Experimental Study Of Bushen Huatan Decoction Affect Inflammatory Factors In Bone Tissue By Regulating TLR4/MyD88/NF-κB Signaling Pathway

PurposeTo explore the internal mechanism of Bushen Huatan Decoction in affecting bone metabolism by affecting the intestinal barrier and changing the expression levels of inflammatory factors in bone tissue.MethodsForty SPF-grade six-month-old female SD rats were randomly divided into a sham operation group,a model group,a estradiol valerate group,and a Bushen Huatan Formula group.After adaptive feeding for 1 week,food and water deprivation for one night,the rats were anesthetized by intraperitoneal injection of 10% chloral hydrate at 30 m L kg-1,and skin was prepared.The rats were fixed in the prone position on the rat board and the skin around the operation area was sterilized.An incision of about 1.5cm was made at the side of spine under the bilateral rearmost ribs.All layers of tissues were successively cut into the abdominal cavity,and the ovaries were found near the lower pole of the kidney.The model group,estradiol valerate group and Bushen Huatan Formula group tied and removed the bilateral ovaries with No.4 thread.The sham group only removed the adipose tissues around the ovaries with the same weight as the bilateral ovaries.The incisions were sutured in layers,and each animal was given intraperitoneal injection of penicillin200,000 U kg-1 for three consecutive days and reared separately.After one week,gavage was performed.The rats in the sham operation group and the model group were orally administered with 0.9% normal saline 1ml/100 g per day.The estradiol valerate group was prepared into estradiol valerate suspension with normal saline and gavage was performed at 0.184 mg kg-1.The rats in the Bushen Huatan Decoction group were intragastrically administered with 9.4 g kg-1 of the concentrated decoction of Bushen Huatan Decoction.After12 weeks of intragastric intervention,the rats were sacrificed and the samples were taken for detection.Micro-computed tomography(Micro-CT)was used to scan the microstructures of the humerus bones in rats and restore the three-dimensional structure of the trabecular bone in the region of interest,to calculate the BV/TV,Tb.N,Tb.TH,Tb.SP values of the humerus.The expression levels of TLR4,My D88,and p-NF-κB p65 proteins in bone tissues were detected by ELISA and Western Blot,respectively.The mRNA expression levels of TLR4,My D88,and NF-κB p65 as well as the mRNA expression levels of pathway-related inflammatory factors IL-1βand IL-6 in bone tissues were detected by reverse transcription-polymerase chain reaction(RT-PCR).ResultMicro-CT scan of the humerus bone microstructure showed that compared with the sham operation group,the BV/TV,Tb.N and Tb.Th values of the trabecular bone in the model group were significantly decreased,and Tb.Sp was significantly increased(P<0.05),indicating that the bone microstructure was disordered.Compared with the model group,the BV/TV,Tb.N and Tb.Th values of trabecular meshwork in the estradiol valerate group and Bushen Huatan Decoction group were increased,while Tb.Sp was significantly decreased(P<0.05),and the bone microstructure was improved.The detection of serum LPS level by ELISA showed that compared with the sham operation group,the serum LPS concentration of rats in the model group was significantly increased(P<0.05).Compared with the model group,the serum LPS levels of estradiol valerate group and Bushen Huatan Decoction group were decreased significantly to different degrees(P<0.05).The serum LPS level of estradiol valerate group was closer to that of the sham operation group.The protein expressions of TLR4,My D88 and p-NF-κB p65 in bone tissue detected by WB method showed that compared with the sham operation group,the protein expressions of TLR4,My D88 and p-NF-κB p65 in bone tissue of the model group were significantly increased(P < 0.05).Compared with the model group,the protein expression levels of TLR4,My D88 and p-NF-κB p65 in the estradiol valerate group and Bushen Huatan Decoction group were decreased significantly(P<0.05).The mRNA levels of TLR4,My D88,NF-κB p65,IL-1β and IL-6detected by RT-PCR in bone tissue showed that compared with the sham operation group,the mRNA expression levels of TLR4,My D88,NF-κB p65,IL-1β and IL-6 in bone tissue of the model group were significantly increased(P < 0.05).Compared with the model group,the expression levels of TLR4,My D88,NF-κB p65,IL-1β,and IL-6mRNA in the estradiol valerate group and Bushen Huatan Decoction group were decreased to different degrees(P<0.05).ConclusionBushen Huatan Decoction could reduce the serum LPS content of OVX rats,regulate the over-activated TLR4/My D88/NF-κB pathway in bone tissue,reduce the expression level of inflammatory factors in bone tissue,affect the bone microenvironment,improve the bone metabolism imbalance,and inhibit the progression of PMOP.