Introduction
Subacute
ruminal acidosis (SARA) is a common nutritional metabolic disease involved in
ruminant production. It has a great impact on the long-term health and
production efficiency of animals (Danscher et al. 2015). In
recent years, in order to improve the production efficiency of ruminants and
the quality of animal products, researchers have conducted extensive research
on the adverse effects of SARA on intensive ruminant production systems.
Reports indicate that ruminants fed rapidly fermentable carbohydrates for a
long time will develop an excessive accumulation of organic acids in the rumen,
and a dramatic decline of rumen pH, further producing a variety of abnormal
metabolites such as HIS and LPS (Liu et al. 2013). These toxic and
harmful substances can be absorbed into the blood, which in turn causes a
systemic inflammatory response (Sun 2017) and ultimately induces SARA with loss
of appetite, laminitis and diarrhea (Plaizier et al. 2012). Evidence
suggests that rumen LPS is produced by Gram-negative bacteria (Khafipour et
al. 2009; Wang et al. 2015). When ruminants suffer from SARA,
Gram-negative bacteria in the rumen rupture and cell lysis releases a large
amount of LPS, which can compromise rumen epithelial barrier
function (Liu et al. 2013; Sato 2016). The free LPS are then translocated
from the rumen into the blood across the rumen epithelial barrier, increasing
the concentration of blood LPS, further activating the inflammatory and acute
phase responses (Dong et al. 2011). Therefore, the accumulation and
translocation of LPS might cause disruption of epithelial barrier integrity in
the gastrointestinal tract (Tao et al. 2014), which results in an
increase in the permeability of LPS. Many researchers accept that the increase
of ruminal LPS is often accompanied by the grain-induced SARA challenge (Gozho et
al. 2007). HIS is an important bioactive substance and also an important
mediator of the inflammatory response and immune challenge (Khafipour et al.
2009). Aschenbach et al. (1998) were the first to show that application
of HIS in relevant dosages (10 and 100 μm)
impaired differentiation of rumen epithelial barrier integrity and function. A
recent in vitro study indicated HIS could activate the inflammatory
pathway of cultured rumen epithelial cells via
NF-κB (Sun 2017), which has consequences for rumen epithelial integrity
and function (Aschenbach et al. 2019). Taken together, SARA is known to
be characterized by an increased VFA concentration, low pH, hyperosmolarity and
elevated LPS and HIS concentrations in the rumen, and these variables have some
detrimental effects on the ability of the rumen epithelium to facilitate the
translocation of toxic compounds such LPS and HIS (Penner et al. 2011).
Several studies conducted in
cow and goat reported that SARA increased ruminal epithelial permeability and
compromised rumen epithelial barrier function (Sun et al. 2018b).
Previous studies have investigated the effects of low pH (Gaebel et al.
1989; Penner et al. 2011), hyperosmolarity (Lodemann and Martens 2006),
or an exposure to toxins (Emmanuel et al. 2007) on ruminal epithelial
barrier function in vitro. Greco et al. (2018) and Meissner et
al. (2017) used chambers to demonstrate that a low pH in combination with
SCFA induces damage to the rumen epithelial barrier function. While any one or
a combination of these factors may affect epithelial barrier function, the
extent to which LPS and HIS contribute to disruption of rumen epithelial
permeability at low ruminal pH has not been systematically investigated.
Therefore, the present study was designed to elucidate the effects of LPS and
HIS on the permeability of the ruminal epithelium at physiological and
acidotic luminal pH values, with a special focus on determining whether the
co-presence of LPS and HIS can aggravate the damage of the rumen epithelial
barrier elicited by low pH.
Materials
and Methods
The animal
experiment protocols were approved by the Animal Care and Use Committee of The
Inner Mongolia Academy of Agricultural & Animal Husbandry Sciences and were
in accordance with relevant guidelines formulated by the Ministry of
Agriculture of the People’s Republic of China.
Animals, experimental
design and treatments
Nine 2.5-year-old
female lactating Saanen dairy goats (42.79 ± 5.61 kg of BW; Mean ± SD) were
placed in individual stalls with free access to water. Goats were fed a diet
containing a non-fiber carbohydrate to neutral detergent fiber ratio (NFC/NDF)
of 1.40 (NRC 2007). The nutrient compositions of the diets are presented in
Table 1. The diet (800 g dry matter per animal per day) was provided in equal amounts
at 0830 h and 1830 h daily for 30 days.
