriptional activity , and lps mediated cardiac hypertrophy is reversed by blocking of p300-hat activity . therefore , blocking p300-hat activity may be a viable method for the treatment or prevention of myocardial hypertrophy . the results showed that curcumin attenuated lps induced cardiac hypertrophy in rodents and that the most probable mechanism of action involves inhibiting the p300-hat activity .
to study the ameliorating effects of curcumin in lipopolysaccharide ( lps ) induced cardiac hypertrophy , mice were assigned to 4 groups ( 3 males and 3 females in each group ) : ( a ) control , ( b ) curcumin : 100  g / kg of body weight by intraperitoneal route ( ip ) , ( c ) lps : 60  mg / kg ( ip ) , and ( d ) lps + curcumin : both at previously stated concentrations by ip route . 
 all mice were sacrificed as 12  hr and 24  hrs groups accordingly after lps injection . 
 the hearts were collected , photographed for cardiomegaly , and weighed to compare heart weight / brain weight ( hw / bw ) in mg / mg . for immunohistochemistry , the tissue sections were exposed to histone h3 , h4 and acetylated histone h3 , h4 antibody . 
 lps induced a significant increase in histone acetylation as shown by intense staining . in curcumin 
 + lps treated mice nuclear staining was similar to the control group indicating that curcumin traversed the histone acetylation activity of the lps . to further check the mechanism of action of curcumin , p300 protein acetylation levels were analyzed . 
 this study suggests that the probable mechanism of action of curcumin is via the reduction of p300 hat activity .