Rumen
tissue sampling
The dairy
goats were killed by exsanguination, and ruminal tissue from the ventral sac
was harvested for subsequent Ussing chamber experiments. Six ruminal epithelial
tissues were collected from each goat, and every set of three ruminal
epithelial tissues were included in one treatment group.
Ussing Chamber Measurements
The
electrophysiological properties and permeability of the ruminal epithelium were
determined for the intact ruminal epithelium using the Ussing chamber technique
(Physiologic Instruments, America). Firstly, for preparation of the electrode,
2 g of Agarose was weighed and inserted into 50 mL centrifuge tubes, then KCl
(150 Table 1: Composition and nutrient levels of experimental diets
|
Ingredients,
% |
|
Nutrient
levels2, DM bases |
|
|
Alfalfa |
30.72 |
NEL,
MJ/kg |
7.12 |
|
Hay |
18.57 |
ME, MJ/kg |
9.68 |
|
Corn |
37.88 |
CP, % |
12.45 |
|
Soybean
meal |
1.47 |
NFC3,
% |
44.45 |
|
Wheat bran |
8.20 |
NDF, % |
31.78 |
|
NaCl |
0.46 |
ADF, % |
21.33 |
|
Limestone |
0.19 |
Ca, % |
0.54 |
|
Premix1 |
2.51 |
P, % |
0.32 |
|
Concentrate:
forage |
51:49 |
Ca: P |
1.68 |
|
Total |
100.00 |
NFC/NDF
ratio |
1.40 |
Legend:1 One kilogram of Premix contained
the following: MnSO4·5H2O 1560 mg, FeSO4·7H2O
6240 mg, ZnSO4·7H2O 3500 mg, KI 17 mg, NaSeO3
130 mg, Co2Cl·6H2O 206 mg, CuSO4·5H2O
300 mg, VA 1620 000 IU, VD3 324 000 IU, VE 540 IU, VB12
0.9 mg, VB5 450 mg, VK3 150 mg, folic acid 15
mg/kg, calcium pantothenate 750 mg/kg
2 Ca and P
were tested values and NFC, DM and ME were calculated values
3 NFC (%) ＝1-NDF-CP-EE-Ash
Table 2: Composition
of the buffer solution used in the Ussing Chamber
|
Component |
Content
(mmol/L) |
|
NaCl |
80.0 |
|
KCl |
5.0 |
|
NaH2PO4
× H2O |
0.40 |
|
Na2HPO4
× 2H2O |
2.4 |
|
C3H5NaO2 |
10.0 |
|
C2H3NaO2
× 3H2O |
25.0 |
|
C4H7NaO2 |
5.0 |
|
MgCl2
× 6H2O |
1.2 |
|
CaCl2
× 2H2O |
1.2 |
|
NaHCO3 |
25.0 |
Table 3: Compounds
with different pH values of VFA and lactic acid mixture
|
Item |
pH = 7.4 |
pH = 5.5 |
pH = 5.2 |
|
Acetate, mM |
30 |
60 |
90 |
|
Propionate,
mM |
30 |
60 |
90 |
|
Butyrate,
mM |
10 |
20 |
30 |
|
Lactate, mM |
0.5 |
1.0 |
1.5 |
%2079-86_files/image002.jpg)
Fig. 1: Effect of SARA on expression
levels of intracellular junction proteins in the epithelium of dairy goats
mL, 3
mol/L) solution was added, and the centrifugal tubes were placed into 100°C
water for 90 min, until the liquid had a consistency of transparently sticky
and there were no bubbles. The KCl-Agar solution was drawn with a 5 mL syringe,
and a 0.5 ~ 1 cm length of KCl-Agar was injected into the tip of the electrode
sleeve, and then placed into KCl (3 mol/L) solution (Fig. 1).
A piece of ruminal epithelial tissue from the ventral sac
(~100 cm2) was rinsed by immersion in the buffer solution (Table 2).
The time from the goat slaughter to mounting the epithelium was 30~45 min. The ruminal
epithelium was removed from the muscle layer, placed quickly in a buffer
solution kept at 37°C, gassed with 95% O2 and 5% CO2 and
then cut into squares (about 1 cm × 0.5 cm) and mounted in the Ussing chamber (EM-CSYS-6). The aperture area of sliders in
the Ussing chamber was 0.5 cm2, which provided sufficient contact area
for the ruminal epithelium and buffer. Both halves of the chambers were
immediately filled with buffer solution (Table 2) and gassed with 95% O2/5%
CO2 at 37°C. Glucose was added to the serosal and mucosal sides for
a final concentration of 10 mmol/L. The buffer temperature was kept constant at
37°C throughout the measurement.
Chemicals and reagent
Six rumen
epithelial tissues were collected from each goat and
treated in two groups with 3 replicates per group. Finally, 18 groups were
completed with different mucosal incubation solutions as follows: pH (7.4, 5.5
and 5.2), HIS (0, 0.5 and 10 ng·mL-1) and LPS (0, 30 and 60 KEU·mL-1),
each alone or in combination. The different mucosal pH values were adjusted by
adding VFA and lactic acid according to Table 3, and then using HCl to adjust
the final pH value. In total, repeated measurements were made on three
different incubation chambers per group. After a 20 min equilibration period,
the 8 μL FITC (final concentration 0.2 mmol/L) and 8 μL
HRP (final concentration 2 μmol/L) were added to the mucosal side
of each chamber. After a 20 min equilibration period, transepithelial
conductance (Gt, as a measure for passive ion permeability) and
short-circuit current (Isc, as a measure for active electrogenic
electrolyte transport) data were continuously collected with the aid of a
computer-controlled voltage-clamp device (voltage/current clamp) (Wang et al. 2021).
Mucosal-to-serosal fluxes of HRP and FITC were measured by sampling 200 μL of solution from the serosal side at
20-min intervals over a 100-min period. The volume from the serosal side was
replenished with 200 μL of fresh standard buffered solution to
maintain a constant volume. The concentrations of HRP and FITC in the serosal
samples were measured as described previously (Cheng 2016).
Statistical Analysis
Each replicate served as an experimental unit.
Data for pH × LPS and pH × HIS were obtained for the analysis in the double
factors MIXED model in S.A.S. Version 9.3 (S.A.S. Institute Inc.,
Cary, NC). There are three levels of pH factor, LPS factor
and HIS factor. Data for pH × LPS × HIS were analyzed by one way-ANONA for a
single-factor variance analysis. Duncan's test was used to test the
significance of multiple differences, and the data are presented as means ± SD.
P < 0.05 was considered the level of significance.
Results
Interaction of pH and LPS on rumen
epithelial permeability
As shown in Table 4, rumen epithelial Isc
and Gt values were greatest at mucosal pH
5.2 and lowest (P < 0.05) at mucosal pH 7.4. In the pH × LPS group, the effect of treatment was significant (P < 0.05).
With LPS as the main factor, Isc and Gt of ruminal epithelium
incabated at different pH levels in combination
with LPS 60 were greater (P < 0.05) than those in LPS 30. When pH as the main factor, Isc and Gt of ruminal
epithelium incubated with mucosal
addition of LPS-containing solution were the highest (P
< 0.05) at pH 5.2, and PD at both pH 5.5 and pH 5.2 were lower (P
< 0.05) than that at pH 7.4. Overall, the Isc and Gt of incubated ruminal epithelium were the highest (P
< 0.05), while the PD value was lowest in the pH 5.2-LPS 60 group, which indicated the highest permeability of the ruminal
epithelium.
Table 5 summarizes
data for the mucosal-to-serosal fluxes of FITC and HRP. The fluxes of FITC and HRP through
the ruminal epithelium at mucosal pH 5.2 were greater than those at mucosal pH 7.4. At mucosal
pH 5.2, the HRP flow rate was higher than that at mucosal pH 5.5, while FITC
flow rate was lower (P < 0.05). The effect of treatment was
significant (P < 0.05) for the FITC flow rate. When LPS as the main
factor, the flow rates of HRP and FITC (P < 0.05) at different pH
values in combination with LPS 60 were greater than those in LPS 30; in addition,
the concentration of LPS in the serosal side at different pH levels in
combination with LPS 60 was greatest (P < 0.05). When pH as the main
factor, the FITC flow rate of ruminal epithelium incubated with mucosal addition
of
LPS-containing solution was greatest (P < 0.05) at mucosal pH 5.2. The mucosal-to-serosal fluxes of
HRP and FITC of ruminal epithelium incubated at the mucosal pH 5.2-LPS 60
were greatest (P < 0.05) (Table 5).
Interaction
of pH and HIS on rumen epithelial permeability
As shown in
Table 6, the interaction between pH and HIS had significant effects on Isc,
Gt and PD of incubated rumen epithelium. Compared with mucosal
pH-HIS 0.5 groups, Isc and Gt were significantly
increased (P < 0.05) in mucosal pH-HIS 10 groups with an HIS-based
effect, while PD was reduced (P < 0.05). When pH was the
main Table 4: Effects of different pH × LPS treatments on rumen
epithelial electrophysiological parameters in dairy goats (n = 3)
Table 6: Effects of
different pH×HIS treatments on rumen epithelial
electrophysiological parameters in dairy goats
|
HIS
content/ng·mL-1 |
pH value |
Isc/Ma (cm2·h)-1 |
Gt/mS
(cm2·h)-1 |
PD/mV (cm2·h)-1 |
|
0 |
7.4 |
0.05ef |
3.70d |
1.15d |
|
5.5 |
0.06ef |
4.05c |
1.08d |
|
|
5.2 |
0.31b |
4.14c |
6.66a |
|
|
0.5 |
7.4 |
0.02g |
3.70d |
2.92c
|
|
5.5 |
0.08e
|
4.35b
|
0.73de
|
|
|
5.2 |
0.13d
|
4.84a |
0.86de
|
|
|
10 |
7.4 |
0.16d
|
3.46c |
3.94b
|
|
5.5 |
0.24bc
|
5.93a |
0.24f
|
|
|
5.2 |
0.46a |
4.41b |
3.43b
|
|
|
SEM |
|
0.022 |
0.165 |
0.290 |
|
Main effects |
||||
|
HIS |
0 |
0.14B |
3.96C |
2.96A |
|
0.5 |
0.08B |
4.30B |
1.50C |
|
|
10 |
0.29A |
4.60A |
2.54B |
|
|
pH |
7.4 |
0.08C |
3.62C |
2.67B
|
|
5.5 |
0.13B |
4.78A |
0.68C
|
|
|
5.2 |
0.30A |
4.46B |
3.65A |
|
|
P-value |
pH |
< .0001 |
< .0001 |
< .0001 |
|
HIS |
0.001 |
< .0001 |
< .0001 |
|
|
pH×HIS |
< .0001 |
< .0001 |
< .0001 |
|
Means with different lowercase letters are
significantly different; means with different (P < 0.05). Uppercase
letters within the same column are significantly different
Table 7: Effects of
different pH × HIS treatments on rumen epithelial HRP and FITC flows and HIS
content in the serosal side in dairy goats
|
HIS
content /ng·mL-1 |
pH value |
FITC/mmol
(cm2·h)-1 |
HRP/mol (cm2·h)-1 |
HIS
content/ng·mL-1 |
|
0 |
7.4 |
0.15bc |
0.03c |
- |
|
5.5 |
0.15bc |
0.14a |
- |
|
|
5.2 |
0.21a |
0.10b |
- |
|
|
0.5 |
7.4 |
0.17ab |
0.09b |
0.15 |
|
5.5 |
0.13cd |
0.09b |
0.12 |
|
|
5.2 |
0.12cd |
0.08b |
0.12 |
|
|
10 |
7.4 |
0.14bc |
0.12ab |
0.13 |
|
5.5 |
0.13cd |
0.02c |
0.13 |
|
|
5.2 |
0.09d |
0.14a |
0.14 |
|
|
SEM |
|
0.013 |
0.026 |
0.009 |
|
Main effects |
||||
|
HIS |
0 |
0.17A
|
0.09 |
- |
|
0.5 |
0.14B |
0.09 |
0.13 |
|
|
10 |
0.12C
|
0.09 |
0.14 |
|
|
pH |
7.4 |
0.16A |
0.10 |
0.14 |
|
5.5 |
0.13B |
0.08 |
0.13 |
|
|
5.2 |
0.14B |
0.11 |
0.13 |
|
|
P-value |
pH |
0.001 |
0.052 |
0.275 |
|
HIS |
0.021 |
0.710 |
0.253 |
|
|
pH×HIS |
0.451 |
0.050 |
0.097 |
|
Means with different lowercase
letters are significantly different; means with different (P <
0.05). Uppercase letters within the same column are significantly different
|
LPS
content/KEU·mL-1 |
pH value |
Isc/mA (cm2·h)-1 |
Gt/mS
(cm2·h)-1 |
PD/mV (cm2·h)-1 |
|
0 |
7.4 |
0.05d |
3.70d |
1.15d |
|
5.5 |
0.16c |
4.05bc |
1.08d |
|
|
5.2 |
0.46b/ |
4.14bc |
6.66a |
|
|
30 |
7.4 |
0.03d |
2.71e |
1.74c |
|
5.5 |
0.13cd |
4.41bc |
1.24cd |
|
|
5.2 |
0.16c |
5.05b |
2.54b |
|
|
60 |
7.4 |
0.09d |
3.87bc |
2.75b |
|
5.5 |
0.15c |
4.17b |
2.44bc |
|
|
5.2 |
0.65a |
5.87a |
1.26d |
|
|
SEM |
|
0.043 |
0.228 |
0.125 |
|
Main effects |
||||
|
LPS |
0 |
0.22B |
3.96C |
2.96A |
|
30 |
0.11C |
4.06B |
1.84C |
|
|
60 |
0.30A |
4.64A |
2.16B |
|
|
pH |
7.4 |
0.06C |
3.43C
|
1.88B |
|
5.5 |
0.15B |
4.21B
|
1.58C |
|
|
5.2 |
0.42A |
5.02A |
3.49A |
|
|
P-value |
pH |
< .0001 |
< .0001 |
< .0001 |
|
LPS |
< .0001 |
< .0001 |
< .0001 |
|
|
pH×LPS |
< .0001 |
< .0001 |
< .0001 |
|
Means with different lowercase letters are
significantly different; means with different (P < 0.05). Uppercase
letters within the same column are significantly different
Table 5: Effects of
different pH × LPS treatments on rumen epithelial HRP and FITC flows and LPS
content on the serosal side in dairy goats
|
LPS
content/KEU·mL-1 |
pH value |
FITC/mmo l(cm2·h)-1 |
HRP/mol
(cm2·h)-1 |
LPS
content/KEU·mL-1 |
|
0 |
7.4 |
0.15c |
0.03b |
- |
|
5.5 |
0.15c |
0.12ab |
- |
|
|
5.2 |
0.21bc |
0.10ab |
- |
|
|
30 |
7.4 |
0.11d |
0.04b |
25.92 |
|
5.5 |
0.12cd |
0.05b |
21.94 |
|
|
5.2 |
0.38ab |
0.05b |
27.91 |
|
|
60 |
7.4 |
0.15c |
0.05b |
29.11 |
|
5.5 |
0.18c |
0.06b |
35.75 |
|
|
5.2 |
0.48a |
0.16a |
26.36 |
|
|
SEM |
|
0.056 |
0.042 |
3.730 |
|
Main effects |
||||
|
LPS |
0 |
0.17C |
0.08 |
- |
|
30 |
0.20B |
0.05 |
25.26
B |
|
|
60 |
0.27A |
0.09 |
30.41
A |
|
|
pH |
7.4 |
0.14B |
0.04 |
27.52B |
|
5.5 |
0.15B |
0.08 |
28.85A |
|
|
5.2 |
0.36A |
0.10 |
27.14B |
|
|
P-value |
pH |
<.0001 |
0.099 |
0.041 |
|
LPS |
0.007 |
0.101 |
0.048 |
|
|
pH×LPS |
0.036 |
0.047 |
0.472 |
|
Means with different lowercase
letters are significantly different; means with different (P
< 0.05). Uppercase letters within the same column are significantly
different
Table 8: Effects of
different pH × LPS × HIS treatments on rumen epithelial electrophysiological
parameters in dairy goats
|
pH × LPS ×
HIS treatments |
Isc/Ma (cm2·h)-1 |
Gt/mS
(cm2·h)-1 |
PD/mV (cm2·h)-1 |
|
7.4×60
KEU·mL-1×10 ng·mL-1 |
0.31c |
4.16b |
0.36b |
|
5.5×60
KEU·mL-1×10 ng·mL-1 |
0.76a |
3.79c |
0.28c |
|
5.2×60
KEU·mL-1×10 ng·mL-1 |
0.52b |
5.03a |
1.49a |
|
SEM |
0.004 |
0.108 |
0.114 |
|
P-value |
< .0001 |
< .0001 |
< .0001 |
a-c Means with
different superscript letters differ significantly (P < 0.05)
1 Isc = short-circuit current
2 Gt =
tissue conductance
3 HRP =
horseradish peroxidase
4 FITC =
fluorescein isothiocyanate
Table 9: Effects of
different pH × LPS × HIS treatments on rumen epithelial HRP and FITC flows and
LPS and HIS contents in the serosal side in dairy goats
|
pH × LPS ×
HIS treatments |
FITC/mmol
(cm2·h)-1 |
HRP/mol
(cm2·h)-1 |
LPS/KEU·mL-1 |
HIS/ng·mL-1 |
|
7.4×60
KEU·mL-1 ×10 ng·mL-1 |
0.62b |
0.18 |
41.94 |
0.29 |
|
5.5×60
KEU·mL-1 ×10 ng·mL-1 |
0.68a |
0.25 |
35.83 |
0.19 |
|
5.2×60
KEU·mL-1 ×10 ng·mL-1 |
0.60b |
0.39 |
41.18 |
0.19 |
|
SEM |
0.020 |
0.094 |
2.242 |
0.045 |
|
P-value |
0.012 |
0.275 |
0.09 |
0.241 |
Means with different lowercase
letters are significantly different, means with different (P < 0.05).
Uppercase letters within the same column are significantly different
factor, Isc
of ruminal epithelium incubated with mucosal addition of HIS-containing
solution was greatest (P < 0.05) at mucosal pH 5.2, Gt was
greatest (P < 0.05) at mucosal pH 5.5, and PD was lowest (P
< 0.05) at mucosal pH 5.5. The interaction between pH and HIS showed that
Isc and Gt of incubated ruminal epithelium were the
highest (P < 0.05) at mucosal pH 5.2-HIS 10 and at mucosal pH 5.5-HIS
10, respectively, and the PD was lowest (P < 0.05) at mucosal pH
5.5-HIS 10.
Table 7 summarizes data for the mucosal-to-serosal fluxes
of FITC and HRP. When HIS was the main factor, the FITC flow rate from mucosal
to serosal was greater (P < 0.05) in mucosal HIS 0.5 than that in
mucosal HIS 10, while the
HRP flow rate remained consistent. When pH was the
main factor, the FITC flow rate of ruminal epithelium incubated with mucosal
addition of HIS-containing solution at mucosal pH 5.2 was lowest (P
< 0.05), while the HRP flow rate was highest (P < 0.05). The
interaction between pH and HIS showed that the mucosal-to-serosal flux of FITC
at mucosal pH 5.5-HIS 10 was increased compared with mucosal pH 5.2-HIS 10,
whereas the HRP flow rate was decreased, and the concentration of HIS in the
serosal side showed no significant change (Table 7).
Interactions
of pH, LPS and HIS on rumen epithelial permeability
As seen in
Table 8, 9 the co-presence of pH, LPS and HIS had a significant effect on Isc,
Gt and PD of the incubated ruminal epithelium. Isc
was the highest in pH 5.5-LPS 60-HIS 10, whereas PD was the lowest, and the
difference between the treatments was significant (P < 0.05). Gt
reached the highest level (P < 0.05) in pH 5.2-LPS 60-HIS10.
The FITC flow rate of the incubated rumen epithelium was
greatest (P < 0.05) in pH 5.5-LPS 60-HIS 10. The HRP flow rate was
greater at mucosal pH 5.2 than for the other groups. No significant differences
among the treatments were observed for the concentrations of LPS and HIS in
the serosal side (Table 9).
Discussion
Previous
reports have clearly shown that SARA can compromise the rumen epithelial
barrier and increase rumen epithelial permeability (Steele et al. 2011; Klevenhusen et al.
2013; Meissner et al. 2017; Sun et al. 2018a). One of
our previous studies from the same experiment demonstrated that pH interacts
with both HIS and LPS to decrease the abundance of mRNA for genes involved in
tight junction protein of the ruminal epithelium, which are
probably related to increases in the permeability of the ruminal epithelium
(Sun et al. 2018b). In the
present study, we provide evidence that concurrent
presence of low pH with excessive LPS and HIS in the rumen might the main
trigger for increased rumen epithelial permeability.
The ruminal epithelium is a
stratified squamous epithelium consisting of four distinct strata with a
junctional complex that forms a barrier between the luminal contents and the
internal milieu. As a permeable barrier, its role is to facilitate absorption
of ions, water and nutrients, while at the same time preventing paracellular
permeation of microorganisms and toxic compounds
including LPS (Amaral et al. 2007; Liu et al. 2013). The Ussing
chamber could indicate the permeability of the ruminal epithelium by the
measurement of electrophysiological parameters (Vidyasagar and MacGregor 2016).
Increased Isc indicates an increase in the transport capacity of
ions through the epithelium, and significantly increased Gt after
rumen mucosal acidification indicates impaired rumen epithelial barrier
function and increased epithelial permeability. Since the PD value is
positively proportional to the epithelial resistance, the epithelial resistance
can represent the tight junction of the epithelial intercellular and paracellular permeability and can also be used
to monitor the rumen epithelial activity of ruminants (Ussing and Zerahn 1951).
In the present study, the Ussing chamber technique was used to monitor rumen
epithelial permeability in terms of Isc, Gt and PD of the
incubated ruminal epithelium and the fluxes of HRP and FITC. Our results
indicated that the Isc, as a measure of active electrogenic
electrolyte transport, as well as the Gt, as a measure of passive
ion permeability, significantly increased in the concurrent presence of low
mucosal pH with excessive addition of LPS and HIS, which indicated that the
rumen epithelial barrier functions were profoundly compromised.
Ruminal pH, VFA, osmolarity and LPS concentration have
been suggested as triggers for impairment of the rumen
epithelial barrier, because they are known to be detrimental for the rumen
epithelial barrier (Penner et al. 2011; Greco et al. 2018). Of
these, the ruminal pH clearly plays a crucial role and impairs epithelial
barrier function as indicated by increased permeability of the ruminal epithelium
(Penner et al. 2010). An in vivo study conducted by Klevenhusen et
al. (2013) demonstrated that low pH is a primary event preceding LPS
release and LPS translocation across the rumen epithelial barrier during SARA
(Enemark et al. 2002). In this study,
pH, LPS and HIS were applied to healthy ruminal epithelium in vitro on a
short-term basis. As a result of the single factor of pH, rumen epithelial
permeability increased, and tissue activity decreased with a decrease of
mucosal pH. Moreover, low mucosal pH in combination with 60 KEU·mL-1 LPS
induced a higher permeability of the ruminal epithelium, again
suggesting that the influence of both pH and LPS on the rumen epithelial
permeability was greater than that of pH alone. This may indicate that the combined
effect of LPS and pH in the rumen of goats suffering from SARA aggravated the
destruction of the ruminal epithelium. The ruminal LPS concentration has
been found to increase to 26,915 EU/mL when SARA was induced (Gozho et al.
2006). SARA was the main cause of rumen epithelial barrier dysfunction, which
is associated with low pH and high osmotic pressure (Aschenbach et al. 1998; Dong et al. 2013). In addition, HIS or inflammatory responses during
acidosis can also impair the barrier function of the ruminal epithelium.
HIS is produced by the decarboxylation of histidine
catalyzed by histidine decarboxylase. Under normal physiological conditions,
the body generally contains HIS, but in very small amounts (Slyter 1976; Martens et al. 1987). Trace
amounts of HIS can be involved in the collective regulation of a variety of
physiological functions, such as nerve, endocrine, gastrointestinal and
circulatory regulation (Klingspor et al. 2013). The notion that SARA is
accompanied by the increase of abnormal metabolites, such as HIS, is widely
accepted. When there is so much HIS in the rumen that it exceeds the normal
metabolic capacity of the body, HIS will be transported into the blood
circulation through the damaged ruminal epithelium and cause
inflammation. This further aggravates the SARA and causes the further
destruction of the ruminal epithelium (Gozho et al. 2007).
Cheng (2016) showed that in dairy goats the
concentrations of LPS and HIS in plasma and rumen were significantly increased
during grain-induced SARA. The increased HIS or LPS translocating from the
gastrointestinal tract into the blood can down-regulate the expression of
gastrointestinal tight junction protein and embedded protein, and increase the
apoptosis rate of epithelial cells, resulting in further damage of the
epithelial barrier (Pilachai et al. 2012). Aschenbach et al.
(1998) showed that HIS-induced apoptosis increased cell shedding and interfered
with nuclear division and cell maturation. This might mean that HIS could
interfere with the regeneration of epithelial cells during SARA, thus causing
cell damage and triggering an inflammatory reaction. In the present study, our
data showed that the rumen epithelial permeability was significantly increased,
and tissue activity was reduced in mucosal pH 5.2-HIS 10. This suggests that
lower mucosal pH with excessive HIS induced more severe barrier dysfunction.
Our data are similar with the results of Penner et al. (2010) and
Meissner et al. (2017), which indicated a low pH of 5.2 has only
moderate effects on the ruminal epithelial barrier, whereas concurrent presence of low pH with high SCFA concentrations
can trigger a profound impairment of epithelial barrier function (Hu 2008; Hu
et al. 2015). Therefore,
we concluded that the disruption of the rumen epithelial barrier function was
not caused only by pH, and the concurrent presence of pH-HIS or pH-LPS might
contribute to the more obvious increases of rumen epithelial permeability in
the present study.
Additionally, the permeability of
marker molecules of different sizes (HRP as a large marker,
FITC as a small marker) was also measured in the present study. Compared with
mucosal pH alone or concurrent presence of pH-LPS and pH-HIS, significant
increases in mucosal-to-serosal fluxes of HRP and FITC coupled with enhanced Isc and Gt
were observed in the concurrent presence of
mucosal pH with LPS and HIS. An increased flux of FITC or HRP reflects
increased paracellular permeability and impaired ruminal barrier. Our results
further suggested that the combined treatment of pH,
LPS and HIS contributes to triggering higher epithelial permeability and more
profound barrier dysfunction. Thus, subacute rumen acidosis is a process
involving pH, LPS, HIS and their synergistic interactions. One of our previous
studies in dairy goats reported that a concurrent increase of both HRP and FITC
mucosal-to-serosal flux rates were observed during SARA (Sun et al.
2018b), which is somewhat inconsistent with the results of the present study. In the
short-term pH×LPS×HIS cross-treatment, the small molecule (FITC) permeability
of the incubated ruminal epithelium was increased, but the permeability
to large molecules (HRP) was not increased significantly. This observation may
also be related to the different absorption mechanisms of large and small molecular
markers by ruminal epithelium in the short term. In order to ensure the
health of the animal, the ruminal epithelium may normally prevent penetration of
large molecule toxic substances and only allow small molecules, such as
amino acids and water, to pass through (Oba et al. 2005).
Conclusion
Our results have shown an increased rumen
epithelial permeability during SARA is caused by the combined action of low pH
with high LPS and high HIS concentrations, which is critical for the impairment
of the rumen epithelial barrier. Our study also showed
that concurrent presence of LPS 60 KEU‧mL-1 and HIS 10 ng‧mL-1
at mucosal pH 5.5 can aggravate rumen epithelial barrier dysfunction.
Acknowledgments
This
research was supported by grants from China National Natural Science Foundation
of China (no. 31101739and no. 31472124), Inner Mongolia Natural Science
foundation (no. 2019MS03031), China Agriculture Research System (no. CARS-36),
Innovation Fund of Inner Mongolia Agricultural and Animal Husbandry (no.
2021CXJJM02) and Open Project of Beijing Key Laboratory
of Dairy Cow Nutrition, Beijing University of Agriculture. The
authors declare no conflict of interest in this study.
Author Contributions
We thank study participants for their contribution, Y.Y. Sun:
writing-original draft preparation and writing-reviewing & editing, M. Gao:
supervision and project administration, L.W. Song, M. Xu, C. Li, Y. Li, and L.Q.
Chen: experimental sample and data collation, H.L. Hu: Writing-reviewing and
editing and L.S. Jiang: funding acqusition.
Conflict of Interest
The authors
declare no conflict of interest in this study
Data
Availability
All data
presented in this study are available upon request
Ethics Approval
The
experimental design and procedures were approved by the Animal Care and Use
Committee of the Inner Mongolia Academy of Agricultural and Animal Husbandry
Sciences and were performed in accordance with relevant guidelines formulated
by the Ministry of Agriculture of the People’s Republic of China.
Funding Source
This study was
supported by grants from China National Natural Science Foundation of China
(no. 31101739 and no. 31472124), Inner Mongolia Natural Science foundation (no.
2019MS03031), China Agriculture Research System (no. CARS-36), Innovation Fund
of Inner Mongolia Agricultural and Animal Husbandry (no. 2021CXJJM02) and Open
Project of Beijing Key Laboratory of Dairy Cow Nutrition, Beijing University of
Agriculture. The authors declare no conflict of interest in this study.
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